Enhanced treatment of

coke oven plant wastewater

(ECOWATER)

Research and
Innovation

EUR27075 EN

EUROPEAN COMMISSION
Directorate-General for Research and Innovation
Directorate D Key Enabling Technologies
Unit D.4 Coal and Steel
E-mail: rtd-steel-coal@ec.europa.eu
RTD-PUBLICATIONS@ec.europa.eu
Contact: RFCS Publications
European Commission
B-1049 Brussels

European Commission

Research Fund for Coal and Steel

Enhanced treatment of coke oven
plant wastewater
(ECOWATER)

E Aries, J Chen, D Ciappara, S Pearson

Tata Steel UK Limited
Swinden Technology Centre, Moorgate, Rotherham, South Yorkshire, S60 3AR, United Kingdom

W Huang, J Berry
University of Sheffield
Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom

D Mirabile
Centro Sviluppo Materiali SpA
, Via di Castel Romano 100, 00128 Roma, Italy

Grant Agreement RFCR-CT-2010-00010

1 July 2010 - 31 December 2013

Final report
Directorate-General for Research and Innovation

2015

EUR 27075 EN

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Luxembourg: Publications Office of the European Union, 2015
ISBN 978-92-79-45179-9
doi:10.2777/77100
European Union, 2015
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TABLE OF CONTENTS

1.

Final Summary ................................................................................................................................................6

2.

Scientific and technical description of the results .......................................................................................13

2.1

Project objectives ...................................................................................................................................13

2.2

Management and coordination aspects ..................................................................................................13

2.2.1

Structure of the ECOWATER project .....................................................................................................13

2.2.2

Progress against plan / Gantt chart .......................................................................................................13

2.3

Work Package 1: Fundamental studies ..................................................................................................15

2.3.1 Background on the European Water Framework Directive ...................................................................15

2.3.2 Task 1.1: Identification of key water discharges in cokemaking .......................................................17
2.3.2.1
Inventory of Tata Steel coke oven BET plants in Europe ...............................................................17
2.3.2.2
Typical emission discharges for Tata Steel BET plants in Europe ............................................19
2.3.3 Task 1.2: Identification of appropriate sampling procedures for water characterisation .................20
2.3.3.1
Sampling methods for wastewater effluents...............................................................................20
2.3.3.2
Sampling methods for ambient water (local rivers) ..................................................................21
2.3.4 Task 1.3: Development of analytical methods for the analysis of organic and inorganic PHS and PS
in water matrices .............................................................................................................................................23
2.3.4.1
Analytical methods used for the analysis of PAHs in waste water effluents .............................23
2.3.4.2
Analytical methods for trace metals in water ............................................................................25
2.3.4.3
Analytical methods used for mercury analysis in water ............................................................27
2.3.5 Task 1.4: Development of biosensor arrays for the detection of toxicity in coke oven effluents ........27
2.3.5.1
Biosensors .................................................................................................................................27
2.3.5.2
Development of a toxicity biosensor for PAHs ..........................................................................29
2.3.5.2
Development of of a specific biosensor for n-alkane / oil detection based on Acinetobacter
baylyi ADP1 ................................................................................................................................................32
2.3.6 Task 1.5: Fundamental studies into the interactions between organic pollutants and heavy metals in
coke oven effluents and effects upon toxicity responses ..................................................................................38
2.4
Work Package 2: Characterisation of coke oven and by-product plant effluents using chemical and
biological approaches.........................................................................................................................................43
2.4.1 Task 2.1: Evaluation of the efficiency of waste water treatment steps for the removal of PHS / PS
and the detoxification of coke oven effluents ...................................................................................................43
2.4.1.1
Measurement of trace metal / PAH abatement efficiencies at Tata Steel BET Plant A ............43
2.4.1.2
Measurement of trace metal / PAH abatement efficiencies at Tata Steel BET Plant B .............47
2.4.2 Task 2.2: Determination of the concentrations of PHS/PS and the toxicity of treated water at the
final BET plant discharge points .....................................................................................................................49
2.4.2.1
Concentrations of PHS/PS at Tata Steel Plant A.......................................................................49
2.4.2.2
Concentrations of PHS/PS at Tata Steel Plant B.......................................................................53
2.4.2.2
Direct Toxicity Assessment ........................................................................................................55
2.4.2.2
Results from Direct Toxicity Assessment on Tata Steel effluents (cokemaking / steelmaking) ..56
2.4.3
Task 2.3: Mass inventory releases of PHS / PS in cokemaking .....................................................58
2.4.3.1
Mass inventory releases of PHS / PS at Tata Steel Plant A .......................................................58
2.4.4
Task 2.4: Identification of priority areas for improving the treatment and detoxification of
wastewater from coke making operations .......................................................................................................65
2.5
Work Package 3: Impact studies ............................................................................................................66
2.5.1 Task 3.1: Impact studies of coke oven effluents on the chemical quality of local river basins...........66
2.5.1.1
Impact studies carried out at Tata Steel Plant A .......................................................................66
2.5.1.2
Impact studies carried out near a coke oven BET plant in Italy ................................................72
2.5.2 Task 3.2: Impact studies of coke oven effluents on the biological and ecological quality of local river
basins 74
2.5.2.2
Tests on environmental samples using the n-alkane biosensor Acinetobacter baylyi
ADPWH_alk ................................................................................................................................................75
2.3.5.3
Task 3.3: Assess the impact of cokemaking effluents with respect to transitional areas of
exceedances.................................................................................................................................................77
2.5.3.1
Definition of mixing zones .........................................................................................................77
2.5.3.2
Impact Assessment on the main effluent discharge points at Tata Steel Plant A .......................78

2.6
Work Package 4: Enhance PAH degradation in coke making BET plants using state-of-the-art
molecular biology techniques .....................................................................................................................81
2.6.1 Background on the molecular biology techniques used in the ECOWATER project .....................81
2.6.2 Outline of the microbiological analytical methods used in the ECOWATER project ........................85
2.6.3 Task 4.1: Studies of cultivable PAH-degrading bacterial strains present in wastewater and
biosludges from coke oven BET plants ............................................................................................................85
2.6.4 Task 4.2: Studies of non-cultivable PAH-degrading bacterial strains present in wastewater and
biosludges from coke oven BET plants ............................................................................................................87
2.6.4.1
Qualitative analysis of microbial diversity in coke oven activated sludges using DGGE analysis of
16s rRNA 87
2.6.4.2
Analysis of microbial diversity in coke oven activated sludges using Stable Isotope Probing
and Magnetic Nano Particles (MNPs) ........................................................................................................92
2.6.5 Task 4.3: Revealing controlling factors affecting PAH biodegradation in coke oven waste water ...99
2.6.5.1
Physiological testing of MNP-free cells using the Biolog high- throughput phenotypic assay for
carbon sources ................................................................................................................................................99
2.6.5.2
Physiological testing of MNP-free cells using the Biolog high-throughput phenotypic assay for
nitrogen sources ........................................................................................................................................100
2.6.6 Task 4.4: Enhancement of PAH biodegradation in coke oven BET plants ......................................102
2.7
Work Package 5: Novel abatement solutions Industrial application and transferability ..................103
2.7.1 Task 5.1: Study into the use of heterogeneous photocatalysis using Anatase (TiO2) for the
biodegradation of coke oven waste water .....................................................................................................103
2.7.1.1
Background on photocatalysis using Anatase .........................................................................103
2.7.1.2
Background on zeolites............................................................................................................105
2.7.1.3
Laboratory tests results using photocatalytic treatment using anatase ..................................106
2.7.1.4
Laboratory tests results using zeolite adsorption ....................................................................114
2.7.2 Task 5.2 Scaling up of the anatase (TiO2) photocatalysis process and cost benefit analysis ...........118
2.7.2.1
Design of the photocatalytic reactor - Fluid-dynamic simulations ........................................118
2.7.2.2
Results from the pilot plant tests .............................................................................................121
2.7.3 Task 5.3. Study into the use of improved filtration sorbents for the removal of organic and inorganic
pollutants in wastewater from coke making operations ................................................................................126
2.7.3.1
Background on Powdered Activated Carbon Treatment (PACT) ............................................126
2.7.3.2
Commissioning and optimisation of a pilot scale BET plant by Tata Steel .............................127
2.7.3.3
Activated lignite injection laboratory tests undertaken by Tata Steel .....................................133
2.7.3.4
Activated lignite injection pilot scale tests undertaken by Tata Steel ......................................136
2.7.4 Task 5.4. Study into the use of Fuzzy filtration for the removal of organic and inorganic pollutants in
wastewater from cokemaking operation ........................................................................................................138
2.7.4.1
Background on fuzzy filtration technology ..............................................................................138
2.7.4.2
Pilot scale tests using fuzzy filters realised by Tata Steel ........................................................140
2.7.4.3
Cost benefit analysis of the fuzzy filtration technology ...........................................................146
2.8

3.

Work Package 6 Project management / Transfer of knowledge ........................................................147

2.8.1 Task 6.1 - Project coordination (Tata Steel) ................................................................................147
2.8.2 Task 6.2 Evaluation of results, meeting, reporting and publications ........................................147
2.8.3 Task 6.3 Dedicated workshop organisation ..............................................................................148

Conclusions, exploitation and impact of the research results ..................................................................149

3.1

Challenges addressed by the ECOWATER project...............................................................................149

3.2

Impact of the ECOWATER project on the EU steelmaking industry ....................................................149

3.3

Dissemination of the results / possible patents .....................................................................................153

4.

References ....................................................................................................................................................155

5.

List of abbreviations ....................................................................................................................................160

6.

Appendix 1 ...................................................................................................................................................162

7.

Appendix 2 ...................................................................................................................................................164

8.

Appendix 3 ...................................................................................................................................................166

9.

Appendix 4 ...................................................................................................................................................168

10.

Appendix 5 ...............................................................................................................................................170

List of Figures .......................................................................................................................................................174

List of Tables ........................................................................................................................................................178

ECOWATER: ENHANCED TREATMENT OF COKE OVEN PLANT WASTEWATER

RFCS Grant Agreement Number RFCR-CT-2010-00010
Final report for the period 1st July 2010 - 31st December 2013
1.

Final Summary

With the introduction of the European Union Water Framework Directive (WFD), the steel industry is
facing significant challenges to reduce wastewater emissions of Priority Substances (PS) and Priority
Hazardous Substances (PHS) including trace metals (i.e. Cd, Ni, Pb, Hg) and PAHs (i.e.
Benzo[a]pyrene). With the WFD coming into force, tighter environmental quality standards were
introduced for these substances in local river basins.
The first objective of ECOWATER was to address the challenges faced by the steel industry to fulfil
the requirements of the WFD. At the beginning of the project, there was a significant gap in the
knowledge across the steel industry on the emissions of these substances in wastewater effluents
from cokemaking and ironmaking / steelmaking operations. Research was required in this area to
identify the main emission sources, determine effluent toxicity, quantify emissions and measure the
environmental impacts of steelworks.
The second aspect of ECOWATER was concerned with the investigation of the potential of state-ofthe-art molecular biology techniques for understanding further biodegradation processes in coke
oven biological effluent treatment (BET) plants. In recent years, there has been an explosion in
microbial molecular biology techniques. The environmental sector is well placed to adapt many of
these techniques to gain efficiencies across a range of biological treatment streams. Novel molecular
biology techniques could not only provide the flexibility and adaptability that is required in
treatment but could deliver significant environmental improvements by enhancing the
biodegradation capabilities of bacteria therefore minimising the use of other less cost-effective
abatement solutions. In the project ECOWATER, it was proposed, for the first time, to use a range
of novel state-of-the-art molecular biology techniques to identify unculturable microorganisms that
plays a major role in the degradation of organic pollutants such as phenols and PAHs.
The third important aspect of the ECOWATER project was concerned with the development of
innovative technological solutions for the removal of PS and PHS in wastewater effluents from the
steel industry in order to ensure that integrated steelworks comply with the final discharge consent
in the future. In particular, the potential application of a photocatalytic oxidation process using
anatase (TiO2) was studied. Photocatalytic oxidation is an interesting approach for water treatment
because the process gradually breaks down the contaminant molecules and therefore no sludge
requiring disposal to landfill is produced. In parallel, work was carried out at the pilot scale to
investigate the potential of advanced adsorption techniques using sorbents such as powdered
activated carbon (activated lignite) and zeolites which present high adsorption capabilities,
respectively, for the removal of organic and inorganic pollutants. Finally, pilot scale tests were also
carried out to investigate the potential for coke oven effluent treatment of a new high efficiency
filtration technology (Fuzzy filtration), which has only been used in municipal waste biological
effluent treatment plants and oil refineries to date.
Work package 1 (WP1) focused on the development, validation and application of specialised
analytical methods for the analysis at trace levels (low ppb range) of the WFD substances. This was
achieved by developing and validating a sample preparation and analytical procedure for the
analysis of PAHs using solid phase extraction (SPE) and gas chromatography mass spectrometry
(GC-MS) with very low limit of detection (ca. 10 to 40 ng / L). For trace metals such as Cd, Pb, Ni,
Fe, Al, and As, a procedure was also developed for analysis of steelmaking / cokemaking effluents
using ICP-MS (inductively coupled plasma mass spectrometry) with low limit of detection ranging
from 10 to 200 ng / L. Mercury analyses were carried out separately out using cold vapour atomic
fluorescence (CVAF) because some interferences were observed in ICP/MS. These methods were
used by Tata Steel in work package 2 to characterise over a three-year period the emissions of PAHs
and trace metals at two major integrated steelworks in the UK.
In work package 1, work also focused on the development of biosensors for the environmental
detection of toxicity and chemical compounds in the environment. In particular, two biosensors were
developed by Sheffield University for the detection of PAHs and n-alkanes (ie. oils) in effluents from
the steel industry. The PAH biosensor was constructed by cloning the human cytochrome P450 (CYP)
in a soil bacterium (Acinetobacter baylyi). All PAH compounds are oxidised by CYP which
metabolically converts PAHs into carcinogens. Therefore, the advantage of using CYP was to develop
a biosensor that could respond to all PAHs (including PAH mixtures) rather than having to develop a
biosensor for each individual PAH compound. The alkane bioreporter developed in the project was

also constructed through cloning of the soil bacterium Acinetobacter baylyi. The resulting biosensor
showed a unique ability to adhere to an interface of oil and water and to emulsify mineral and crude
oils into oil droplets at the micrometre level. It also was able to respond to alkanes with various
chain lengths from C7 to C36, the first biosensor reported in environmental microbiology exhibiting
such a wide range of detection.
Finally, studies were carried by CSM out to investigate the potential concomitant and/or inhibitory
effects of PAHs and trace metals upon toxicity responses when using a standard ecotoxicity test
(Microtox) for analysis. The work carried out led to the development of a model enabling the
calculation of the EC50 value as a function of phenol, trace metals and naphthalene concentrations in
the samples.
The objective of Work package 2 (WP2) was to determine the current abatement efficiencies at
several typical coke oven BET plants and to understand the distribution of trace metals and PAHs
between the dissolved phase and the suspended solids. Another objective was to provide emissions
data for all the key WFD substances from all the processes involved in steelmaking / ironmaking and
cokemaking in order to build a robust emissions inventory of sources that have been poorly
characterised in the past.
In the initial phase of the project, analysis of untreated coke oven effluents showed that species
such as Cd, Pb, Al and Zn were almost exclusively associated with the suspended particulate matter
whereas elements such as V, As, Cu and Ni were predominantly present in the dissolved phase. With
regard to PAHs, the results indicated that only low-molecular weight PAHs such as naphthalene,
acenaphthene and fluorene were found in the dissolved phase whereas medium- and high-molecular
weight PAHs were associated with the suspended particulate matter (> 90%). In terms of
abatement, the results obtained indicated that the BET plant treatment efficiency for low-molecular
weight PAHs such as naphthalene was very high (> 99.9%). However, after treatment, there were
some residual medium-molecular weight PAHs (such as pyrene and fluoranthene) and highmolecular weight PAHs (such as benzo[a]pyrene) present in the final discharge effluents. For these
compounds, the abatement efficiency typically ranged from 60% to 80%. For the trace metals, the
abatement efficiencies for Fe, Co, As, Cd, and Pb typically ranged from 60% to 70%, but were
somewhat lower for aluminium (ca. 53%) and chromium (ca. 35%). The lowest abatement
efficiencies were observed for nickel and copper (< 5%) and zinc (ca. 11%).
In the second phase of the project, a significant number of measurements over a 3-year period were
carried out by Tata Steel at two major integrated steelworks in the UK to characterise and quantify
the main emission point sources of trace metals and PAHs in the steel industry. This work led to
identify without ambiguity that cokemaking activities were responsible for >90% of PAH emissions
in wastewater from the steel industry. Interestingly, the two sites investigated exhibited very
different PAH annual mass releases despite the fact that their steel production capacity was similar
(ca. 4 million tons / annum). The results showed that Tata Steel Plant A exhibited much lower
annual mass releases of PAHs (21 to 71 kg / annum) than Tata Steel Plant B (423 to 480 kg /
annum). The reasons for these differences were linked to the fact that much higher concentrations
of suspended solids were measured in the treated effluent of BET plant B (ca. 400 mg / L) compared
to BET plant A (ca. 20 to 50 mg / L). During this investigation, it was found that treatment stability
at BET Plant A was much better than BET plant B. In particular, thiocyanate treatment was relatively
poor at BET plant B owing to very strong coke oven liquor which may inhibit the treatment of
thiocyanate and also the possibility of unwanted nitrification occurring at the plant which may also
inhibit thiocyanate treatment. When the treatment of thiocyanate was poor, suspended solid
concentrations in the treated effluent increased significantly. Since PAHs are mostly bound to
suspended solids, this resulted in much higher PAH releases at plant B. At BET plant B, in order to
reduce significantly PAH emissions, it was therefore recommended that the factors inhibiting
thiocyanate treatment should be tackled first to establish a stable treatment process.
With regard to trace metal emissions, the work carried out in the project showed that the three most
significant trace metals emitted in terms of volume from an integrated steelworks were Fe, Zn and
Pb. Ironmaking (BF) and steelmaking (BOS) operations were the main source of Fe (ca. 70%) and
Pb (ca. 90%). Fe, Pb and Zn were almost exclusively particle-bound. Zn was originating from
ironmaking / steelmaking (ca. 60%) and cokemaking (ca. 40%) activities. With regard to Cd and Ni,
Ni was emitted in greated quantities than Cd. The main sources of nickel were ironmaking /
steelmaking (ca. 60%), cokemaking (ca. 10%) and the rolling mills (ca. 10%). Cd sources were
mostly from ironmaking / steelmaking (ca. 74%) and continuous casting (ca. 15%). In both of these
these streams, cadmium was mostly found in the dissolved phase. Arsenic was emitted from two
major sources: ironmaking / steelmaking (ca. 50%) and cokemaking BET plants (ca. 30%). Finally,
mercury emissions were very low at all discharge points.

Two complete emissions inventory were realised at major integrated steelworks. This work
constitutes the first source of information across the steel industry concerning the emissions of WFD
substances in effluents of the steelmaking / ironmaking and cokemaking processes. The data
reported in the report (ie. generic emission factors / annual mass releases) could be used by other
steelmakers in Europe to carry out preliminary assessment of their releases of PHS / PS. It will also
be useful to other steelmaking companies to compare their current performance / level of emissions
of WFD substances and establish benchmark annual mass releases in the future.
More effort is required in the future to understand the relatively high variations observed in PAH and
trace metal emissions both in the cokemaking and steelmaking / ironmaking effluents. It is thought
that these emissions are greatly influenced by the quality of the raw materials used (ie. coke, iron
ores) as well as process parameters. Investigating the effects of raw material inputs / process
parameters upon the chemical quality of effluents in the steel industry was outside the scope of the
ECOWATER project, but it is clear that more work is required in this area.
Work package 3 (WP3) focused on measuring the environmental impact of steelmaking /
cokemaking effluent in local river basins in respect of the environmental quality standards (EQS) set
by the WFD.
First, a series of toxicity tests were carried out in WP3 to assess the toxicity of cokemaking /
steelmaking effluents. For this work, Direct Toxicity Assessment (DTA) tests using living organisms
for the measurement of the EC50 were carried out. A series of ecotoxicity tests were selected for
freshwater (ie. Daphnia Magna / Algae inhibition of growth), and seawater (ie. Oyster Embryo-larval
/ Tisbe Battagliai / Marine algae inhibition of growth), and they were applied to cokemaking /
steelmaking effluents. Although the DTA tests provided a good estimation of the EC50 value, they
were very expensive and proved extremely difficult to organise / set-up owing to a range of
technical limitations including the need for a minimum of 3-week period for preparation and growth
of the organisms and the necessity of analysing the samples within 12-h of collection.
Another significant development in the ECOWATER project was concerned with the use, for the firsttime in the steel industry, of passive sampling techniques such as DGT (Diffusive gradient in thin
films samplers) and SPMD (Semi-permeable membrane devices) for monitoring the concentrations
of trace metals and PAHs in local rivers near the effluent discharge points. As opposed to standard
spot sampling techniques which only yields an instantaneous measurement of pollutant levels and
require the use of expensive electrically-powered equipment, passive samplers are left unattended
in the environment for periods of approximately three-weeks allowing continuous monitoring of
aqueous contaminant levels. Consequently, the problem of potentially significant short-term
concentration variations is addressed since only time-weighted average (TWA) concentrations are
determined using passive samplers. In addition, they often provide a direct measurement of the bioavailable fraction for the pollutants of concern, which is essential to assess the contamination of
local rivers by trace metals or PAHs. The two types of passive samplers were tested during a major
impact study at a UK integrated Works Tata Steel Plant A). This campaign showed that the results
obtained using DGTs and SPMDs were useful in assessing the ambient concentrations of a range of
trace metals (ie. Ni, Cd, Pb, Fe, As, Zn, Cr and Cu) and PAHs (including B[a]P) in respect of the EQS
proposed in the WFD. For PAHs, naphthalene and anthracene concentrations were much lower than
the EQS at all locations. The EQSs for fluoranthene, benzo[a]pyrene and benzo [b] +
benzo[k]fluoranthene were exceeded at some locations. The EQS for indeno [1,2,3-c,d]pyrene +
benzo[g,h,i]perylene was largely exceeded at all locations. However, the likelihood of being able to
reach concentrations below EQS in water bodies for these compounds appeared very difficult
considering that the EQS defined in the WFD were extremely low, and below typical limits of
detection for the analysis of PAH compounds using modern analytical techniques. For instance, the
LoD defined for indeno[1,2,3-c,d]pyrene + benzo[g,h,i]perylene using the analytical procedure
developed by Tata Steel was 16 ng / L, but the EQS defined in the WFD was 2 ng / L. This research
showed that the EQS defined in the WFD for PAHs were unrealistic for some compounds, however it
is likely that they will be changed to biota EQS in the future.
During the sampling campaign, it appeared that the concentrations of trace metals determined using
the DGTs were below the EQS (ie. for cadmium, lead and nickel) even at the steelworks location
situated downstream from most of the steelmaking discharges. Overall, the DGT results were
typically slightly lower than the results obtained using the spot sampling techniques and the analysis
of the dissolved fraction. This was expected since only weakly bound metal complexes in solution
can be sampled using DGTs. Larger complexes, which can be predominant when humic levels are
high, would not be sampled by DGTs but would be detected in spot sampling. In terms of EQS,
whilst DGT samples did not show any exceedances, cadmium in some spot samples were found to
largely exceed the EQS at some locations.

Overal, passive sampling techniques were much easier to implement and less costly overall than
standard spot sampling techniques. The use of these techniques should be promoted across the
steel industry for impact assessement purposes in the future. These sampling techniques for impact
assessment are generic enough to be directly used by other coke plants in order to ensure that they
will be able to comply with the EQS defined by the Water Framework Directive.
Work package 4 (WP4) focused on the use of state-of-the-art molecular biology techniques to
study the microbial communities involved in the degradation of key pollutants (ie. phenols / PAHs) in
coke oven BET plant biosludges. This was one of the most innovative aspects of the project. With
the development of advanced molecular biology techniques in the field of environmental
microbiology over the past few decades, microorganisms in activated sludge processes can now be
studied in a culture independent manner. This new field of molecular microbial ecology has opened
up new possibilities for studying microbial communities at the genetic level leading to a far greater
appreciation of their diversity in nature. In the project, techniques such as 16s rRNA sequencing,
denaturing gradient gel electrophoresis (DGGE) and stable isotope probing (SIP) have been
employed to study and quantify the microbial diversity in coke oven biosludges. In addition, a
completely new approach using magnetic nanoparticles (MNP Magnetic Nanoparticles) to
functionalise living cells and isolate selectively degraders has been developed for the first time in
this project.
First, standard cultivation methods were tested to isolate the cultivable species that were actually
present in coke oven biosludges and involved in phenol and PAH degradation. Although several
bacteria were isolated and identified after sequencing of their DNA, it was very difficult to obtain
quantitative data from this type of analysis, and therefore it was not possible to ascertain which of
the bacteria isolated were predominantly involved in the degradation of phenols or PAHs. Indeed, it
is thought that typically >90% of the bacteria present in an activated sludge are actually
uncultivable. Therefore, although simple cultivation techniques could provide useful qualitative data
between activated sludges, it was found that there were significant limitations to this type of
approaches.
Further analyses were carried out to study and compare the profiles obtained by DGGE analysis of
the 16srRNA isolated after a total DNA extraction of sludge samples. This type of analysis revealed
clear differences between the microbial communities profiles of all the BET plants investigated.
Clearly, each BET plant exhibited a distinct and unique bacterial community fingerprint. From these
profiles, it was clear that the coke oven biosludges of each site were characterised by a great
diversity in bacterial species including some predominant species. Interestingly, the analysis of
several of these biosludges was repeated after several months and the same fingerprint was
obtained indicating the stability of the bacterial communities at each site. However, this technique
did not allow identifying with certainty phenol and PAH degraders, therefore only qualitative data
were obtained. The DGGE method, however, enabled to identify changes in the composition of
microbial communities when a loss of treatment occurred at the BET plant where a clear change of
the bacterial community fingerprint was observed.
The most successful technique employed for bacterial identification was a method specifically
developed in this project using MNP. The idea was to use MNP to selectively isolate phenol- and
naphthalene-degrading microorganisms, and to recover live bacterial cells in order to carry out
additional studies such as inhibition tests. The technique developed was called MNP-mediated
isolation (MMI). MMI started with functionalising all the cells from the biosludge with biocompatible
MNP of 183 nm. The efficiency of MNP functionalisation was greater than 99.9%, which ensured
that all cells were initially coated with MNP. In a second step, the MNP-functionalised cells were fed
with both 13C- and 12C-phenol and/or naphthalene as the sole carbon source. In presence of phenol,
the active phenol degraders present in the sludge started dividing. As a result, the newly divided
cells were not coated with MNP. After several hours, the only cells without MNP were the cells
actively involved in the biodegradation of phenols, and it was possible to selectively isolate them
using a permanent magnet to separate them from the rest of the cells (non-divisive or inactive cells)
which were attracted and immobilised by the magnet. This approach enabled the separation of live
bacterial cells in a complex activated sludge.
In the case of Tata Steel BET plant A, the bacteria isolated in the MNP-free cells of the biosludge
were identified by DNA sequencing. It showed that only one strain was present and that the isolated
bacterium using the MMI approach was Ralstonia spp., belonging to the Burkholderiales order, which
contains over 200 species. The phenol-degrading capability of the MNP-free cells was confirmed by
Raman microspectroscopy and by SIP using pyrosequencing analysis of the 13C-DNA recovered from
the sludge after incubation with 13C-phenol. All these evidence indicated that the key phenol
degrader in the biosludge collected at Tata Steel BET Plant A was an uncultivable Ralstonia spp.,
which was actually completely different from the species isolated using standard cultivation

technique. This result confirmed that standard microbiology techniques based on simple cultivation
techniques were inadequate for identifying key degraders in complex industrial biosludges. In this
case, although some phenol-degraders were isolated using cultivation techniques, these species
were not the most predominant phenol-degraders in the sludge. Advanced techniques using MMI led
to the identification of an uncultivable bacterium as the most predominant phenol-degrading in the
sludge. Further studies were carried out with MNP to identify naphthalene-degrading bacteria and a
series of uncultivable Pseudomonas bacteria were recovered and identified with this technique.
A remarkable advantage of MMI was that it was able to isolate live bacterial cells for ecophysiological analysis. Therefore, it was possible to study the effects of potential chemicals which
could inhibit or promote phenol degradation. This was carried to investigate the potential cause for a
sudden loss of treatment at Tata Steel BET plant A for thiocyanate and sometimes phenol. It was
observed that the treatment often failed when NO2- concentration in the aeration cells was greater
than a threshold of 15 mg/L. It was therefore concluded that nitrite could act as an inhibitor of
phenol / thiocyanate degradation. Inhibition tests carried out on the Ralstonia sp. isolated from the
sludge revealed that nitrite did not inhibit phenol degradation. However, it was found that
hydroxylamine (NH2OH) exhibited one of the most significant inhibition effects with threshold
between 2 and 5 mg/L. Ammonia-oxidizing bacteria responsible for nitrification in activated sludge
first oxidise NH3 to NH2OH, which is then oxidised to nitrite (NO2-) and finally to nitrate (NO3-). The
results of this study suggested that the accumulation of hydroxylamine in the aeration cells likely
caused by the inefficient conversion of hydroxylamine and nitrite could be responsible for treatment
failure.
The results obtained in the project ECOWATER showed that the application of advanced molecular
biology techniques for identifying key degraders in coke oven activated sludges and potentially
reveal their controlling factors had huge potential. Techniques such as SIP and MNP, in particular,
were the two most promising methods that could unveil the microbial composition and architecture
of the complex communities involved in the treatment process. BET plants are well established as
best available technology (BAT) for coke oven effluent treatment, accordingly the results obtained in
the project will benefit most European steelmakers. Within the time frame of the project, the
significant amount of time spent on adapting DGGE and SIP techniques and developing a completely
new method (MMI) for analysis of coke oven biosludges meant that the work carried out mostly
focused on one particular site and two types of species (phenol- and naphthalene-degrading
bacteria). Clearly, there is now significant opportunities to apply these techniques to other BET
plants in Europe as well as to identify additional species involved in the degradation of other key
compounds such as thiocyanate for instance.
Four water treatment applications were investigated in Work Package 5 (WP5) at the laboratory
and pilot scale in the project ECOWATER to improve the treatment of coke oven effluents. These
were photocatalysis using anatase (TiO2), zeolite filtration, powdered activated lignite adsorption
and fuzzy filtration.
Photocatalytic tests using Anatase showed that the best abatement efficiencies for phenols and COD
in coke oven effluents were obtained when combining the anatase powder (Aeroxide) with 1% H2O2.
In parallel, very good adsorption test results were obtained using filtration of coke oven effluents
over a column packed with a naturally-occurring type of zeolite (Mordenite). Accordingly, a pilot
scale plant was built and commissioned by CSM to carry out combined pilot scale tests using
photocatalysis and zeolite filtration. In addition, tests using two columns of zeolite in series were
also carried out. Overall, the best abatement efficiencies (> 80%) for PAHs and phenols were
obtained at the pilot scale using both types of configuration. For most metals, very good abatement
efficiencies were obtained using two zeolite columns for treatment of the coke oven wastewater. In
particular, very high adsorption was observed for lead (ca. 99%) and cadmium (ca. 96%). However,
zeolite adsorption did not perform so well for the abatament of zinc (ca. 25%), manganese (ca.
42%) and chromium (ca. 25%). A cost-benefit analysis of the zeolite filtration technology was
carried out. It was proposed that the technology could be applied post-treatment as an effluent
polishing technique. For PAHs, the cost of abatement was estimated to be 6.45 Euros / g PAHs, and
the expected abatement was >80%.
Activated lignite injection tests were carried out by Tata Steel using a pilot BET plant commissioned
and optimised during the project. Although very good results were obtained during laboratory tests
in terms of colour reduction and trace metal abatement, the tests carried out at the pilot scale were
disappointing overall. The use of activated lignite in the aeration cell (HOK Super) at 400 mg / L
did not result in significant reduction of the colour of the final effluent. PAH emission reduction was
only ca. 10%. With regard to the trace metals, there was no effect of lignite upon the emissions of
Cd and Pb. The only trace metal for which a significant abatement improvement was found was
nickel (ca. 20% improvement). The main benefit of adding activated lignite was a slight

10

improvement in the settleability of the activated sludge which resulted in slightly lower and less
variable suspended solid emissions. In terms of cost, the cost of abatement was estimated to be 1.0
Euros / g PAHs, but the expected abatement was only 10%. For nickel, the cost of abatement was
2.5 Euros / g nickel with 20% abatement.
Another application tested at the pilot scale in the project was fuzzy filtration, an innovative and
cost-effective compressible media filter, suitable for the removal of suspended solids from waste
water. It has been developed and is currently commercialised worldwide by BOSMAN, a water
treatment company based in the Netherlands. A fuzzy filtration unit is equipped with a compressible
filter medium composed of fuzzy filter balls and is a modular system that is typically used posttreatment of effluent wastewater as a polishing technique. It has been used so far principally at
municipal wastewater treatment plant and oil refineries, therefore the work presented in the project
constitute the first application to cokemaking. The results showed that with the highest compression
rate (47.5%) and a relatively low flow rate of 7 m3 / h, this technology led to significant abatement
of suspended solids (>50%) and PAHs (>50%). It is also possible that the abatement efficiency
could be further improved by adding additional fuzzy filter balls in the filtration tower for the same
compression rate; however it was not possible to carry out these tests during the project. A costbenefit analysis of the technology was carried out using Tata Steel BET plant A with total capacity of
126 m3 / h as a basis for calculations. The results showed that the abatement costs for the first year
were relatively high for PAHs (ca. 21 Euros / g PAHs reduced) and suspended solids (ca. 30 Euros /
kg of suspended solids reduced). From the second year onwards, when considering only the energy
and maintenance costs, the abatement costs using fuzzy filtration were much lower (ca. 1.5 Euros /
g PAHs reduced) and (ca. 2.2 Euros / kg of suspended solids reduced). Therefore, the technology
could potentially be beneficial for treatment of coke oven effluents leading to significant reduction of
PAH and suspended solid emissions (ca. both 50%) in the future.
In this project, the two most promising technologies identified were the fuzzy filtration and the
zeolite filtration (alone or in combination with photocatalysis). The fuzzy filtration is currently the
most advanced and the most mature solution and therefore, building on the experience of
BOSMAN in previous applications (municipal wastewater), fuzzy filters could be installed at coke
oven plants relatively easily. Further work would be needed, however, to evaluate the zeolite
filtration technology and refine the cost analysis.

11

2.

Scientific and technical description of the results

2.1

Project objectives

The main objectives of ECOWATER are:

to reduce discharges of priority substances (PS) and priority hazardous substances (PHS) in
coke oven effluents in order to meet the objectives of the European Union Water Framework
Directive (WFD).
to characterise the behaviour of PS and PHS in the coke oven wastewater treatment process.
to study the chemical and ecological impact of coke oven effluents upon the quality of local
river basins.
to enhance the efficiency of biological effluent treatment plants through better
understanding of the sludge treatment characteristics using novel molecular biology
approaches.
to investigate the use of advanced photo-oxidation, filtration and adsorption techniques for
the abatement of PS and PHS in coke oven effluents.

2.2

Management and coordination aspects

2.2.1

Structure of the ECOWATER project

The project consortium is composed of three institutions, namely Tata Steel UK Limited (United
Kingdom), Centro Sviluppo Materiali SpA (Italy) and the University of Sheffield (United Kingdom).
The project is structured into six work packages as shown in Table 1. A detailed description of the
work packages is provided in Section 12 (Project Technical Annex).
Table 1. Work packages for ECOWATER.
Title

WP Leader

WP1

Fundamental studies

Tata Steel UK Ltd.

WP2

Characterisation of coke oven and byproduct plant using chemical and biological Tata Steel UK Ltd.
approaches

WP3

Impact studies

WP4

WP5
WP6

2.2.2

Tata Steel UK Ltd.

Enhance PAH degradation in coke oven

biological effluent treatment plants using
state-of-the-art molecular biology
techniques
Novel abatement solutions Industrial
application and transferability
Project management Transfer of
knowledge

University of Sheffield
Centro Sviluppo
Materiali SpA
Tata Steel UK Ltd.

Progress against plan / Gantt chart

During the course of the project, two managerial changes were carried out including a transfer of
the project to a new Technical Group and a revision of the Technical annex.
Transfer of the project from TGC2 to TGS9 technical group
The first annual report of the ECOWATER project was submitted on 30th March 2011 and an oral
presentation was given to the members of the TGC2 Technical Group in Oviedo (Spain) on the 25th
May 2011. The two members of TGC2 Technical Group assigned to review the project (ie. Dr.
Kyumcu: Berlin University and Mrs. Borrego: Instituto Nacional del Carbon in Oviedo) suggested
that the project would be much better monitored by TGS9 committee (Technical Group for Steel
encompassing environmental aspects), owing to the environmental aspects of the project. The
chairman of the TGS9 Technical Group accepted the request, and the project was transferred on the
10th January 2012.
Revision of the technical annex
A request to revise the technical annex of the ECOWATER project was submitted to Mr. H Von Bose
(Director DG research Industrial Technologies) on the 6th December 2011. The requested change

13

was concerned with the replacement of one of the proposed application (Macro-porous Polymer
Extraction: MPPE) included in the original proposal in Work Package 5 with an alternative
technology: fuzzy filtration. The change was requested since initial results obtained in the project
showed that the MPPE technology would not be applicable to the treatment of coke oven effluents.
However, the fuzzy filtration technique could potentially improve the filtration of suspended solids in
coke oven effluents, and therefore constituted an interesting technology to try at the pilot scale
during the project. Accordingly, it was proposed to include it in the Technical Annex instead of
MPPE. The costs and man hours remained unchanged as it was estimated that the funding allocated
initially for MPPE would cover the costs of the fuzzy filtration application. After revision of the
Technical Annex, the request was accepted by Mr. Von Bose. Two milestones and deliverables were
affected by this change: milestone N. 20 and deliverable N. 20.
The project progressed well according to plan throughout the period 2010-2013. In work package 1,
most of the activities were completed according to plan. In work package 2, a significant amount of
work has been carried out well ahead of schedule (ie. Task 2.3). This was made possible owing to
the fact that the development of sampling and analytical methods for PAHs and trace metals by Tata
Steel was completed ahead of schedule enabling monthly monitoring of emissions at UK integrated
works to take place earlier for emissions inventory. Work package 3 and 4 progressed according to
plan. In work package 5, there were some delays with regard to Task 5.3 owing to the fact that the
construction and commissioning of the pilot scale BET (Biological Effluent Treatment) plant
necessary to carry out the work in this activity was postponed. Work started on the pilot scale in the
second half of the year 2011, and the pilot scale BET plant was fully commissioned by June 2012.
Despite this delay, milestones and deliverables for Task 5.3 were completed on time.

14

2.3

Work Package 1: Fundamental studies

2.3.1

Background on the European Water Framework Directive

The Water Framework Directive (WFD) constitutes the most important EU (European Union) legislation
relating to the quality of fresh and coastal waters across Europe. The aim of the WFD is to attain good
ecological and chemical status for all surface and groundwater by 2015 [1]. The chemical status of
Europes surface waters is addressed by the Environmental Quality Standards Directive [2], a daughter
Directive of the WFD, proposed in 2006 and amended in 2008, which sets out Environmental Quality
Standards (EQS) in fresh and coastal waters for a range of pollutants designated as Priority Substances
(PS). In total, 33 PS have been designated thus far including some trace metals, herbicides, insecticides,
fungicides, volatile organic compounds, polycyclic aromatic hydrocarbons (PAHs) and phtalates. Of these,
twenty substances have been classed as hazardous and designated as Priority Hazardous Substances
(PHS) owing to their toxicity, their persistence in the environment and bioaccumulation in plant and
animal tissues. Besides the PS, the WFD also highlights a series of substances which may be of concern
at local or national level but not as PS or PHS at the EU level. For these substances which are designated
as Specific Pollutants (SP), environmental standards are currently set by national governments. However,
the European Commission review the list of PS every four years and identify where appropriate whether
some of the SP should be included as new PS or PHS and any need to revise the EQS.
In the iron and steel industry, very large quantities of water are used in a wide range of thermal
processes as process water or to clean off-gases in wet waste gas treatment systems (wet scrubbers) or
for the cooling of materials / steel products. These operations can give rise to significant wastewater
emissions which may contain trace amounts of PHS and PS identified in the WFD. Although several PS
from the WFD are not directly relevant to the steel industry (ie. herbicides, pesticides, insecticides), other
substances are emitted from the iron and steelmaking operations. The substances directly relevant to the
steel industry are summarised in Table 4 together with their associated EQS, where available. In addition,
the SP which are also important in the steel industry but for which no specific EQS are currently available
are listed. With regard to the EQS, two types of standards are proposed:

the annual average value or concentration of the substance concerned calculated over a one-year
period (AA-EQS). The purpose of this standard is to ensure the long-term quality of the aquatic
environment.
the maximum allowable concentration (MAC-EQS) of the substance measured specifically. The
purpose of this second standard is to limit short-term pollution peaks.

The EQS are differentiated for inland surface waters (rivers and lakes) and other surface waters
(transitional, coastal and territorial waters). For some substances, only annual average standards have
been set out. If the EQS of a priority substance is breached in the vicinity of a discharge then there may
be pressure to tighten the discharge consent. However there is allowance for exceeding an EQS in an
area close to the discharge, an area known as a mixing zone.
As shown in Table 2, the substances classed as PHSs in the WFD include two trace metals (ie. mercury
and cadmium) and a set of six PAHs (ie. benzo[a]pyrene, anthracene, benzo [b and k], benzo [g,h,i]
perylene, indeno [1,2,3-cd pyrene]). In addition, four substances are classed as PS including two trace
metals (lead and nickel) and two PAHs (naphthalene and fluoranthene).

15

16

SP

PS

PHS

20 (dissolved)

2.4

0.1

20 (dissolved)

1.2

7.2 (dissolved)

Iron (Fe) ; Arsenic (As) ; Chromium (Cr) ; Iron (Fe) ; Free cyanide ; Zinc (Zn)

Nickel and its compounds

Naphthalene

7.2 (dissolved)

0.1

0.002

0.002

Lead and its compounds

0.03

0.03

Benzo (b) fluoranthene + Benzo (k)

fluoranthene
Benzo (g,h,i) perylene + Indeno (1,2,3cd) pyrene
Fluoranthene

0.05

0.05 (dissolved)

0.25
(dissolved)

0.1

AA-EQS
other surface waters
(g/l)

0.05

(dissolved ;
dependant upon
water hardness)
0.05 (dissolved)

0.08 0.25

10

AA-EQS
inland surface
waters
(g/l)
0.1

Benzo(a) pyrene

Mercury and its compounds

Cadmium and its compounds

Benzene

Anthracene

Substance classification in the WFD

0.1

0.07

0.45 1.5
(dissolved ; dependant
upon water hardness)

50

MAC-EQS
inland surface
waters
(g/l)
0.4

0.1

0.07

0.45 1.5
(dissolved ; dependant
upon water hardness)

50

0.4

MAC-EQS
other surface waters
(g/l)

Table 2. Environmental Quality Standards (EQS) for the PHS and PS identified in the WFD and list of the specific pollutants (SP) which are of relevance for
the iron and steelmaking industry.

2.3.2

Task 1.1: Identification of key water discharges in

cokemaking

2.3.2.1

Inventory of Tata Steel coke oven BET plants in Europe

Tata Steel currently operates three BET plants (designated as plant A, B and C in this report) to treat
coke oven effluents from its major integrated steelmaking sites in Europe. The BET plants exhibit
different features. In the project ECOWATER, emission from these sites have been characterised in Work
Package 2 to study their respective performance in terms of abatement of PHS and PS. This section
provides a description of the design of the three plants selected for these investigations.
Tata Steel BET plant A
Tata Steel BET plant A was originally built in 1974 and is currently treating the waste water effluent from
two major coke oven plants supplying coke for the integrated steelworks. Effluent is collected at both
coke oven sites and is pumped to an intermediate reservoir. From there, the effluent is pumped through
a 200 mm diameter pipeline across a distance of 6.5 km to the inlet reservoir of the BET plant. In
addition, effluents from an external tar processing company are pumped into the inlet reservoir of the
BET plant for treatment. Originally, the BET installation was designed to allow for a flow through the plant
of 38 000 m3 per week from a combination of three coke oven plants, two tar distillation plants and
contaminated ground water from the coke ovens. Currently, however, owing to restructuring and plant
closures, the effluent volume to be treated has decreased to below 20 000 m3 per week from two coke
plants and one tar distillation works. The remaining tar distillation plant is now being closed so that the
concentration and volume of liquor to be treated will further reduce.
A process flow diagram of the BET plant is provided in Fig. 1. The BET plant is composed of six aeration
cells, each of 576 m3 in volume, and three clarifiers arranged in three waste water streams. The plant
does not include any nitrification or denitrification installations. The plant therefore consists of 6 standard
aerobic basins operating in parallel with pairs of basins sharing a common clarifier. The oxygen required
by the biomass is supplied by injecting pure oxygen into the basins using a Vitox oxygenation system
developed and marketed by BOC. The Vitox aeration system presents several advantages over
standard mechanical aerators. By using pure oxygen instead of air, a far higher population of biomass
can be maintained in a given volume, thereby allowing a higher degree of treatment. The process also
reduces heat losses compared with mechanical surface aerators because it eliminates the surface
agitation and fine spray caused by surface aerators. The Vitox system also reduces the loss of organic
compounds and odours to air caused by the stripping action of large volumes of air passing through the
wastewater. Finally, surface foaming generation is also reduced. Fig. 2 depicts the six treatment cells
during oxygen injection.
Fig. 1: Process flow diagram of Tata Steel BET plant A.
BIOLOGICAL EFFLUENT TREATMENT PLANT

INLET RESERVOIR

TREATMENT
TREATMENT
CELL
CELL

TREATMENT
CELL

CELL

N. 2
4
N.

N. 5

N. 6

TREATMENT
TREATMENT

TREATMENT

TREATMENT

CELL
CELL

CELL

CELL

N.11
N.

N. 2

N. 3

TREATMENT

3
5

2 1

1
No1
O
O
CLARIFIER

STREAM 1

3
No2
O
CLARIFIER

STREAM 2

No3
O
CLARIFIER

STREAM 3
OUTLET
PENSTOCK

Tidal
reservoir
DISCHARGE
TO RIVER

17

Fig. 2: Picture of the aeration basins at Tata Steel BET plant A during oxygen injection in the treatment
cells using the Vitox system.

After treatment, the effluent and the sludge are allowed to settle into three clarifiers, and the
supernatant effluent is gravity fed using a pipeline to a tidal reservoir. From there, the effluent is
discharged under gravity using a 450 mm pipeline of 2.3 km to the tidal section of a major river for
discharge. Discharge occurs one hour after high tide for a period of four hours to ensure optimum
dispersion in the tidal section of the river, in accordance with the site environmental permit.
Tata Steel BET plant B
Tata Steel BET plant B was commissioned in 1981 to treat the waste water effluent from the coke oven
batteries providing coke to Tata Steel integrated steelworks B. The treatment plant capacity is 99.8 m3/h.
The plant was built with significant overcapacity as it was designed to serve an additional battery of 84
coke ovens that was never built. As a result, typical flows at Tata Steel BET plant currently range from
14 to 20 m3/h. A lay-out of the BET plant is shown in Fig. 3. Both the coke oven liquor from the ammonia
stills and waste water used in the benzole plant are treated in two aeration basins, each of 1305 m3 in
volume. In 1986, a Vitox system similar to the system installed at Tata Steel Plant A was installed.
However, since 2002, aeration using the Vitox system has been stopped and conventional mechanical
aerators are currently used. After clarification, effluents are transferred to N.2 sump where the treated
coke oven liquor is mixed with all the other treated effluents from the integrated steelmaking site. The
combined effluent is discharged into the sea using a long sea outfall.
Fig. 3: Lay-out of Tata Steel BET plant B.

Benzole plant cooling

system effluents

Aeration basin
N.1
(1305 m3)

Clarifier
(350 m3)

Ammonia stills
coke oven effluents

Aeration basin
N.2
(1305 m3)

All other effluent

streams from integrated steelworks

N.6 Sump

N.2 Sump

Long sea outfall

Tata Steel BET plant C

Tata Steel BET plant C is equipped with a Carrousel 2000 system, a waste water treatment plant based
upon the general oxidation ditch technology [3]. This water treatment installation was built in 1999 and
includes an internal pre-denitrification basin. The Carrousel basin is shaped like a race track and has a

18

central, longitudinal partition wall. While the wastewater is circulating around the channel at a speed of
approximately 0.3 - 0.4 m/s, micro-organisms break down the organic compounds and nitrogencontaining compounds (Fig. 4). At Tata Steel Plant C, nitrification and COD removal take place
simultaneously in the aerobic part of the installation where aerators are controlled by measuring
dissolved oxygen continuously and comparing it with a set point of 1.5 to 2 mg/l. The end products from
this conversion are CO2, water and nitrate. Denitrification is the biological process where nitrate is
converted by bacteria into nitrogen gas. This process takes place under anoxic conditions. In this process,
denitrifying bacteria need to be supplemented with a carbon source. This is achieved by circulating part
of the influent and the nitrified wastewater at the same time in the anoxic part of the installation, which
is called the pre-denitrification area.
Fig. 4: Schematic of the Carrousel 2000 system waste water treatment plant at Tata Steel BET plant C.

As opposed to Tata Steel BET plants A and B, the composition of the influent liquor to be treated at Tata
Steel BET plant C is complex since it is composed of five very different streams including:

Waste water from the coke plant (ca. 90 m3/h);

Waste water from blast furnaces gas scrubber system (ca. 140 m3/h);
Waste water from the Airfine gas cleaning system at the sinter plant (ca. 50 m3/h);
Polluted groundwater with benzole (ca. 40 m3/h);
Sanitary waste water (ca. 3 m3/h).

2.3.2.2

Typical emission discharges for Tata Steel BET plants in Europe

Work was carried out to gather information about discharges from BET plants operated by Tata Steel that
were selected for investigation in the ECOWATER project. Effluent discharge limits for Tata Steel BET
plants differ from one plant to another according to the nature of the receiving water and the water
quality objectives for that water body. The effluent discharge limits for both plants are summarised in
Tables 3 and 4, respectively.
Table 3: Effluent discharge limits for Tata Steel Plant A.
Parameter
Volumetric flow
pH maximum
pH minimum
Biological oxygen demand (BOD)
Suspended solids
Ammoniacal nitrogen
Thiocyanate (as SCN-)
Uncomplexed (free) cyanide (as CN-)
Monohydric phenols
Total mineral oil and hydrocarbons

Discharge limit
5,000 m3/d
9
5
100 mg/l
150 mg/l
200 mg/l
10 mg /l
0.3 mg/l
5 mg/l
5 mg/l

19

Table 4: Effluent discharge limits for Tata Steel Plant B.

Parameter
Suspended solids
Oil and grease
Uncomplexed (free) cyanide
Ammoniacal nitrogen
Phenol
Soluble zinc
Soluble lead
Soluble chromium
PAHs as (B[a]P)
Volumetric flow

Discharge limit
Concentration (mg/l)
150
25
0.2
27.5
2
2.5
0.3
0.2
1
6,000 m3/h
70,000 m3/d
40C (Daily mean)
45C (Hourly maximum)

Discharge rate
27.500 kg/d
3.150 kg/d
45 kg/d
4,000 kg/d
350 kg/d
2.100 kg/month
300 kg/month
200 kg/month
250 kg/month

Temperature

At Tata Steel BET plant C, a mixture of four different effluent streams is treated. Typical chemical
composition of the different streams is summarised in Table 5 including total suspended solids (TSS),
thiocyanate (CNS), total Kjeldahl nitrogen (TKN), phenols and total cyanide (CN-total). The effluent
discharge limits at Tata Steel BET plant C are summarised in Table 6.
Table 5: Chemical composition of the four effluent streams treated at Tata Steel BET plant C.
Effluent
BF
SP
CP

Flow
m3/h
140-150
50-55
80-90

GW

35-40

COD
mg/l
65-120
250-450
30003500
150-350

TKN
mg/l
130-150
200-300
200-300

CN-total
mg /l
5-20
20-60

TSS
mg/l
25-35
10-25
20-50

Phenols
mg/l
500-750

CNS
mg/l
200-250

100-200

10-20

< 10

BF: blast furnaces scrubber water blowdown; SP: Sinter Plant airfine gas cleaning system;
CP: coke plant effluent; GW: polluted groundwater.

Table 6: Wastewater discharge limits for Tata Steel BET plant C.

Parameter
Volumetric flow (m3/h)
TSS (mg/l)
COD (mg/l)
TKN (mg/l)
Phosphate (mg/l)
Cyanide (mg/l)
Thiocyanate (mg/l)
Cd (mg/l)
Hg (mg/l)
As (mg/l)
Cr + Cu + Pb +Ni + Zn (mg/l)
PAH (mg/l)

Maximum
400
60
100
30
10
10
4
0.01
0.005
0.025
0.8
0.005

Average
340
30
150
15
5
6
2

2.3.3

Task 1.2: Identification of appropriate sampling procedures for water

characterisation

2.3.3.1

Sampling methods for wastewater effluents

For monitoring the concentrations of PHS and PS identified in the WFD, there are currently no continuous
monitoring techniques available. Therefore, only two sampling methods can be employed for such
determinations. These are spot and composite sampling techniques.
When collecting a spot sample, the whole sample volume is taken at one time. Spot sample are useful for
determining the chemical quality of the waste water at a certain time and where the composition of the

20

treated waste water is relatively constant. However, in cases where compliance is judged on the basis of
the mean effluent quality, composite samples should always be used for better representativity.
For the project ECOWATER, work was carried out to identify suitable sampling equipment for the
monitoring of waste water effluents from coke oven BET plants. The Aquacell automatic composite
sampler developed by Aquamatic (Manchester, UK) was selected. This composite sampler device is fully
compliant with the ISO International Standard 5667 for the sampling of waste water effluents [4]. The
sampler is composed of an air vacuum pump for suction, a battery, an electronic programmable
controller and a 25 l polyethylene or stainless steel composite container (Fig. 5).
Fig. 5: Photograph of the Aquacell automatic composite sampler used in the project ECOWATER for
sampling coke oven waste water effluents.

The Aquacell composite sampler has several advantages such as:

The possibility to take a series of discrete samples taken at fixed intervals (intervals ranging from
1 min up to 99 h).
The sampling line from the intake has a minimum internal diameter of 9 mm to minimise
clogging.
The precision and accuracy of delivered volumes are equal to or less than 5% of the intended
volume.
The intake liquid velocity is higher than 0.5 m/s to prevent phase separation in the sampling line
and measuring chamber.

2.3.3.2

Sampling methods for ambient water (local rivers)

The most universally accepted technique for carrying out ambient water monitoring is spot sampling
followed by a laboratory-based extraction and determination of compounds of interest. This technique
yields only an instantaneous measurement of pollutant levels and the problem of potentially significant
short-term concentration variations is not addressed. An increase in sampling frequency or the use of
time-weighted automatic samplers may reduce these errors and give a more accurate picture of timeintegrated pollutant levels; however, the associated increase in costs can prove prohibitive. Because they
have to be left unattended for long periods, automated samplers are prone to vandalism. For these
reasons, passive sampling devices have been used extensively in recent years since they allow
continuous monitoring of aqueous contaminant levels. They also do not have the disadvantages of using
expensive electrically-powered equipment and are less likely to be vandalised. In addition, they often
provide a direct measurement of the bio-available fraction for the pollutants of concern, which is essential
to assess the contamination of local rivers by trace metals or PAHs. In the ECOWATER project, passive
sampling methods were selected to monitor for the chemical quality (ie. PAH and trace metal
concentrations) of local rivers.
Passive sampling systems typically consist of a receiving phase with a high affinity for organic or
inorganic pollutants, separated from the aquatic environment by a diffusion-limiting membrane. They rely
on the partitioning of the pollutants from the aqueous phase to a receiving phase by means of diffusion
through a suitable membrane. For most passive samplers, calculation of the concentration of the target
compounds in the water body is based on the accumulated concentration of the target species on the
receiving medium. Depending on the accumulation regime, there are two types of in situ samplers: either
kinetic or equilibrium based. Most samplers have a slow uptake and are exposed during a linear phase
regime (kinetic samplers), whereas equilibrium samplers are exposed until the thermodynamic
equilibrium is reached between the water and the receiving medium.

21

Passive samplers for trace metals: Diffusive gradient in thin films

Diffusive gradient in thin films samplers (DGTs) were developed by Davison and Zhang in 1994 at
Lancaster University (UK) [5]. The devices use a layer of Chelex resin impregnated in a hydrogel to
accumulate metals. The resin-layer is overlaid by a diffusive layer of hydrogel and a filter (Fig. 6). Ions
diffuse through the filter and diffusive layer to reach the resin layer and it is the establishment of a
constant concentration gradient in the diffusive layer that forms the basis for measuring metal
concentrations in solution quantitatively. When deployed in water, DGTs sample labile species. Data are
available for Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn and can be used in almost all natural waters.
Interestingly, time-weighted average (TWA) concentrations of labile metals may be determined based on
the mass of metal accumulated in the resin layer, the known metal diffusivities in the gel and the
thickness of the gel layer. Typical deployment times are two weeks and upon recovery, the mass of metal
on the resin layer can be measured after elution with acid by ICP-MS. DGTs are commercially available
(DGT Research Ltd., Lancaster, UK) and owing to its simple, robust plastic construction, it is readily
deployed in situ.
Fig. 6: Photograph of a DGT and schematic of a section through the DGT assembly.

In the environment, the determination of the free metal concentration is essential for the interpretation
of the bioavailability of a trace metal since it is widely accepted that only free soluble forms of metals can
pass through cell membranes. DGTs allow the determination of the labile fraction of dissolved metals in
water composed of free inorganic metals and weak labile organic complexes. The labile metal fraction is
very similar to the bio-available fraction, therefore its determination is essential for environmental risk
assessment and impact studies. As a result, passive sampling techniques, and particularly DGT for
metals, are gaining interest from environment agencies in charge of regulatory monitoring programmes.
Passive samplers for PAHs: Semi permeable membrane devices (SPMDs)
For several years, the time-weighted average concentration of organic contaminants such as PAHs has
been monitored in ambient water using semi-permeable membrane devices (SPMDs). SPMDs have been
the most widely used technique for passive sampling of organic compounds since they were first
developed in 1990 [6]. SPMDs are designed to sample lipid- or fat-soluble (non-polar or hydrophobic)
semi-volatile organic chemicals from water. The SPMD consists of a neutral, high molecular weight lipid
(> 600 Da) such as triolein which is encased in a thin-walled (50-100um) polyethylene membrane tube
(Fig. 7). The semi-permeable membrane allows non-polar chemicals to pass through to the lipid where
they are concentrated. Larger molecules (>600 Da), suspended particulate matter and microorganisms
are excluded. A standard SPMD is 2.5 cm wide by 91.4 cm long containing 1 ml of triolein. SPMDs are
typically deployed for one month using a stainless steel canister that can contain several SPMD devices.
Although SPMDs have been widely used for ambient water monitoring, they present a major drawback
since the analysis stage is relatively complicated. Organic contaminants are recovered using a dialytic
extraction step into an organic solvent such as n-hexane. Following dialysis, an additional clean-up
sample preparation step is required using gel permeation chromatography prior to GC/MS analysis.

22

Fig. 7: Schematic of a Semi Permeable Membrane Device for passive sampling of organic contaminants in
ambient freshwaters.

2.3.4

Task 1.3: Development of analytical methods for the analysis of organic

and inorganic PHS and PS in water matrices

2.3.4.1

Analytical methods used for the analysis of PAHs in waste water effluents

For drinking water, the analysis of PAHs is typically performed by liquid-liquid extraction followed by gas
chromatography/mass spectrometry (GC-MS) analysis or high performance liquid chromatography
(HPLC) coupled with fluorescence detection analysis, as described in the US EPA standard method 550
[7], or following the international standard method US EPA 525 [8]. These methods were used as a
starting point for the development of analytical methods suitable for the analysis of PAHs in both coke
oven or steelmaking effluents. For this type of sample (as opposed to drinking water samples),
wastewater may contain PAHs that are present both in the dissolved phase and suspended particulate
matter. Therefore, it was important to develop an analytical procedure where both the PAHs present in
the dissolved and the particulate phase could be analysed either separately or in combination.
Accordingly, a versatile method was developed to analyse both the total and the dissolved concentrations
of PAHs in the effluents (i.e. dissolved + particle-bound PAHs). The method is summarised in Fig. 8.
Briefly, effluent samples were filtered to remove the suspended particulate matter. The filtrate was
extracted using solid phase extraction (SPE). For the SPE step, reverse phase SPE was employed using a
cartridge packed with a solid sorbent containing non-polar functional groups such as octadecyl (C18) or
octyl (C8) bonded silica that exhibit a very high affinity for PAHs. Separately, the filters and residues from
the initial filtration were extracted using accelerated solvent extraction (ASE) to extract the PAHs bound
to the suspended solids. At this stage, both extracts were either combined and analysed by GC-MS to
determine the total PAH concentration in the samples or analysed separately if necessary. Quantification
was achieved using a set of surrogate, internal and recovery deuterated PAH standards.

23

Fig. 8: Analysis of dissolved and particle-bound PAHs in cokemaking and steelmaking effluents.

Effluent
sample
Filtration 0.5 - 1L

Total suspended
matter

Filtrate

Surrogate
standard

Extraction
Accelerated solvent
extraction (ASE) with DCM

Combination

Spiking

Extraction 50-500ml

Solid phase extraction (SPE)

using THF and Hexane
Recovery
standard

Spiking

GC/MS
analysis

Spiking

Internal
standard

PAH concentration in
water sample

The method was validated using a certified standard effluent sample (QCO-259) containing the 16 US
EPA PAHs at low concentrations ranging between 0.5 - 2.0 g/l. In total, twelve replicate analysis of the
certified effluent sample were carried out. The results are summarised in Fig. 9.
Fig. 9: Validation data for the analytical procedure for PAHs in cokemaking /steelmaking effluents. The
graph compares the mean concentrations of the 16 US EPA PAHs (N = 12 replicate analysis) with the
certified values.
QCO-259 PAH Standard
2.750
2.500

Mean values ug/L

2.250

Concentration ug/L

Certified values ug/L

2.000
1.750
1.500
1.250
1.000
0.750
0.500
0.250
N
ap
ht
Ac
ha
en
le
ne
ap
ht
hy
Ac
le
ne
en
ap
ht
he
ne
Fl
uo
Ph
re
ne
en
an
th
re
An
ne
th
ra
ce
Fl
ne
uo
ra
nt
he
Be
ne
nz
o
Py
C
[
a
hr
] a re
ys
ne
nt
en
hr
e
ac
+
e
Be
ne
tri
ph
nz
en
o
[b
yl
]f
en
Be
lu
e
nz
or
an
o
[k
th
]f
en
lu
e
or
an
Be
th
In
n
e
de
zo
ne
D
no
ib
[a
en
]p
[1
zo
,2
yr
,3
en
[a
-c
e
,h
d]
+
py
a,
re
c]
ne
Be
an
th
nz
ra
o
ce
[g
,h
ne
,i]
pe
ry
le
ne

0.000

As may be seen from Fig. 9, very good agreement was obtained between measured and certified values.
Typically, the relative standard deviation was less than 10% for most PAHs. After submission to the
United Kingdom Accreditation Service, the analytical procedure became ISO 17025 accredited in
December 2010.
The limit of detection (LoD) of the method was determined by analysing a series of nine low level spiked
blank samples. For each compound of interest, LoDs were obtained by applying the following formula:
LoD = SD t0.99 where SD is the standard deviation of the replicate measurements and t0.99 is the tdistribution factor for 99% confidence with a degree of freedom (N-1), in that case t0.99 = 3. The results
are presented in Table 7.

24

Table 7: Limit of detection (LOD) for the procedure developed by Tata Steel to analyse for 16 US EPA
PAHs in cokemaking and steemaking effluents.
PAH

Limit of Detection (LoD : ng / L)

Naphthalene (Naph)

28

Acenaphtylene (Acy)

Acenaphthene (Ace)

Fluorene (Fluor)

Phenanthrene (Phen)

42

Anthracene (Ant)

2.3.4.2

Fluoranthene (Flant)

25

Pyrene (Pyr)

18

Benzo [a] anthracne (B[a]ant)

Chrysene (Chry)

Benzo [b] fluoranthene (B[b]flant)

Benzo [k] fluoranthene (B[k]flant)

Benzo [a] pyrene (B[a]P)

Indeno [1,2,3-cd] pyrene (IDP)

Dibenzo [a,h] anthracene (D[a,h]ant)

Benzo [g,h,i] perylene (B[g,h,i]per)

Analytical methods for trace metals in water

Inductively coupled plasma mass spectrometry (ICP-MS) is nowadays the best analytical technique for
determining trace multi-elemental and isotopic concentrations in liquid samples. It combines an ion
generating argon plasma source with the sensitive detection limit afforded by mass spectrometry
detection. It offers better sensitivity and leads to significantly less interference than other analytical
techniques such as graphite furnace atomic absorption or inductively coupled plasma optical emission
spectroscopy (ICP-OES) that have been used extensively in the past. For instance, atomic absorption
techniques suffer from severe matrix effects requiring the addition of matrix modifiers and the use of the
standard addition method to quantify some elements. ICP-OES suffers from many overlapping spectral
interferences from other elements and a very high background emission from the plasma itself, limiting
detection limits. Interferences may occurr using ICP-MS, however they are generally easy to detect and
handle using collision and reaction cell technology. One of the key advantages of ICP-MS is extremely low
detection limits (part per trillion levels) for a wide variety of elements. Both national and international
standard methods for the determination of trace metals in water have been published and are readily
available to be used as a basis for method development [9, 10].
Accordingly, an analytical method based on ICP-MS was developed for the determination of trace metals
in steelmaking effluents. As previously described for PAHs, trace metals in steelmaking effluents may be
present both in the dissolved phase and suspended particulate matter. Therefore, separate analytical
procedures were developed to analyse both the dissolved and the total concentrations. The analytical
procedures developed included the following twelve trace elements: Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As,
Cd, Pb. For determination of the dissolved concentration of trace metals, samples were filtered through a
0.45 m pore diameter membrane filter, pre-digested on a hotplate/hotblock using nitric acid at 90C for
30 minutes, and digested with nitric acid using a microwave instrument. For determination of the total
concentration of trace metals, water samples were pre-digested without filtration on a hotplate/hotblock
using nitric acid at 90C for 30 minutes. Subsequently, samples were digested with nitric acid using a
using a microwave instrument. All samples were analysed using an Agilent 7500cx ICP-MS equipped with
an Octopole Reaction System (Fig. 10). The Octopole Reaction System is operated in 3 different modes,
standard mode, helium mode and hydrogen mode, and can be combined in a single acquisition. The
standard mode is typically used to analyse for elements such as mercury or lead that do not suffer from
spectral interferences. The helium collision mode is used for all analytes that suffer matrix-based
interferences (for instance 35Cl16O on 51V, 40Ar12C on 52Cr, 23Na40Ar on 63Cu) and also to reduce plasmabased interferences (such as 40Ar16O or 40Ar38Ar). The hydrogen reaction mode is used only for intense
plasma-based interferences.

25

Fig. 10: Schematic of the Agilent 7500cx ICP/MS used by Tata Steel for the analysis of trace metals in
steelmaking effluents.

The method was validated by analysing replicate samples (N = 40) of a waste water effluent supplied by
LGC Standards part of an International Proficiency Scheme (Aquacheck) in which typically around 50
laboratories are participating across Europe.
The validation data (dissolved concentrations) are
summarised in Table 8.
Table 8: Validation data for the analytical procedure used to analyse trace metals in steelmaking
effluents. The graph compares the mean concentrations of trace metals with the assigned values.
Assigned value
Mean value
Waste water effluent
(Dissolved, mg/l)
(Dissolved, mg / l)
International Proficiency
(N = 40)
Scheme Certified Sample
27
Al
5.73
5.47
51
V
3.18
3.03
52
Cr
2.05
2.05
55
Mn
4.42
4.38
56
Fe
3.90
3.11
50
Co
3.77
3.69
60
Ni
4.65
4.81
63
Cu
1.79
1.70
66
Zn
4.98
5.18
75
As
0.37
0.38
111
Cd
166.0
167.8
208
Pb
2.50
2.45
SD: Standard deviation ; RSD: Relative standard deviation.

SD
mg/l

RSD
(%)

0.13
0.17
0.11
0.26
0.21
0.15
0.06
0.10
0.09
0.00
4.29
0.11

2.4
5.6
5.6
5.9
6.8
4.0
1.2
5.9
1.8
0.8
2.5
4.4

As may be seen from Table 8, a very good agreement was obtained between measured and certified
values. Typically, the relative standard deviation was less than 5%. After submission to the United
Kingdom Accreditation Service, the analytical procedure became ISO 17025 accredited in December
2010. Limits of detection (LOD) for all elements were determined by performing 10 replicate
measurements of an experimental blank. The LOD was obtained by multiplying the standard deviation for
each element by 3, as previously described in Section 2.1.4.1. LODs for each element are summarised in
Table 9.

26

Table 9: Limits of detection (LODs) for the procedure developed by Tata Steel to analyse for selected
trace metals in cokemaking and steemaking effluents.
Trace metal

2.3.4.3

Limits of detection (LoD : ng / L)

27

Al

150

51

50

52

Cr

100

55

Mn

100

56

Fe

200

50

Co

100

60

Ni

100

63

Cu

100

66

Zn

300

75

As

50

111

Cd

50

208

Pb

50

Analytical methods used for mercury analysis in water

Cold vapour atomic fluorescence spectrometry (CVAF) is the preferred technique for mercury analysis
over ICP-MS, as it is more sensitive (able to detect in the sub part-per-trillion concentration range) and
exhibits good linearity over a large concentration range. It utilises the phenomenon of mercury vapour
generation at room temperature. A problem with mercury analysis by ICP-MS is the adsorption of
mercury onto the plastic introduction systems inside the instrument. Accordingly, all the mercury
analyses carried out in the project ECOWATER were realised using CVAF.
Briefly, atomic fluorescence is an emission process in which atoms are excited by the absorption of an
electromagnetic radiation. The instrument contains a lamp which emits ultra-violet radiation at 253.7 nm.
Mercury atoms are excited by this radiation, and relax back to their ground electronic configuration,
giving up their excess energy as photons. The instrument measures the intensity of these photons
(fluorescence), and converts it to a concentration. Prior to analysis, mercury in the samples is reduced
from Hg2+ to elemental Hg0 vapour using tin (II) chloride as a reductant. The vapour is then purged from
the solution using argon as a carrier gas into the measurement cell and is detected by atomic
fluorescence. There are two standard methods that can be used for the analysis of mercury using CVAF,
these are the US EPA method 1631 and the British Standard EN ISO 17852 [11, 12]. The latter one was
used in the project ECOWATER.
For mercury analysis, two additional problems must be considered: the adsorption of mercury onto
container walls and the potential volatilisation of mercury. To limit the adsorption of mercury species onto
the walls of sample containers, the use of glass container must be avoided. Instead, FEP (fluorinatedethylene-propylene) or PTFE (polytetrafluoroethylene) must be used. These fluorinated containers also
prevent the diffusion of atmospheric mercury vapour through the bottle. Prior to analysis, samples must
be preserved by addition of a potassium bromide/potassium bromate solution, prepared in hydrochloric
acid, to avoid the volatilisation of Hg0 vapour. This leads to the formation of the highly oxidising species
bromine monochloride (BrCl), which oxidises all the mercury present in the sample to Hg2+, a stable form
of mercury which can volatilise.

2.3.5

Task 1.4: Development of biosensor arrays for the detection of toxicity in

coke oven effluents

2.3.5.1

Biosensors

The assessment of environmental risks associated with aqueous and soil contamination requires a rapid
and efficient method to evaluate their potential toxic impact on the environment and human health.
Whole cell biosensor technique employs living cells to sense environmental contaminants and toxicity. It
establishes a direct link between environmental pollutants and living organisms and has been approved
to be a simple, rapid, reliable, sensitive and low-cost approach.
The principle of biosensors is to design a genetic circuit in bacteria, using cellular sensory and regulatory
components to generate a quantifiable reporter output in response to chemical detection or toxicity

27

(Fig. 11). Essentially, a reporter protein is brought under the control of a sensory-regulatory network
with the desired specificity. Common reporter proteins include the bacterial luciferase (lux) found in
bioluminescent bacteria and also Green Fluorescent Protein (GFP). First, the regulatory protein usually
patrols in the cellular cytoplasm and remains inert until it interacts with an inducer (e.g. naphthalene).
Once the regulatory protein is activated by the inducer, it rapidly changes its configuration which makes
it able to bind to a promoter and switch on a reporter gene that is fused with the promoter. Whole cell
biosensors are ideally suited for use in ecotoxicity assessments of coke making effluents for a number of
reasons. First, they offer rapid testing in situ, second they are biologically relevant by detecting the
bioavailable fraction in complex environments and finally, they are robust and very cost-effective.
Fig. 11: Principles of whole cell biosensors.

Two types of biosensors can be constructed: specific and non-specific biosensors [13].
Specific
biosensors can directly detect chemicals / pollutants whereas non-specific biosensors can detect toxicity /
genotoxicity.
Recently, the University of Sheffield has constructed a novel toxicity biosensor [14], and 12 specific
biosensors including toluene/xylene, salicylate, phenol, naphthalene, oil (crude oil, alkanes/alkenes),
benzoate, 1,2,4-trichlorobenzene, dinitrotoluene, catechol [15-18]. For instance, as shown in Fig. 12, a
biosensor specific to toluene was able to detect increasing concentrations of toluene in water whereas the
toxicity biosensor was able to detect DNA damage caused by a mutagen molecule (Mytomycin C: MMC)
or UV light.

28

Fig. 12: Specific / non specific biosensors for the detection of contaminants and toxicity. (A) toluene
biosensor quantitatively detected toluene in water. (B) Toxicity biosensor detected DNA damage caused
by a mutagen (Mytomycin C : MMC) or UV light.

The toxicity biosensor constructed by Sheffield University [14], was developed for toxicity assays on
environmental samples based on the Acinetobacter baylyi ADP1_recA_lux system [13]. For the
construction of this biosensor, Acinetobacter baylyi ADP1 strain, a genetically malleable soil bacterium,
was selected because it exhibited a remarkable competence for transformation. In addition, it could be
manipulated by homology directed recombination through linear DNA fragments. These unique features
make Acinetobacter baylyi ADP1 very interesting for environmental and biotechnological applications.
Furthermore, it was demonstrated that Acinetobacter baylyi was more robust than E. coli concerning
viability, maintenance, and storage [14]. Accordingly, from the start of the ECOWATER project, it was
thought that Acinetobacter baylyi was a promising bioreporter for use on coke oven effluents as it was
robust and could operate over a wide temperature range. Additionally, it was shown to be capable of
detecting both genotoxicity and cytotoxicity, qualitatively and quantitatively. Therefore, it was selected as
a starting point for the development of a biosensor for PAHs in the project ECOWATER. In addition, work
was also carried out in parallel to build a specific biosensor for the detection of n-alkanes / oils also based
also on the Acinetobacter baylyi ADP1_recA_lux system.

2.3.5.2

Development of a toxicity biosensor for PAHs

In the Agency for Toxic Substances and Disease Registry document [19], it has been stated that a
number
of
PAHs,
including
benzo[a]anthracene,
benzo[a]pyrene,
benzo[b]fluoranthene,
benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, dibenzo[a,h]anthracene, and indeno [1,2,3c,d]pyrene can cause tumours in studied animal models in laboratory conditions, however there is little
information about their impact on humans. Generally, owing to the well-known genotoxicity action of
benzo[a]pyrene (B[a]P), this compound has been widely used as a positive control in evaluating the
sensitivity of various bioassays determining the genotoxic activity of unknown substances. Many genetic
toxicological studies have been carried out using B[a]P as the main PAH compound because it has been
established via in vitro studies that B[a]P can induce genetic damage in both prokaryotic and eukaryotic
systems including mammalian cells [19]. There is a wide range of genotoxic effects exerted by B[a]P
including: mutation in somatic cells, chromosomal damage in germinal and somatic cells, formation of
DNA adducts, unscheduled DNA synthesis (UDS), sister chromatid exchange, and neoplastic cell
transformation. In human cell line culture, it binds to DNA and leads to mutations of genes, chromosomal
defects, sister chromatid exchange, and UDS [19].
However, a number of epidemiological studies have also observed an increased mortality rate,
particularly due to lung cancer, in people who have been exposed to a source of mixed PAHs rather than
an individual PAH compounds. For this reason, one cannot measure individually the contribution of each
PAH to the total carcinogenicity. Therefore, a global approach which would take into consideration the
presence of several PAH compounds and would provide a measure of the overall toxicity from PAH
mixtures would be advantageous. In the project ECOWATER, it was decided that the work would focus on
the development of a toxicity biosensor able to detect toxicity from PAH mixtures rather than the
development of several biosensors specific to each individual PAH compounds. Accordingly, the approach
selected was to focus on the human cytochrome P450 (CYP) because all PAH compounds are oxidised by
CYP which metabolically converts PAHs into carcinogens [20]. Indeed, PAHs only become carcinogenic
once they have been metabolised into epoxides. The P450 CYP1A1 enzyme is believed to play an
important role in the activation of these procarcinogenic PAHs. Indeed, CYP oxidises PAHs to epoxides,

29

and it is thought that these compounds could potentially also activate the bacterial recA gene in the SOS
system of the biosensor Acinetobacter ADP1_recA_lux that was previously constructed by the University
of Sheffield [14].
Accordingly, the process selected for constructing the PAH biosensor included two steps:

Cloning the human P450 1A1 into ADP1 which converts procarcinogenic PAHs into DNA
mutagens;
The resultant oxidised PAHs induce the recA gene that is fused with reporter cassette luxCDABE
to express bioluminescence.

The approach choosed to develop the PAH biosensor is depicted in Fig. 13.
Fig. 13: Use of human cytochrome P450 activation of PAHs into carconogens (epoxides) for construction
of a PAH biosensor using Acinetobacter baylyi ADP1_recA_lux system.

After cloning of the human cytochrome P450 into Acinetobacter ADP1, experiments were carried out
using several PAHs to detect a response of the newly created biosensor, designated as Acinetobacter
ADP1-P450-lux. PCR amplification and sequencing of the P450 gene was used to confirm the construction
of the new biosensor. The first tests were carried out using pyrene. The results are summarised in
Fig. 14. Initial results indicated that the PAH biosensor cloned with the cytochrome P450 exhibited a
positive response in presence of pyrene, as opposed to the former biosensor without the cytochrome
P450. A series of tests using Mytomycin C (MMC) were also used as positive control.
Fig. 14: Detection of pyrene by the PAH toxicity biosensor Acinetobacter ADP1_P450_lux. Mitomycin C
(MMC) was used as a positive control.

To test the performance of ADWPH_recA_sal450, several tests were carried to optimise cell culture times
and temperatures for toxicity detection. For these tests, the performance of the ADWPH_recA_sal450
biosensor was evaluated against the ADPWH-recA biosensor (without P450) used as a control. Tests were
realised using three PAHs including pyrene, B[a]P, and naphthalene, and a standard toxic compound (ie.

30

MMC). Data are summarised in Figs. 15 and 16. In the first series of experiments, the results obtained
showed that the expression level and protein folding of cytochrome P451A1 were significantly affected by
temperature. For pyrene, a strong signal was only obtained at 30C with the biosensor
ADWPH_recA_sal450 after two days of cell culture time, whereas weak bioluminescence responses were
measured at 20C and 37C. Interestingly, only the biosensor ADWPH_recA_sal450 responded to the
exposition to pyrene. There was no response from the ADPWH-recA biosensor (Fig. 15). In the second
series of experiments carried out at 30C, very good responses were obtained for biosensor
ADWPH_recA_sal450 for the three PAHs investigated after two days of cell culture time, as previously
found for pyrene. Again, there was no response for the biosensor ADPWH-recA which did not contain the
P450, proving that the ADWPH_recA_sal450 could detect PAHs and genotoxicity in the samples.
Fig. 15: ADWPH_recA_sal450 and ADPWH-recA response to genotoxic compounds (pyrene and MMC) at
three different temperatures as a function of the cell culture time.

31

Fig. 16: ADWPH_recA_sal450 and ADPWH-recA response to genotoxic compounds (pyrene, B[a]P,
Naphthalene and MMC) at 30 as a function of the cell culture time.

In conclusion, with the human P450 CYP 1A1 gene cloned into the chromosome of Acinetobacter baylyi
ADP1, the new biosensor ADPWH_recA_Sal450 (former name ADP1-P450-lux) had therefore the
capability of specifically sensing genotoxic PAH chemicals. The optimum temperature for detection was
30C and two days of cell culture time was required. The activity of human P450 CYP 1A1 was further
confirmed through Promega human P450 Kit and the relative protein through Western Blot, proving that
human P450 1A1 was successfully expressed in the bacterial cells. This biosensor will be further tested
and discussed in Work Package 3 on real environmental samples. This biosensor could be used as a
warning system and this could have future applications for commercialisation of the biosensor for online
and in situ purposes.

2.3.5.2

Development of of a specific biosensor for n-alkane / oil detection

based on Acinetobacter baylyi ADP1

The second part of the biosensor activity was concerned with the construction of a whole cell biosensor
for specific chemicals detection, in particular n-alkanes and oils. Amongst the Acinetobacter alkane
degraders, Acinetobacter baylyi ADP1 is able to utilise n-alkanes and the gene regulation for alkane
degradation is well characterised [21-23]. Acinetobacter baylyi ADP1 also exhibits AraC/XylS-like
transcriptional regulatory protein ALKR that regulates the n-alkane hydroxylase gene alkM in the ADP1
chromosome involved in the initial step of alkane oxidisation [21-22]. Accordingly, the Acinetobacter
baylyi ADP1 strain was selected for the study.
To date, only a few alkane bioreporters have been developed [24-26], and those bioreporters have been
shown to detect short and medium (i.e. C5-C10) carbon chain of alkanes and alkenes. One of the main
challenges to overcome for alkane bioreporters is to extend their alkane detection range by constructing
biosensors that can respond to alkanes and alkenes with various carbon chain lengths ranging from C7 to
C36. In the ECOWATER project, an alkane bioreporter designated as ADPWH_alk that can respond to
alkanes and alkenes with various carbon chain lengths ranging from C7 to C36 was constructed. This
biosensor, based on Acinetobacter baylyi ADP1, was able to adhere to an interface of oil and water and to
emulsify mineral and crude oils into oil droplets at the micrometre level. These unique properties enabled
ADPWH_alk to overcome alkanes low solubility and accessibility, and to actively search for oil / n-alkanes
in water samples.

32

Biosensor ADPWH_alk development

For the construction of the biosensor, the luxCDABE was inserted into a site at alkM in the chromosome
of Acinetobacter baylyi ADP1 by mixing the vector pAlkRM_lux_km and ADP1. The transformants showing
brightest bioluminescence in the presence of mineral oil were chosen and these were the biosensors
designated as ADPWH_alk. The vector pAlkRM_lux_km was constructed on pGEM-T backbone which
cannot replicate in ADP1. It suggests that the luxCDABE cassette was at the chromosome of ADPWH_alk.
Southern blotting confirmed that a single copy of luxCDABE was at ADPWH_alk. ADPWH_alk was able to
grow on LB agar plates with 300 g/ml ampicillin, suggesting that the whole vector pAlkRM_lux_km had
been inserted into the chromosome by Campbell-like integration. The DNA sequences of colony PCR
products, which used ADPWH_alk colony as DNA template and ADP1_alk_for/luxC_rev and
alk_P_up/ADP1_alk_rev as primer pairs, confirmed the genetic structure of ADPWH_alk construct as
shown in Fig. 17. The DNA sequence also indicated that the plasmid pAlkRM_lux_km introduced three
point mutations to the promoter region within the intergenic region between alkM1-luxCDABE and alkR
(Fig. 17). The genetic structure of the ADPWH_alk biosensor is depicted in Fig. 18. Those mutation
points may affect alkR regulatory binding and RNA transcription in response to alkanes.
Fig. 17: A. Schematic outline of construction of alkane bioreporter Acinetobacter baylyi ADPWH_alk (DNA
lengths are not scaled).

33

Fig. 18: Genetic structure of alkane regulation part of ADPWH_alk. There are three mutation points at
promoter region of alkRM. The mutation points are highlighted in bold. The inverted repeats with two
mismatches are marked by arrows under the sequence.

Biosensor ADPWH_alk characterisation

Characterisation of the alkane bioreporter ADPWH_alk induction was conducted by exposing the
bioreporters to 100 mM suspension solution of a series of n-alkanes including: hexane (C6), dodecane
(C12), tetradecane (C14), octadecane (C18), tetracosane (C24), triacontane (C30) and tetracontane
(C40). The timecourse of ADPWH_alk activation and the bioluminescence expression are plotted in Fig.
19. As may be seen from Fig. 19, the bioreporter was able to detect a broad range of alkanes and
alkenes with carbon chain length from C7 to C36 and responded to the alkanes in about 30 minutes,
independent to the cell growth phase.
Fig. 19: Dynamic ADPWH_alk response to n-alkanes with 100 mM of each compound.

To study whether the biosensor ADPWH_alk response was affected by complex hydrocarbon mixtures and
by the salinity in sea water environments, further tests were carried out by exposing the biosensor to a
Brent crude oil in pure water and sea water. The results are depicted in Fig. 20.

34

Fig. 20: Dynamic ADPWH_alk response to 100 mg / L Brent crude oil in pure water (PW) and seawater
(SW).

As may be seen from Fig. 20, ADPWH_alk was also rapidly induced by Brent crude oil in pure water and
seawater and the ADPWH_alk sensing performance to Brent crude oil was not affected by salt and other
impurities in seawater.
Further tests were carried out to identify the range of compounds that could be detected by the
ADPWH_alk biosensor. Accordingly, tests were carried out by exposing the biosensor to various n-alkanes
ranging from C6 (n-hexane) to C40 (tetracontane). The results are presented in Fig. 21.
Fig. 21: Response of ADPWH_alk and ADPWH-lux to 100 M of n-alkanes ranging from n-hexane (C6) to
tetracontane (C40).

As may be seen from Fig. 21, ADPWH_alk was sensitive to n-alkanes with carbon chain length ranging
from C7 to C36. In comparison, ADPWH_lux, a biosensor previously developed by Sheffeld University to
detect salicylate [17], and used as control in these experiments, did not exhibit any significant response.
This confirmed the specificity of the n-alkane ADPWH_alk biosensor. For a series of alkanes and alkenes,
the biosensor induction ratios were determined to study which compounds induced the highest response
of the biosensor. The results are depicted in Fig. 22.

35

Fig. 22: Response of ADPWH_alk (induction ratios) to 100 M of n-alkanes /n-alkenes ranging from nhexane to tetracontane (C40).

Analysis of the results showed that ADPWH_alk induction ratios varied for n-alkanes and n-alkenes
depending upon carbon chain lengths, however similar induction ratios between alkanes and alkenes
exhibiting identical carbon chain lengths were observed. Overall, the induction ratios of C12, C18 and
C24 alkanes or alkenes were significantly higher than alkanes/alkenes with other carbon chain lengths
(Fig. 22), suggesting that alkanes or alkenes with specific carbon chain lengths had effects on the
complex of ALKRpromoterRNAP and favoured alkM transcription initiation.
Properties of ADPWH_alk at the interface oil /water
The solubility of alkane and crude oil in water are usually extremely low; hence n-alkanes and crude oils
are inaccessible to cells, which may hamper oil detections in complex water and soil environments.
However, in the case of Acinetobacter baylyi ADP1 and its derivative ADPWH_alk, the biosensor was
found to adhere to an oilwater interface and to emulsify oils into small droplets as opposed to E. Coli
(Fig. 23).

36

Fig. 23: Acinetobacter baylyi ADPWH_alk natural affinity to oil droplets. Unlike E. coli which was unable to
emulsify and attach to oil droplets (A), ADPWH_alk, a subclone from ADP1, was able to emulsify and
attached to the surface of mineral oil (B, C) and crude oil (D) droplets. The scale bar is 5 mm.

Examination of Fig. 23A showed that in the Escherichia coli DH5aoil mixture, it was difficult to observe
small oil droplets unless vigorous shaking was applied. However, in the ADPWH_alkoil mixture,
ADPWH_alk emulsified both mineral and crude oils into 1080 mm oil droplets and the cells were found
attached to the surface of oil droplets but were absent from the water phase (Fig. 23 BD). It indicated
that A. baylyi was able to recognize alkanes and switch their movements to the surface oil droplets. The
cell density of ADPWH_alk on the surface of mineral and crude oil droplets was estimated to be 1.4 0.2
1010 cells m-2, implying that cells occupied a single layer on the oil surface.
In conclusion, Acinetobacter baylyi ADP1 was found to tolerate seawater and has a special ability of
adhering exclusively to an oil-water interface of 10 - 80 m emulsified mineral and crude oil droplets.
These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to
actively search and sense oil spills in water and soils. Here Acinetobacter baylyi bioreporter ADPWH_alk
was developed and applied to the detection of alkanes and alkenes in water and seawater. Bioreporter
ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to
C36. This bioreporter responded to the n-alkanes in about 30 minutes, independent to the cell growth
phase. In work package 3 (impact studies), further work will be carried out to tests this biosensor on a
range of contaminated waters and soils.

37

2.3.6

Task 1.5: Fundamental studies into the interactions between organic

pollutants and heavy metals in coke oven effluents and effects upon
toxicity responses

Complex effluents can contain a wide variety of chemical substances, both of organic and inorganic
origin. The interactions between these pollutants can lead to a detectable toxic response. To overcome
the limitations associated with the traditional chemical-specific approach, toxicity testing was used to
determine the effects of mixtures of substances in effluents on a living organism and to evaluate the
environmental impact.
The testing methodology for interactive toxicity tests of organic pollutants and heavy metals refers to the
variation of light outputs by the organisms involved in the Microtox test (a preparation of specifically
selected strains of the marine bacterium Vibrio fischeri (Fig. 24), formerly known as Photobacterium
phosphoreum). Microtox is an ASTM Standard method (D-5560, 1995) for determining the toxicity of
aqueous wastes before and after treatment [27]. Briefly, the test exposes living luminescent organisms
to aqueous samples and measures the increase or decrease in light output by the test organisms. The
test system measures the difference in light output between the sample and the control and attributes it
to the effect of the sample on the organisms. Each test cuvette contains about one million individual test
organisms; therefore, variations among individual organisms become statistically insignificant.
Fig. 24: Bioluminescens bacteria (Vibrio fischeri)

For this study, the Microtox toxicity testing was selected by CSM to research possible interactions
between heavy metals and organic contaminants and the effects upon toxicity. The Microtox test is very
sensitive with organic compounds meaning that only short experiments (ca. 5 min) were required.
However, for heavy metals, longer experiments were necessary (ca. 30 min).
To allow the establishment of statistically valid relationships between factors affecting a process and its
outputs, a Design of Experiment (DOE) methodology was used. DOE isa structured and efficient method
for planning experiments in order to maximize the amount of information obtained with a minimum
number of experiments. A structured DOE approach has several benefits including:

identification of the most important factors;

analysis of simultaneous factors interaction;
determination of optimal operating conditions.

Often, experiments are simplified by using only two levels for each factor to determine the real effects of
each individual factor. For this type of experiments, there are statistical approaches to analyse the data.
However, simple and effective ways to interpret the results are also to plot the average values of each
factor and level, both individually and in combination, and visualise them in a bar chart, as illustrated in
Fig. 25.

38

Fig. 25: Interpretation of the possible interaction between two factors using two levels.

For this, the DOE approach was used for evaluating the environmental impact (toxicity response) caused
by the interaction in water of organic and inorganic pollutants. For these tests, synthetic solutions
exhibiting different substances were prepared in order to test their toxicity. These solutions contained
phenol, salicylic acid and naphthalene as organic compounds and salts (or standard solutions) of As, V,
Cd, Cr and/or Fe as inorganic compounds. Salicylic acid was chosen because several studies have
reported that it was formed as an intermediate in the degradation of PAHs such as naphthalene,
acenaphthene and phenanthrene [1-5]. Therefore, its effect was also taken into consideration for the
toxicity tests. The treatment time was 1 h. Table 10 provides a summary of the experimental conditions.
Table 10: Experimental conditions used to study the effects of interactions between organic compounds
(phenols, naphthalene, salicylic acid) and heavy metals upon toxicity responses.
Case
1
2
3
4
5
6

Phenol
(mg/l)
10-20
10-20
5-1
1
1-5
100-200

Salycilic acid
(mg/l)
0
0
0
0
1-5
0

Naphthalene
(mg/l)
0
0
0
0
0
1-5

pH
7-9
7
7-9
7
7
7

Heavy metals
(g/l)
10000-50000
10-50
1-5
1-5
1
250-500

Fe (mg/l)
0
0
0
1-5
0
0

Six different cases were analysed. First, synthetic solutions exhibiting both high concentrations of phenols
and heavy metals were characterised (Case 1). In cases 2 and 3, solutions with lower concentrations of
heavy metals but high concentrations of phenols were examined. In case 4, the possible interaction
between iron and heavy metals in presence of phenol was investigated. Finally, the interactions between
phenol and salicylic acid or naphthalene and heavy metals were studied in cases 5 and 6. Full details for
each experiment carried out and the results obtained are reported in Table 64 (Appendix 1).
The main results obtained during these tests are summarised below.
Case 1: During the trials, precipitation of some metals was observed. This could be an effect of pH and
the high concentration. DOE data indicated that in the presence of high organic and metal concentrations,
the Microtox toxicity test response did not seem to recognise the concomitant presence of different
toxicants. The higher effects were actually detected when the chemicals were used alone rather than in
combination (Fig. 26).

39

Fig. 26: Comparison of the effects - single and combined parameters

(Case 1: phenols and heavy metals at high concentrations, pH)

Case 2: The higher Microtox toxicity response was detected in the presence of heavy metals only (Fig.
27). No precipitation of heavy metals was observed. The data reported are only the data obtained after 5
mins since, after 30 min, the Microtox values were very variable and they have not been considered
reliable.
Fig. 27: Comparison of the effects - single and combined parameters
(Case 2: phenols at high concentration and heavy metals at low concentration)

Case 3: The higher Microtox toxicity response, after 30 min treatment, was detected with pH and the
three combined parameters (Fig. 28). This result showed that the organisms in water feel the effect of
different equilibriums that often are often not stable over time.
Fig. 28: Comparison of the effects - single and combined parameters
(Case 3: pH, phenols and heavy metals at low concentration)

40

Case 4: Iron was selected as additional heavy metal, since its concentration in the coke oven wastewater
was higher in comparison to other metals. The effect of iron alone seems to be negligible, nevertheless
its presence in water in conjunction with other metals showed an increase of the toxicity (Fig. 29). This
increase , however, is not easily predictable.
Fig. 29: Comparison of the effects - single and combined parameters
(Case 4: iron and heavy metals at low concentration)

Case 5: As already observed in the previous case, the concomitant presence in wastewater of similar
toxicants resulted in a higher toxicity owing to synergetic effects between the components. In fact, the
toxic effect of phenol and salicylic acid together always appeared to be always higher than the effects
obtained using the chemicals alone (Fig. 30).
Fig. 30: Comparison of the effects - single and combined parameters
(5case: phenols and salicilic acid at low concentration)

In summary, the results obtained showed:

Siginificant effects of heavy metals upon toxicity (after 30 min);

Importance of the discharge pH value (affecting both phenol and metals solubilisation);
Evidence of possible synergetic effects due to the concomitant presence of chemicals in solution;
Complex equilibria in water responsible for the detected toxicity.

The effect of single and combined parameters was examined in order to select the most important
variable to be used for the modeling. Data have been analysed only on EC50 detected after 30 min. The
experiments and data used for the model are summarized in Table 11.

41

Table 11: Scheme of factorial experimentation.

solution

phenol (mg/l)

heavy metals (g/l)

naphthalene
(mg/l)

200

500

8.38

200

250

16.38

3
4

200
200

250
500

5
1

8.05
13.61

5
6

100
100

500
250

5
1

12.29
24.9

100

250

13.6

100

500

19.02

A model has been created by means of a linear regression, using the SAS software:
EC50(30-min)=a+b*[phenol]+c*[heavy-metals] +d*[naphthalene]

EC50
(30 min)

(1)

The steps followed during the modeling were:

formulation of the null hypothesis: all coefficients are contemporarily zero. If this hypothesis is
accepted, the model is discarded. If the hypothesis is rejected, the model has a significance value
of 85%1,

calculation of R-Square factor (R2) as an indicator of the reliability of the model adaptation and
the calculation of standardized coefficients to comprehend the variables weights, considered in
the model,

error verification by means of analysis of the residue behavior.

The data in Table 12 indicates that the model gives a significant contribution to describe the phenomena
variability (Pr>F is <0,05). Moreover, the variability, described by the model, indicated as R-Square (R2)
and Adj R-sq, is about 0.9, a number close to 1 which verifies the good correlation between the observed
and the predicted values, and provides the excellent relationship between the independent variables
(factors) and the response (EC50).
Table 12: Variance analysis data
F
21,09

Pr>F
0,0065

R-Square
0,9405

Adj R-sq
0,8959

In Table 13, the model variables and the corresponding estimated coefficients are shown.
Table 13: Parameters Estimation.
Variable
Intercept
phenol
heavy-metals
naphthalene

Parameter
Estimate
32.83
-0.058
-0.0096
-1.97

t Value

Pr > |t|

11.20
-4.60
-1.89
-6.21

0.0004
0.010
0.13
0.0034

Standardized
Estimate
0
-0.56
-0.23
-0.75

Variance
Inflation
0
1.00
1.00
1.00

An analysis of these data showed that:

the estimated coefficients, corresponding to naphthalene and phenol variables, were significantly
different from 0 (Pr > |t| are all <0,05): the coefficient associated to the heavy-metals variable
caused the decrease of model significance to 0.85 (i.e. 85%),

the variable with the most significant correlation with EC50 was that with the highest (absolute)
value in the standardized estimate, which in this case was naphthalene. In comparison, the
heavy-metals had a minor effect,

the variance inflation was < 10, this meant that the co-linearity between the regressors was
under control.
Therefore, the resulting EC50 model value (after 30 min) was:

42

EC50 (30 min) = 32,8-0,058*(phenol)-0,0096*(heavy-metals)-1,97*(naphthalene)

within the validity concentration ranges:
100 mg/l < phenol < 200 mg/l
1 mg/l < naphthalene < 5 mg/l
250 g/l <heavy metals < 500 g/l
The comparison between real and estimated data can be observed in Fig. 31. In this graph, a good fit
among estimated and real data was shown.
Fig. 31: Comparison between real (x-axes) and estimated (y-axes) data for the variable EC50(30 min)

In conclusion, the modelling results showed that:

Each of the three independent variables had a significant contribution to the explanation of the
phenomenon variability;
The most significant variable was naphthalene;
There was an inverse relationship between EC50 after 30 min and the three independent
variables, i.e. if one of the variables increased, the value of EC50 decreased.

2.4

Work Package 2: Characterisation of coke oven and by-product plant

effluents using chemical and biological approaches

2.4.1

Task 2.1: Evaluation of the efficiency of waste water treatment steps for
the removal of PHS / PS and the detoxification of coke oven effluents

In the first phase of the project, work focused on determining the distribution of PAHs and trace metals in
coke oven effluents between the dissolved phase and the suspended solids. It also considered the
abatement efficiency of the BET plants operated by Tata Steel in the UK for these species. PAHs and trace
metals.

2.4.1.1

Measurement of trace metal / PAH abatement efficiencies at Tata

Steel BET Plant A

Sampling campaigns were carried out at Tata Steel BET plant A in October and November 2010 to
determine the concentrations of PAHs and trace metals before and after treatment. In order to measure
the abatement efficiency, simultaneous sampling at the inlet and outlet of the BET plant was undertaken.
Composite samplers were used to allow a representative sample collection every hour for several 24-h
periods. For each campaign, the BET plant was monitored over three days (triplicate samples/24h).
Samples were collected from the inlet reservoir (untreated liquor), clarifiers 1 and 3 (treated liquor,
streams 1 and 3) and the final discharge point (combined treated effluent/tidal reservoir) (See Fig. 1).
All the samples were analysed for dissolved and total analyte concentrations. The results obtained for the
untreated coke oven liquor are summarised in Figs. 32 and 33 for trace metals and PAHs, respectively. As
may be seen from Fig. 32, results showed that Cd, Pb, Al and Zn were almost exclusively associated with
the suspended particulate matter whereas elements such as V, As, Cu and Ni were predominantly present

43

in the dissolved phase. With regard to PAHs, the results indicated that only low-molecular weight PAHs
such as naphthalene, acenaphthene and fluorene were found in the dissolved phase whereas mediumand high-molecular weight PAHs were associated with the suspended particulate matter (> 90%).

Percent present in the dissolved phase (%)

Fig. 32: Percentage of various trace metals found in the dissolved phase in the untreated coke oven
liquor at Tata Steel BET Plant A.
100.0

80.0

60.0

40.0

20.0

Iro
n

M
an
ga
ne
se

Al
um
in
iu
m

Le
ad

C
ad
m
iu
m

A
rs
en
ic

Zi
nc

C
op
pe
r

N
ic
ke
l

C
ob
al
t

C
hr
om
iu
m

V
an
ad
iu
m

0.0

Fig. 33: Percentage of individual PAHs found in the dissolved and suspended particulate matter phase in
the untreated coke oven liquor at Tata Steel BET plant A.
Inlet Reservoir (25/10/2010) Percent in the liquid phase
Inlet Reservoir (25/10/2010) Percent in the particulate phase

100.0
90.0
80.0
70.0

60.0
50.0
40.0
30.0
20.0

B [g,h,i] Per

D [a,h + a,c] Ant

IDP

B [a] Pyr

B [k] Flant

B [b + j] Flant

Chry + Trip

B [a] Ant

Pyr

Flant

Ant

Phen

Fluor

Ace

Acy

0.0

Naph

10.0

The concentration of the 16 US EPA PAHs for the untreated coke oven effluents and the treated effluents
(i.e. clarifiers 1 and 3, tidal reservoir) are depicted in Fig. 34. As may be seen from Fig. 34, naphthalene
was the most predominant PAH compound in the untreated coke oven liquor with a mean concentration
over the sampling period of about 450 g/l. Medium- and high-molecular weight PAHs were found at
much lower concentrations in the untreated effluent (ca. < 150 g/l). After treatment, the PAH
concentrations of the treated effluent in the two clarifiers were very similar indicating that similar
abatement performance between stream 1 and streams 2 and 3 combined was achieved. As shown in
Fig. 33, treatment efficiency for low-molecular weight PAHs such as naphthalene was very high (>
99.9%). After treatment, there were some residual medium-molecular weight PAHs (such as pyrene and

44

fluoranthene) and high-molecular weight PAHs (such as benzo[a]pyrene) present in the final discharge
effluents. For these compounds, the abatement efficiency typically ranged from 60% to 80%.
Fig. 34: PAH concentrations in the untreated coke oven liquor and the treated effluents
(Clarifiers 1 and 3, tidal reservoir) at Tata Steel BET plant A.
500.0
450.0

Inlet Reservoir (22 - 24th November 2010)

Clarifier 1 (22 - 24th November 2010)
Clarifier 3 (22 - 24th November 2010)
Tidal reservoir (22 - 24th November 2010)

400.0

ug / L

350.0
300.0
250.0
200.0
150.0
100.0
50.0
B [g,h,i] Per

D [a,h + a,c] Ant

IDP

B [a] Pyr

B [k] Flant

B [b + j] Flant

Chry + Trip

B [a] Ant

Pyr

Flant

Ant

Phen

Fluor

Ace

Acy

Naph

0.0

The total concentrations of trace metals in the untreated coke oven liquor and the treated effluents (ie.
clarifiers 1 and 3, tidal reservoir) are depicted in Fig. 35. As may be seen from Fig. 35A, Fe, Mn and Al
were found at higher concentrations than other trace metals such as Cd, Pb or As (Fig. 27B). Iron was
the most predominant trace element in the untreated coke oven liquor with a mean concentration over
the sampling period of about 4500 g/l. Among the trace metals selected as PS in the WFD, Ni was
present in the untreated coke oven liquor at the highest concentration (ca. 80 g/l), followed by Pb (ca.
20 g/l). Cadmium was present at very low concentrations in the untreated coke oven liquor (ca. < 1
g/l). After treatment, it appeared that abatement efficiencies varied significantly from one trace element
to another. For instance, only small decreases were observed in Zn, As and Pb concentrations between
the untreated coke oven liquor and the final discharge effluent (tidal reservoir), as opposed to Ni for
instance (Fig. 35B).

45

Fig. 35: Trace concentrations in the untreated coke oven liquor and the treated effluents
(clarifiers 1 and 3, tidal reservoir) at Tata Steel BET plant A.
5000
Inlet reservoir (Total)
Clarifier 1 (Total)
Clarifier 3 (Total)
Tidal Reservoir (Total)

4000

ug / L

3000

2000

1000

0
Aluminium

Manganese

Iron

120.0

Inlet reservoir (Total)

Clarifier 1 (Total)
Clarifier 3 (Total)
Tidal Reservoir (Total)

100.0

ug / L

80.0

60.0

40.0

20.0

0.0
Vanadium Chromium

Cobalt

Nickel

Copper

Zinc

Arsenic

Cadmium

Lead

These differences are illustrated in Fig. 36 where the abatement efficiencies for the trace metals in the
samples collected in October 2010 are depicted. These results showed that the lowest abatement
efficiencies were found for Cu, Zn, As and Pb (ca. < 50%).

46

Fig. 36: Percentage abatement of trace metals at Tata Steel BET plant A.
Percent abatement (25th October)
Percent abatement (26th October)

100.0

Percent abatement (%)

90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0

2.4.1.2

Iro
n

Le
ad
Al
um
in
iu
m
M
an
ga
ne
se

C
ad
m
iu
m

Ar
se
ni
c

Zi
nc

C
op
pe
r

N
ic
ke
l

C
ob
al
t

Va
na
di
um
C
hr
om
iu
m

0.0

Measurement of trace metal / PAH abatement efficiencies at Tata Steel

BET Plant B

Sampling campaigns were carried out at Tata Steel BET plant B throughout 2011 to study the
concentrations of PAHs and trace metals before and after treatment. Sampling of both the treated and
untreated effluents was carried out using composite samplers by collecting samples every hour for
several 24-h periods. For each campaign, the BET plant was monitored over three days (triplicate
samples/24h). Samples were collected simultaneously prior treatment (untreated coke oven effluent) and
after treatment (Sump N.6; See Section 2.1.2.1) to determine the current performance of the treatment
plant.
Table 14 summarises the PAH concentrations obtained. The sum of the total 16 US EPA PAHs and the
concentrations of selected PAHs identified as PHS in the WFD are reported. For each compound, the
abatement efficiency is calculated. As may be seen from Table 14, the PAH abatement efficiency typically
ranged from 72% to 81% with regard to the total US EPA PAHs. This result was in good agreement with
the abatement efficiencies obtained at Tata Steel Plant A (ie. 60 to 80%).
Table 14: PAH concentrations in the untreated coke oven liquor and the treated effluents at Tata Steel
BET plant B and abatement efficiency.
Sampling
periods

PAHs

PAH
concentrations
after treatment
(g / l)
2526
446
490

Abatement
efficiency
(%)

July 2011
August 2011
September
2011
November
2011

Total 16 US EPA PAHs

Total 16 US EPA PAHs
Total 16 US EPA PAHs

PAH
concentrations
before treatment
(g / l)
10243
1644
2594

Total 16 US EPA PAHs

991

276

72

August 2011

B[b]flant
B[k]flant
B[a]P
IDP
B[g,h,i]per

246
80
218
97
103

61
21
53
34
38

75
74
76
65
63

75
73
81

Typically, the abatement efficiency for high-molecular weight PAHs such as indeno[1,2,3-cd]pyrene,
benzo[g,h,i]perylene and dibenzo[a,h]anthracene was lower than low- and middle-molecular weight PAHs
such as anthracene and phenanthrene as depicted in Fig. 37 where the mean abatement efficiency (+/SD) of the sampling campaigns carried out is summarised.

47

Fig. 37: Percentage abatement of PAHs at Tata Steel BET plant B.

100

N=3

Abatement (%)

90
80
70
60
50
40
30
20
10
pyre
ne
ben
zo [
a] a
nth
rac
ene
chr
yse
ne +
trip
hen
ylen
ben
e
zo [
b+
j] flu
ora
nth
ene
ben
zo [
k] fl
uor
ant
hen
e
ben
zo [
a] p
yren
Ind
e
eno
[1,2
,3-c
d] p
dibe
yre
nzo
ne
[a,h
+a
,c] a
nth
rac
ene
ben
zo [
g,h
,i] p
ery
lene
Tot
al 1
6U
SE
PA
PAH
s

fluo
ran
then
e

ant
hra
cen
e

phe
nan
thre
ne

The total concentrations of trace metals in the untreated coke oven liquor and the treated effluents are
depicted in Fig. 38. Analysis of the data showed that:

Fe, Mn and Al were found at much higher concentrations than other trace metals such as Cd, Pb
or As.
Fe was the most predominant trace metal in the untreated coke oven liquor with a mean
concentration over the sampling period of ca. 5750 g/l.
Amongst the trace metals selected as PS in the WFD, Ni was present in the untreated coke oven
liquor at the highest concentration (ca. 80 g/l), followed by Pb (ca. 20 g/l).
Cadmium, a PHS in the WFD, was present at very low concentrations in the untreated coke oven
liquor (ca. < 1 g/l).
In terms of abatement, there was virtually no abatement for copper, nickel and zinc
(concentrations before and after treatment were within the same range).

From Fig. 38, it is clear that the BET plant provides some abatement in its current operating conditions
for a range of trace metals. The abatement efficiencies measured for a range of trace metals are
summarised in Table 15. It showed that the abatement efficiencies for Fe, Co, As, Cd, and Pb typically
ranged from 60% to 70%, but were somewhat lower for aluminium (ca. 53%) and chromium (ca. 35%).
The lowest abatement efficiencies were observed for nickel and copper (< 5%) and zinc (ca. 11%).
Fig. 38: Trace metal concentrations (ie. Al, Fe, Zn, Cr, Co, Ni, Cu, As, Cd and Pb) in untreated coke oven
liquor and the treated coke oven effluent at Tata Steel BET plant B.
8000
Untreated coke influent
Treated coke effluent

PAH concentration ug/l

7000
6000
5000
4000
3000
2000
1000
0
Aluminum

Iron

48

Zinc

Untreated coke influent

Treated coke effluent

PAH concentration ug/l

35
30
25
20
15
10
5

Le
ad

ad
m
iu
m
C

A
rs
en
ic

op
pe
r
C

ic
ke
l
N

C
ob
al
t

hr
om
iu
m

Table 15: Summary of abatement efficiencies for trace metals at Tata Steel BET plant B.
BET plant B

Trace
metals

2.4.2

Abatement efficiency (%)

N=3

Trace metals
Cadmium
Nickel
Lead
Copper
Zinc
Arsenic
Iron
Chromium
Cobalt
Aluminium

PHS
PS
PS
SP
SP
SP
SP
SP

72
<5
58
<5
11
64
69
35
67
53

Task 2.2: Determination of the concentrations of PHS/PS and the toxicity

of treated water at the final BET plant discharge points

During the project, work in this task focused on determining the typical concentrations of PHS and PS in a
wide range of steelmaking and cokemaking effluents and the toxicity of coke oven effluents using Direct
Toxicity Assessment tests in comparison with steelmaking effluents.

2.4.2.1

Concentrations of PHS/PS at Tata Steel Plant A

During the period 2010 - 2013, effluent emissions monitoring campaigns were carried out at Tata Steel
Plant A, a major integrated steelworks located in the UK with production capacity of ca. 4 million tonnes
of liquid steel per annum. At this steelworks, there are two coke making plants, four blast furnaces and
one basic oxygen steelmaking plant. Effluents from the two coke plants are combined and treated in the
same BET plant. In order to characterise the annual mass emissions of PAHs and trace metals from the
coke making process and put these emissions into perspective when compared with effluent discharges
originating from other steelmaking operations, a collection of samples for all the major effluent discharge
points at Tata Steel plant A was organised. The effluent discharge points of the site are described in
detail in Table 16 where the source of each effluent discharge point (process activity and/or site
drainage) and the volume flow are indicated.

49

Table 16: Description of the 10 major effluent discharges at Tata Steel Plant A.
Wastewater
streams
classification
Iron /
steelmaking

Rolling
Concast

Cokemaking
Site drainage

Discharge
Point
(DP)
DP1

Source
Iron and Steelmaking
waste gas cleaning
system wastewater
overflow

DP3

Rolling mill water bleeds

DP4

Concast process water

DP6

Continuous casting
process water

DP10

Coke oven biological

treatment plant

DP2

Site drainage from

rolling mill area

DP7

Site drainage from

former cokemaking site

DP9

Site drainage from

rolling mill area

DP11

Waste water from a

landfill site

DP12

Underground mine
water

Average Daily Discharge

(m3/d)
2013
2012
2011
2010
8,702

14,797

12,633

13,521

940
72
567
1307

1463

1485

1887

29
2049
79

1482
214

2630
1780

100
307

362

2104

1452
1533

As shown in Table 16, the four most significant discharge points in terms of flow rate were DP1 (iron and
steelmaking operations effluents), DP11 (land fill site drainage), DP12 (underground mine water) and
DP10 (treated coke oven effluents). Other discharge points such as DP7 (former cokemaking site
drainage), DP3 (rolling mills water bleeds), DP6 (major concast process water) and DP9 (rolling mill site
drainage) were less significant. Finally, the discharge points exhibiting the smallest flow rates were DP4
(concast plant drainage) and DP2 (drainage from rolling mill operation). All the effluents (except DP10)
are discharged primarily into a small river running within the boundary of the site, which flows into a
major tidal river. At DP10, combined treated effluents from coke making operations are released at high
tide into a major river situated outside the boundary of the steelworks. During 2010 and 2013, the
effluents from DP10 were monitored monthly between January and December. DP1 effluents were
monitored quarterly while the other discharge points were monitored twice per annum. All the effluent
samples were collected during normal plant operations.
PAH results
A summary of the PAH data obtained for the ten discharge points is provided in Table 64 (Appendix 2).
For each discharge point, the mean individual concentrations of each PAH identified as PHS or PS in the
WFD and the sum of the 16 toxic US EPA PAHs are reported. Throughout 2010 and 2013, the results
showed that DP10, which originates from the BET plant, exhibited by far the highest PAH concentrations
in the range of ca. 35 g / l and ca. 94 g / l. In contrast, PAH concentrations in other effluents were
significantly lower, typically within the range 0.1 - 4.1 g / l, the total 16 US EPA PAH concentrations in
most of other discharge points were lower than 1 g / L. Amongst the PAHs identified as PHS or PS in the
WFD, it appeared that benzo[b]fluoranthene exhibited the highest concentrations in the effluents from
DP10 (within the range 7 - 20.3 g / l) whereas anthracene was detected at the lowest concentrations
(less than 0.2 g / l).
Since a large majority of PAH emissions were attributed to coke making activities, work was carried out
to investigate their concentrations and variations in the final effluent in respect with the specified PAH
BAT-AEL value. Indeed, a BAT-AEL for cokemaking was proposed in the BREF document regarding the
emission of PAHs to water. BAT for treating wastewater from cokemaking is to use a biological effluent
treatment plant with integrated nitrification / denitrification stages (for ammonia treatment). Using this

50

treatment technique, a BAT-AEL of 50 g / L has been proposed for the sum of 6 PAHs including
benzo[a]pyrene, fluoranthene, benzo [b and k] fluoranthene, benzo [g,h,i] perylene and indeno [1,2,3-cd
pyrene]. In the UK, although this emission limit is not currently applied, it is expected that the authorities
will introduce a discharge consent limit based on this BAT-AEL in the future.
Accordingly, work was carried out to determine how the concentrations of PAHs in the final discharged
coke oven effluent compared with the BAT-AEL proposed for PAHs. Table 17 summarises the individual
concentrations (expressed in g / l) of the 6 PAHs identified in the BREF document for coke making and
the sum of these concentrations for which a BAT-AEL of 50 g / l has been proposed. In addition, Fig. 39
depicts the sum of the 6 PAH emission concentrations in respect to the BAT-AEL for all the samples
collected at the UK integrated steelwork over the period 2010-2013. Analysis of the data showed that the
mean PAH emissions ( 6 BAT-AEL PAH compounds) was 32.1 g / l with a standard deviation of 23.
Emissions ranged from 8.5 to 99.4 g / l. Overall, emissions were usually below the proposed BAT-AEL
limit. As may be seen from Fig. 39, in 2011 and 2013, no sample was in excess of the proposed BAT-AEL
value. However, a significant variation in the data was observed in 2010 when some values were above
the proposed BAT-AEL value. At that time, coke production levels were higher. At present, the variability
observed is difficult to explain, but could be associated with operational parameters at the coke ovens,
natural variability of coal or BET plant performance. Although the BAT-AEL limit has not been breached in
recent years during low production periods, it is possible that this limit could be breached again in the
future if coke production levels increase.

Table
Table 17: Emission concentrations (expressed in g / l) of the 6 PAHs identified in the BREF reference
document for which a BAT-AEL value of 50 g / l has been proposed (sum of the 6 PAHs).
PAHs
Fluoranthene
Benzo [b + j] fluoranthene

Mean

Range

SD

3.7

0.5 14.9

2.8

10.5

2.0 31.4

7.7

Benzo [k] fluoranthene

3.3

0.6 11.4

2.5

Benzo [a] pyrene

5.7

1.0 19.4

4.2

Indeno [1,2,3-cd] pyrene

4.4

0.9 12.5

3.1

Benzo [g,h,i] perylene

4.5

0.9 14.6

3.4

32.1

8.5 99.4

23.0

50

6 BAT-AEL PAHs
BAT-AEL value

Fig. 39: Sum of the individual PAH emission concentrations at Tata Steel BET plant A for the 6 PAHs
identified in the BREF document with a BAT-AEL value of 50 g / l ( 6 PAHs).
110

PAHs (Flant + B[b]Flant + B[k]Flant + B[a]P + IDP + B[g,h,i]Per

BAT- AEL value

100

Concentration g / L

90
80
70
60
50
40
30
20
10
0
0
0
0
0
0 10 10
0 10 11 11 11 11 11 11 11 12 12 12 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13
0
0
/
01 201 5/1 6/1 201 201 201 /20 /20
01
0 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
/2
/
/
/
/ 0
/2 /11 /2
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/0
/0
0
01 /01 13 04 /07 /08 /09 6/1 7/1
11 -23 /02 /04 /05 /06 /08 /11 /12 /02 /03 /04 /05 /06 /07 /08 /09 /10 /11 /01 /02 /03 /06 /08 /09 /10 /11 /12
/
/
2
27 27
06 04 02 - 2
02 22
25 07 03 27 10 08 12 02 05 27 15 25 11 21 19 29 26 29 14 14 04 08 16 17 20 13
26
25

51

Trace metal results

The concentrations of trace metals determined in the effluents from all major discharge points at Tata
Steel Plant A have been summarised in Table 65 for the year 2012 (See Appendix 3). For each metal,
both the total and the dissolved concentrations are reported, except for Hg for which only the dissolved
concentrations were measured in this study. As may be seen from Table 65, Fe, Al, and Mn were the
trace metals found in highest concentrations in Scunthorpe effluents. Figure 40 summarises the total and
dissolved concentrations of three metals that have been identified in the WFD as PHS (i.e. Cd) and PS
(i.e. Ni and Pb). In addition, Fe and As were also plotted. As may be seen from Fig. 40, Cd was detected
predominantly in DP4 and DP6 effluents, and to a lesser extent DP1, and DP3. The highest concentration
of Pb was found in DP1, almost entirely in the particulate phase. The presence of Ni was identified in all
discharge points, and it was found almost exclusively in the dissolved phase. Arsenic was present
predominantly in the dissolved phase (ca. more than 75%) for all effluents. Iron was found
predominantly in most effluents in the suspended solids fraction, except W10 effluent, exhibiting 20% of
Fe in the dissolved phase.
Fig. 40: Total and dissolved concentrations of Cd, Pb, Ni (c), Fe and As in effluents from Tata Steel Plant
A in 2012.

45.0

Total Pb

40.0

Dissolved Pb

7.0

Total Cd

6.0

Dissolved Cd

Concentration ug/l

C o n cen tratio n u g /l

35.0
30.0
25.0
20.0
15.0

5.0
4.0
3.0
2.0

10.0
1.0

5.0
0.0

14000
12000

P1
DP
2
D
P3
D
P4
DP
6
D
P7
D
P9
D
P1
0
DP
11
D
P1
2

12

11

DP

DP

10
DP

DP

DP

DP

DP

DP

DP

DP

0.0

Total Fe

20.0

Dissolved Fe

18.0

Total Ni
Dissolved Ni

8000
6000
4000

Concentration ug/l

10000

14.0
12.0
10.0
8.0
6.0
4.0

2000

2.0

P7

DP
9
D
P1
0
D
P1
1
DP
12

P6
D

P3

DP
4

P1
D

52

DP
2

0.0

DP
1
DP
2
DP
3
DP
4
DP
6
DP
7
DP
9
DP
10
DP
11
DP
12

Concentration ug/l

16.0

14.0

Total As
12.0

Concentration ug/l

Dissolved As
10.0
8.0
6.0
4.0
2.0

2.4.2.2

D
P
12

D
P
11

D
P
10

D
P
9

D
P
7

D
P
6

D
P
4

D
P
3

D
P
2

D
P
1

0.0

Concentrations of PHS/PS at Tata Steel Plant B

A series of effluent emissions monitoring campaigns were carried out from 2010 to 2013 at a second
major integrated steelworks (Tata Steel Plant B) located in the UK with production capacity of ca. 4.0
million tonnes of liquid steel per annum. At this steelworks, there is only one effluent discharge point
which is composed of treated coke oven effluents, drainage from the site, storm water, effluents from
continuous casting and cold mill, effluents from the BOS gas cleaning system and from the blast furnace
blowdown. The combined effluent is directly discharged to the sea at high tide (long sea outfall).
At this site, monthly samples were collected between 2010 and 2013. The PAH data obtained for all the
samples collected is summarised in Table 18. In this study, the concentrations reported are the total PAH
concentrations (ie. particulate + dissolved fractions). Both the mean and the concentration range are
reported for each of the PAHs classified as PHS and PS in the WFD as well as the sum of the 16 US EPA
PAHs.
Table 18: PAH annual mean concentrations (expressed in g / l) in effluents from the main effluent
discharge point (long sea outfall) at Tata Steel Plant B.

PAHs

2013
(n =12)

2012
(n =12)

2011
(n =12)

2010
(n = 9)

Benzo [a] pyrene (B[a]P)

PHS

2.63

1.62

2.13

3.35

Benzo [b] fluoranthene

PHS

3.86

3.15

3.49

6.38

Benzo [k] fluoranthene

PHS

1.59

1.30

1.20

1.91

Indeno [1,2,3-cd] pyrene

PHS

2.08

1.48

1.45

2.22

Benzo [g,h,i] perylene

PHS

2.10

1.47

1.78

2.56

Anthracene

PHS

0.56

0.36

0.39

0.10

Naphthalene

PS

8.61

0.61

1.92

0.84

Fluoranthene

PS

6.91

2.87

4.38

2.64

50.3

24.6

28.8

30.3

Total 16 US EPA PAHs

As may be seen from Table 18, most of the PAHs listed as PHS or PS in the WFD were found in similar
concentrations range, typically between 1 to 8 g / l, except anthracene which was the PAH found at the
lowest concentrations in the final effluent (ca. < 1 g / l). Benzo[b]fluoranthene exhibited the highest
concentrations in effluents in the four year period, and ranged from ca. 3.2 g / l to ca. 6.4 g / l.
The concentrations of trace metals determined in the effluents from the combined final effluent are
summarised in Table 19. For each metal, both the total and the dissolved concentrations are reported,
except for Hg for which only the dissolved concentrations were measured in this study.

53

Table 19: Concentrations of trace metals (total and dissolved concentrations (expressed in g/l) in
the combined final effluent (long sea outfall) at Tata Steel Plant B.
2013
N=12

Dissolved

Total

Trace metals

2012
n=12

2011
n=12

2010
n=9

Cd

PHS

1.2

1.2

1.1

2.0

Pb

PS

62.7

69.4

63.0

86.7

Ni

PS

9.4

9.9

14.2

10.9

Cr

SP

11.7

11.0

24.4

10.6

Fe

SP

7690

8414

11586

9039

Cu

SP

19.8

20.4

18.4

15.3

Zn

SP

2191

2721

3208

3135

As

SP

4.4

3.9

4.4

3.5

Cd

PHS

0.03

0.1

0.1

0.21

Pb

PS

1.0

0.9

0.9

6.2

Ni

PS

4.9

7.9

7.6

5.6

Hg

PHS

0.09

Cr

SP

0.5

5.1

4.2

0.6

Fe

SP

98.9

69.3

255

46.9

Cu

SP

8.4

7.5

5.7

6.3

Zn

SP

370

561

909

1347

As

SP

2.3

1.7

1.7

1.4

* Not analysed in 2011 and 2013.

As may be seen from Table 19, among the metals identified as PHS, PS and SP in the WFD, Fe and Zn
were the two trace metals exhibiting the highest concentrations ranging from 7690 g / l to 11586 g / l
and 2191 - 3208 g /l, respectively. In contrast, all the other trace elements analysed in this study were
found at much lower concentrations (ca. < 100 g/l), with Pb exhibiting the highest concentrations
(within the range 62.7 86.7 g / l). Cd and Hg exhibited relatively low concentrations. Fig. 41 shows
the percent of metals found in the dissolved phase for the key trace metals which overall shows a good
agreement for the results between 2010-2013. Cr, Fe, Cd and Pb were found almost exclusively in the
suspended solids, whereas a significant proportion of Ni and As were found in the dissolved phase (ca. 50
-60%).
Fig. 41: Percent of trace metals found in the dissolved phase in the combined final
effluent (long sea outfall) of Tata Steel Plant B.

Percentage present in dissolved phase %

80

2013

2012

Zn

As

2011

2010

60

40

20

Cr

Fe

Ni

Cu

54

Cd

Pb

2.4.2.2

Direct Toxicity Assessment

Effluent toxicity testing is also referred as Direct Toxicity Assessment (DTA) in the UK by the England and
Wales Environment Agency. These methods were originally developed as a complementary approach to
the chemical-specific analysis of effluents for controlling the impact of industrial effluents on surface
waters.
DTA involves the testing of the toxicity of an effluent on a standard suite of organisms (algae and
invertebrates). In the UK, two DTA tests have been selected as reference methods for testing effluents
discharging to freshwater, namely Daphnia Magna and the Algae inhibition of growth, whereas three DTA
tests have been selected as reference methods for marine environments, namely Oyster Embryo-larval,
Tisbe Battagliai and Marine algae inhibition of growth. Fig. 42 shows a picture of the different organisms
used in these tests. The different types of tests and their characteristics are also summarised in Tables
20 and 21.
Fig. 42: Picture of organisms used in Direct Toxicity Assessment tests of industrial effluents for
freshwater or marine waters in the UK. (A) Daphnia Magna : 0.5 to 0.5 mm in length ; (B)
Pseudokirchneriella subcapitata freshwater algae ; (C) Tisbe battagliai and (D) Skeletonema costatum
marine algae (size:43 70 m).

Table 20: Direct Toxicity Assessment for industrial effluents discharged to freshwater.
Methods

Species

Duration of study
(hours)

Endpoint

Daphnia Magna
immobilisation

Invertebrate
Daphnia magna

48

immobilisation

Freshwater Algae
inhibition of growth

Algae
Pseudokirchneriella
subcapitata

72

growth inhibition

55

Table 21: Direct Toxicity Assessment for industrial effluents discharged to marine waters.
Methods

Species

Duration of study
(hours)

Endpoint

Oyster Embryo-Larval
development

Invertebrate
Crassostrea gigas (Pacific
oyster)

24

larvae
development

Tisbe battagliai
lethality

Invertebrate
Tisbe battagliai

48

mortality

Marine algae inhibition of

growth

Algae
Skeletonema costatum

72

growth
inhibition

2.4.2.2

Results from Direct Toxicity

(cokemaking / steelmaking)

Assessment

on

Tata

Steel

effluents

A series of DTA assessments were carried out on cokemaking and steelmaking effluents by an external
laboratory (Cheshire Eco Solutions) accredited under the England and Wales Environment Agency
Monitoring Certification Scheme (MCERTS). Full regulatory DTA testing are generally very costly and
provide an exact measurement of the EC50, the effective concentration that causes 50% inhibition,
mortality or immobilisation of the organisms considered (depending on the tests). To achieve this,
analysis with several levels of dilution of the effluent must be carried out. However, it is also possible to
carry out non- regulatory screening tests by selecting only two levels of dilution (ie. 1% and 10%). This
allows faster comparison of the toxicity of different effluents without incurring excessive costs. It also
provides an indication of the value of the EC50 (ie. below 1% and 10%). All the non-regulatory tests are
based on well recognised published methods and the results are comparable to those generated by full
regulatory studies [28-31].
For this study, four effluent discharges were selected at Tata Steel plants A and B. These were discharge
points 1, 6 and 10 at Tata Steel plant A and the long sea outfall discharge at Tata Steel Plant B. Details of
the process effluents which contribute to these discharges are given in Table 22. DP1 and DP6
wastewater are discharged into freshwater therefore these effluents were assessed using freshwater
species. P10 and the long sea outfall discharges were assessed using marine species. These effluents
were tested at concentrations slightly lower than 1, 10 and 100% v/v, owing to the addition of a brine
solution required to adjust the salinity. For the tests, replicate spot samples were collected in 2 litre glass
bottles and transported to Cheshire Eco Solutions in a cool box at a temperature of less than 4 C. To
ensure sample integrity, samples were analysed within 24 hours of receipt.
Table 22: Details of the effluent samples submitted for Direct Toxicity Assessment.

Discharge
DP1

DP6
DP10
Long Sea
Outfall

Description

Test Species

Effluent discharge containing process water

from the blast furnaces (BF) and basic oxygen
steelmaking (BOS) operations and site
drainage waste water.
Concast caster process water and site drainage
from soaking pits, material off-loading area
and the mould preparation bay.
Effluent discharge from the BET plant
Combined effluent from the BET plant and
steelmaking emissions (BF / BOS)

(i) Pseudokirchnereilla Subcapita

Algal Growth Inhibition test
(ii) Daphnia Magna
immobilisation test
(i) Skeletonema Costatum Algal
growth inhibition test
(ii) Tisbe Battagliai
immobilisation test

The results obtained are summarised in Table 23 (freshwater tests) and Table 24 (marine tests). With
regard to DP1 (BF and BOS process water effluents), no toxic effect was observed on either freshwater
algae or daphnia magna, i.e. EC50 >100%. For DP6, DP10 and the Long Sea Outfall (LSO) samples, a
toxic effect was observed on both algae and invertebrate test species. For DP6, Pseudokirchnereilla
Subcapitata algae was found to be the most sensitive species. For LSO, the marine invertebrate (Tisbe

56

battagliai) was the most sensitive species. The toxicity results observed for DP10 suggested that both
test species were similarly sensitive, i.e. EC50 >1%.
Table 23: Results from freshwater ecotoxicity tests on steelmaking effluents.

Discharge
DP1

Sample
date
04/07/201
1

Pseudokirchnereilla
subcapitata Algal
(72 hr)
Test
concentration
(% v/v)

(48 hr)

Growth
Inhibition (%)

Control

04/07/201
1

Immobilisatio
n (%)

Control

10

20

10

10

15

100

30.8

100

35

> 100.0% v/v

Test
concentration
(% v/v)

> 100.0% v/v

Growth
Inhibition (%)

Control

EC50

Test
Concentration
(% v/v)

EC50
DP6

Daphnia magna

Test
Concentration
(% v/v)

Immobilisati
on (%)

Control

10

10

10

>100

10

40

100

>100

100

75

> 1.0% v/v

> 10.0% v/v

Table 24: Results of marine ecotoxicity tests on cokemaking and steelmaking effluents.

Discharge
DP10

Sample
date
04/07/2011

Skeletonema
Costatum Algal

Tisbe Battagliai

(72 hr)

(48 hr)

Test
Concentration
(% v/v)

Growth
Inhibition (%)

Control

04/07/2011

0.85

45

8.5

90

8.5

95

84.7

>100

84.7

100

> 1.0% v/v

Test
Concentration
(% v/v)

> 1.0% v/v

Growth
Inhibition (%)

Control

EC50

Immobilisation
(%)

0.85

EC50
LSO

Test
Concentratio
n
(% v/v)
Control

Test
Concentratio
n
(% v/v)
Control

Immobilisation
(%)
3

0.83

1.6

0.83

25

8.3

10.5

8.3

65

83.3

>100

83.3

100

> 10.0% v/v

> 1.0% v/v

Although, an exact measurement of the EC50 was not possible as only selected tests on three different
levels of dilution were carried out, the results showed that DP10 (cokemaking effluent) exhibited the

57

highest toxicity with an expected EC50 ranging from 1% to 8.5% (v/v). This work led to identify several
limitations of the DTA approach. First, DTA analysis was very expensive. Each sample analysis costed well
over 1000 Euros to obtain an estimation of the EC50. If the full DTA assessment was carried out in order
to obtain an exact measument of the EC50, the price of analysis per sample would have risen to 1500
Euros. Second, DTA assessment requires a significant amount of planning prior the realisation of the
tests. Indeed, for most tests, the organisms must be prepared at least three weeks in advance prior
being exposed to the effluents for testing. In addition, it is also essential that samples are tested within
12-h after sample collection. Therefore, this work was extremely difficult to organise. Although all the
parameters were fulfilled for the tests described above, in practise it would be too costly and almost
impossible to carry out this type of toxicity assessment on a regular basis.

2.4.3

Task 2.3: Mass inventory releases of PHS / PS in cokemaking

2.4.3.1

Mass inventory releases of PHS / PS at Tata Steel Plant A

PAH annual releases and emission factors (Tata Steel Plant A)

The analytical data previously reported in Section 2.4.2.1 were used to calculate annual mass releases
and emission factors to water for the integrated steelworks. The mass releases of the specified
substances to water were calculated annually as recommended by the Environment Agency of England
and Wales. The first step was to multiply the measured emission concentration by the volumetric flow
rate of the emission source at the sampling time. These emission rates were then combined together for
the annual operating period.
Equation 1 was used to calculate emissions based on sample concentration expressed in g / l (ie. mg /
m3), where the pollutant concentration was consistent over the averaging period (i.e. one year):

(1) E = C x Q x 365 x 10-6

Where: E = emission rate of pollutant in kg/annum
C = pollutant concentration (mg/m3)
Q = volumetric flow rate of the emission (m3/d)
365 = number of operating days of the activity per year
In addition, for each emission source, emission factors were calculated. These are usually expressed as
the mass of a substance emitted divided by the mass, volume, distance, or duration of the activity
emitting the substance. In this study, equation 2 was used to calculate the emission factors.

(2) EF = E x 106 / M
Where: EF = controlled emission factor of pollutant per activity in mg/tonne
E = emission rate of pollutant (kg/annum)
M = mass of production in the year (tonnes/annum)
For the effluents originating exclusively from coke-making operations (i.e. DP10), the emission factors
were expressed in mg / tonne coke, while for all other discharge points, the emission factors were
expressed in mg / tonne liquid steel (LS).
For the calculation of annual mass releases, the annual mean flow rates (m3 / day) of the 10 discharge
points were used (See Table 18). With regard to DP10 discharge point, for which effluents originate
exclusively from coke making activities, the emission factors were expressed in mg / tonne coke and
were calculated using the annual coke production of Tata Steel Plant A (ca. 1,173,235 tonnes in 2010, ca.
1,187,436 tonnes in 2011, ca. 1,062,918 tonnes in 2012 and ca. 1,042,829 tonnes in 2013,
respectively). For all other discharge points, the emission factors were expressed in mg / tonne liquid
steel (LS) and were calculated using the 2010 and 2011 annual steel production of Tata Steel Plant (ca.
3,108,872 tonnes in 2010, ca. 3,396,364 tonnes in 2011, ca. 3,165,747.95 tonnes in 2012 and ca.
3,149,345 tonnes in 2013, respectively).
The previous study indicated that most of PAHs originated from DP10, and a small proportion of PAHs
originated from DP1 and DP11. Accordingly, a focus was given to these three major emission sources for

58

which a complete emissions inventory was compiled for the period 2010-2013. The annual mass releases
for the three most significant discharge points of the steelworks and total PAH emissions are presented in
Table 25. For each discharge point, the mean and the range of annual mass releases are reported
individually for each PAH identified as a PHS or PS in the WFD. In addition, the sum of the 16 US EPA
PAHs (i.e. total PAHs) is reported. The total PAH emissions (kg / annum) are expressed as the sum of the
16 US EPA PAHs for all ten effluent discharges (sum of DP1 to DP12).
As may be seen from Table 25, the mean total PAH annual release to water was ca. 35 kg ranging from
21 kg to 71 kg between 2010 and 2013. Over the period, a large proportion of the annual release
originated from DP10, the coke oven BET plant with a mean value of ca. 31 kg / annum, ranging from 20
kg to 65 kg. In comparison, annual mass releases for DP1 were only ca. 2.5 kg. DP1 is composed of
wastewater originating from the gas cleaning systems of the BF and the BOS plant. Although it
represented by far the largest volume of wastewater discharged to the environment (See Table 17), the
contributions to the total PAH emissions of the site were relatively low. Finally, the only other significant
source of PAHs was from DP11 (site drainage) which accounted for ca. 1.3 kg / annum. The individual
PAH exhibiting the highest annual releases was benzo [b] fluoranthene (ca. 7.0 kg / annum). Amongst
the PAHs identified as PHS and PS in the WFD, anthracene exhibited the lowest mass release (ca. 0.16 kg
/ annum).
Table 25: Annual mass releases (kg/annum) of PAHs at Tata Steel Plant A (Period 2010-2013).

PAHs
Anthracene

PHS

Benzo [b]
fluoranthene
Benzo [k]
fluoranthene
Benzo [a]
pyrene
Indeno [1,2,3cd] pyrene
Benzo [g,h,i]
perylene
Naphthalene

PHS

Fluoranthene

PHS
PHS
PHS
PHS
PS
PS

DP1
(n = 16)

DP10
(n =40)

DP11
(n = 10)

Iron /
steelmaking

Cokemaking

Site drainage

Total emissions
from the
Steelworks
All discharge
points

Mean

Range

Mean

Range

Mean

Range

Mean

Range

0.03

0.02 - 0.03

0.09

0.07 - 0.13

0.04

0.001 - 0.10

0.16

0.09 - 0.23

0.23

0.1 - 0.3

6.5

3.8 - 14

0.15

0.003 - 0.32

7.0

3.9 - 14.7

0.08

0.04 - 0.12

2.0

1.1 - 4.2

0.05

0.002 - 0.10

2.2

1.1 - 4.5

0.09

0.05 - 0.12

3.6

2.2 - 7.5

0.07

0.002 - 0.14

3.8

2.2 - 7.9

0.10

0.05 - 0.18

2.7

1.5 - 5.5

0.05

0.003 - 0.11

2.9

1.5 - 5.8

0.15

0.08 - 0.23

2.8

1.7 - 5.9

0.06

0.005 - 0.12

3.0

1.8 - 6.2

0.37

0.26 - 0.47

0.22

0.12 - 0.32

0.07

0.036 - 0.11

0.75

0.53 - 0.88

0.28

0.19 - 0.34

2.5

1.6 - 4.8

0.20

0.012 - 0.47

3.0

1.9 - 5.6

1.8 - 2.9

30.8

18.5 - 65.0

1.3

0.15 - 2.9

35.3

21.2 - 70.9

Total 16 US
2.5
EPA PAHs
(n = number of measurements)

Analysis of the data presented in Table 25 also showed a relatively high variability in the annual mass
releases of PAHs, particularly with regard to cokemaking. For instance, total PAH emissions from
cokemaking operation (DP10) ranged from 18.5 to 65.0 kg / annum. In order to ensure that these
variations were not only associated with variations in the coke production, emission factors were
calculated for DP10. Analysis of the data showed that PAH emissions from 2011 to 2013 were very
similar with emission factors of 15.8, 19.7 and 17.7 mg / tonne of coke produced, respectively. In 2010,
however, PAH emissions were relatively higher with an emission factor of 55.4 mg / tonne of coke. These
differences were difficult to explain. These may be linked to different operational parameters at the coke
ovens. It could also be associated with the use of different coals which can contain varying amounts of
PAHs. Indeed, it has been suggested that the PAH content in coals could vary as a function of their origin
and maturity [32]. Further studies would be needed to understand the variability observed in PAH
emissions in the future however this was outside the scope of the ECOWATER project. The data obtained
over the 4-year period were used to calculate a mean generic emission factor. The mean emission factors
are summarised in Table 26. As may be seen from Table 26 which summarises the mean emission
factors, total PAH and B[a]P emissions from coke making operation were 27.2 mg / tonne coke and 3.2
mg / tonne coke, respectively. In comparison, the emission factors for total PAHs obtained from the
other iron and steel making processes were relatively low, ranging from 0.002 mg / tonne liquid steel to
0.78 mg / tonne liquid steel.

59

60

PS

Fluoranthene

Total 16 US EPA PAHs

PS

PHS

PHS

PHS

PHS

PHS

PHS

Naphthalene

perylene

Benzo [g,h,i]

pyrene

Indeno [1,2,3-cd]

Benzo [a] pyrene

fluoranthene

Benzo [k]

fluoranthene

Benzo [b + j]

Anthracene

PAHs

0.78

0.087

0.116

0.047

0.033

0.028

0.026

0.073

0.045

0.006

0.007

0.003

0.005

0.005

0.004

0.009

0.001

0.12

0.016

0.015

0.010

0.007

0.011

0.006

0.016

0.002

27.2

2.2

0.20

2.5

2.4

3.2

1.8

5.7

0.08

0.43

0.065

0.022

0.018

0.016

0.029

0.015

0.049

0.015

0.002

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

(n = 8)

(n = 8)

(n = 16)

0.008

(n = 8)

(n = 10)

casting)

mills)

Steelmaking)
(n =40)

DP2

DP11

DP10
(Cokemaking)

DP6
(Continuous

DP3
(Rolling

DP1

(Ironmaking /

0.003

0.001

0.002

<0.001

<0.001

<0.001

<0.001

<0.001

<0.001

(n = 8)

DP4

0.054

0.006

0.008

0.001

0.002

0.002

0.002

0.005

0.001

(n = 9)

DP7

Site drainage

0.018

0.003

0.002

0.002

0.002

0.002

0.002

0.004

<0.001

(n = 9)

DP9

0.072

0.006

0.015

0.002

0.002

0.002

0.002

0.003

0.001

(n = 8)

DP12

Table 26: Mean emission factors of PAHs (all expressed in mg / tonne liquid steel, except for DP10: expressed in mg / tonne coke) for all the wastewater
effluent discharges at Tata Steel Plant A. Releases from the cokemaking process are highlighted in grey.

Trace metal annual releases and emission factors (Tata Steel Plant A)
The mean concentrations determined from 2010 to 2013 for each discharge point were used to estimate
the annual mass releases of each trace metal investigated in this study. The calculation method used for
annual mass releases and emission factors is identical to the one described in the previous section for
PAHs. Initial results obtained during the first year of the project showed that the most significant sources
were DP1, DP3, DP6, DP10 and DP11. Accordingly, a focus was given to these five major sources for
which a complete emissions inventory was compiled. The mean and the range of annual mass releases
for these five most significant discharge points are presented in Table 27 together with the total trace
metal emissions from the steelworks for which all discharge points were taken into consideration.
As may be seen from Table 27, the most significant trace metal releases from waste water effluent
discharges were Fe (ca. 12 to 16 ton / annum) and Zn ( ca. 0.78 to 1.1 ton / annum). The total annual
mass releases of Pb ranged from ca. 74 to ca. 277 kg / annum. The total annual mass releases of Ni were
lower and ranged from ca. 40 to 114 kg / annum. Both the emissions of Cd (ca. 2 kg / annum) and, in
particular Hg (ca. 0.3 kg / annum) were very low.
As pointed out in the previous section with regard to PAH releases, some variability was observed in the
trace metal annual releases. For some elements, such as Cd, Hg, As or Pb, relatively narrow emission
ranges were obtained over the period 2010 2013. For instance, Cd emissions for the integrated
steelworks ranged from 1.3 to 2.3 kg/annum. However, for other trace metals such as Fe and Zn, much
wider emission ranges were obtained. For instance, Fe emissions ranged from 12 to 16 tonnes / annum.
It is difficult to explain in detail the reasons behind such variability for some trace elements because
several parameters can play a role including the type of raw materials used in the ironmaking /
steelmaking processes, the annual steel production and the operational parameters. Investigating the
variability of trace metal emissions in wastewater from an integrated steelworks was outside the scope of
this project, but further work is needed to try and correlate process parameters / raw material inputs
with effluent quality.
Table 28 summarises the trace metal mean emission factors calculated for four years over the period
2010 -2013. For the cokemaking process, emissions are reported in mg / tonne of coke whereas for all
other processes or site drainage sources, emission factors are reported in mg / tonne liquid steel (LS).
Analysis of the data showed that the main emissions of Fe originated from the combined ironmaking and
steelmaking effluents with the emission factor of ca. 3197 mg / tonne LS, and the cokemaking effluents
(ca. 1327 mg / tonne coke). Pb was mostly emitted from ironmaking and steelmaking operations (ca.
47.3 mg/tonne LS). Zinc and As were predominantly emitted from the cokemaking process (ca. 341
mg/tonne coke and ca. 5.4 mg/tonne coke, respectively). These data constitute the first detailed report
of wastewater trace metal emissions from the processes used in integrated steelmaking.

61

62

SP

SP

SP

SP

Fe

Cu

Zn

As

10.5

615

56.3

10247

31.4

0.14

42.6

150

1.4

8.0 - 15.9

321 - 925

43.9 - 79.8

7187 - 12478

9.6 - 69.7

0.1 - 0.16

28 - 77.1

65 - 265

1.1 - 1.8

DP1
(Ironmaking /
Steelmaking)
(n = 16)
Mean
Range

0.85

7.1

1.7

573

5.5

0.02

5.4

1.4

0.03

Mean

0.52 1.5

2.1 - 17.0

0.39 - 3.6

225 - 1074

0.9 - 17.4

0.001 - 0.03

1.1 - 13.1

0.54 - 2.3

0.01 - 0.04

Range

DP3
(Rolling mills)
(n = 8)

0.53

11.8

1.7

666

2.3

0.004

2.2

1.8

0.24

Mean

0.23 - 0.77

7.0 - 15.9

1.1 - 2.6

368 - 883

1.9 - 3.2

0.003 - 0.004

1.3 - 3.2

1.3 - 2.7

0.03 - 0.40

Range

DP6
(Continuous casting)
(n = 8)

6.1

374

2.3

1487

12.5

0.10

6.4

5.2

0.07

Mean

4.9 - 9.8

105 - 535

1.9 - 4.0

1066 - 1761

8.6 - 18.8

0.08 - 0.12

4.8 - 8.0

4.8 - 5.5

0.04 - 0.13

Range

DP10
(Cokemaking)
(n =40)

1.4

4.3

0.72

198

0.49

0.04

4.4

0.36

0.01

Mean

0.97 - 1.9

0.22 - 9.7

0.34 - 1.1

76 - 450

0.04 - 1.3

0.02 - 0.05

1.3 - 8.6

0.18 - 0.89

Range

DP11
(Site drainage)
(n = 10)

21.2

1020

64.4

14244

69.2

0.32

69.6

160

1.9

Mean

18.7 - 24.3

780 - 1129

50.9 - 87.5

11987 - 15940

28.3 - 112

0.29 - 0.37

40.2 - 114

74.2 - 277

1.3 - 2.3

Range

Total integrated
steelworks emissions
(All discharge points)

PHS

PS

PS

PHS

SP

SP

SP

SP

SP

Cd

Pb

Ni

Hg

Cr

Fe

Cu

Zn

As

(Ironmaking /

metals

3.3

194

17.6

3197

9.8

0.04

13.3

47.3

0.43

(n = 16)

Steelmaking)

DP1

Trace

0.26

2.2

0.52

176

1.7

0.01

1.7

0.43

0.01

(n = 8)

(Rolling mills)

DP3

0.44

9.4

1.4

515

2.0

0.003

1.7

1.3

0.24

(n = 8)

casting)

(Continuous

DP6

5.4

341

2.1

1327

11.2

0.09

5.8

4.6

0.06

(n =40)

(Cokemaking)

DP10

0.44

1.3

0.23

63.0

0.16

0.02

1.4

0.12

0.004

(n = 10)

DP11

0.01

0.05

0.01

11.0

0.08

<0.001

0.01

0.02

<0.001

(n = 8)

DP2

0.02

0.21

0.07

2.7

0.10

<0.001

0.11

0.01

0.04

(n = 8)

DP4

0.42

1.9

0.28

273

1.3

0.01

1.2

0.22

0.01

(n = 9)

DP7

Site drainage

0.07

0.29

0.07

21.5

0.22

<0.001

0.10

0.07

0.002

(n = 9)

DP9

0.25

0.44

0.28

80.9

4.7

0.02

1.8

0.20

0.02

(n = 8)

DP12

Table 28: Mean emission factors (all expressed in mg / tonne liquid steel, except for DP10: in mg/tonne coke) of trace metals at Tata Steel Plant A.

Hg annual mass releases were calculated using the dissolved emission concentrations.

SP

Cr

PHS

PS

Ni

Hg

PS

Pb

PHS

Cd

Trace
metals

Table 27: Annual mass releases (expressed in kg / annum) of trace metals at Tata Steel Plant A (Period 2010-2013).

In order to understand further the contribution of each source, in particular the steelmaking and
cokemaking process, to the overall emissions of trace metals at Tata Steel plant A, the mean
percentage contribution of each discharge point to the total emissions for eight metals including
Cd, Pb, Ni, Cr, Fe, Cu, Zn and As were plotted for the year between 2010 and 2013 as depicted in
Fig. 43.
Fig 43: Percent contribution of each effluent discharge point to the total trace metals emissions at
Tata Steel Plant A. Metals considered are Cd, Pb, Ni, Cr, Fe, Cu, Zn and As.

% contribution of each discharge point

100%

DP12
DP11
DP10

80%

DP9
DP7

60%

DP6
DP4
DP3

40%

DP2
DP1

20%

0%
Cd

Pb

Ni

Cr

Fe

Cu

Zn

As

Analysis of the data showed:

DP1 (process water from the blast furnaces and the BOS cleaning systems) appeared to be
the most significant emission source.
Cadmium emissions from Tata Steel Plant A were significantly lower than Ni and Pb, and
mostly originated from DP1 (74%), the rest being emitted from DP6, DP4, DP10 and DP12,
in a percentage of 12%, 6%, 4% and 2%, respectively.
With regard to Pb, approximately 92 % of total Pb annual releases were emitted from DP1
discharge point and only 4% from DP10.
Nickel was emitted from various sources, with DP1 the most significant (ca. 60%), followed
by DP10 (ca 10%).
Zinc was discharged in almost equal quantities from DP1 (iron and steelmaking operations)
and DP10 (BET plant / cokemaking process).
A significant proportion of Cr originated from DP10 (20%) and DP12 (10%).
Arsenic was emitted from various sources, with DP1 the most significant (ca. 50%),
followed by DP10 (ca 30%).

PAH annual releases and emission factors (Tata Steel Plant B)

At Tata Steel Plant B, the main discharge point (Long Sea Outfall) was monitored monthly over the
period 2010 2013. The PAH concentrations were reported previously in Section 3.4.2.1. These
data were used to calculate annual mass releases for the integrated steelworks. For calculations,
monthly flow rates of the discharge point were combined with PAH concentrations in order to
determine annual mass releases. Emission factors, expressed in mg / tonne liquid steel (LS), were
also calculated using the annual steel production (ca. 4,126,957 tonnes in 2010; ca. 3,718,840
tonnes in 2011; ca. 2,778,642 tonnes in 2012; ca. 4,150,042 tonnes in 2013). The results are
summarised in Table 30.

63

As may be seen from Table 29, the annual mass releases for the total 16 US EPA PAHs from 2010
to 2012 were very similar with total emission ranging from 425 kg to 480kg. In 2013, however,
PAH emissions were relatively higher with a value of 837 kg, approximately double the value
obtained between 2010 and 2012. Anthracene was the compound exhibiting the lowest annual
mass release amongst all the other PHS or PS. Again, the relatively high variability in PAH
emissions could be associated with operational parameters of the coke ovens or natural variability
in the PAH content of the coals processed, however further work would be needed to confirm this.
Of significance is that this study provided for the first time an estimation for the total PAH
emissions (ca. 147 mg / tonne LS) and for B[a]P (ca. 9.2 mg / tonne LS) to water from integrated
steel works.
Table 29: Annual mass releases of PAHs (expressed in kg / annum) and emission factors
(expressed in mg / tonne liquid steel) for the main effluent discharge point (long sea outfall) at
Tata Steel Plant B in 2010 and 2013.
Annual mass releases
(kg/annum)

PAHs

Mean emission factor

(mg/ tonne liquid steel)

2013

2012

2011

2010

Between 2010 and 2013

Anthracene

PHS

8.6

6.3

6.8

1.4

1.6

Benzo [b + j]
fluoranthene

PHS

43.2

53.4

57.8

89.7

16.7

Benzo [k] fluoranthene

PHS

63.8

21.9

19.7

26.9

8.8

Benzo [a] pyrene

PHS

25.9

27.3

35.8

46.2

9.2

Indeno [1,2,3-cd]
pyrene

PHS

34.4

25.3

24. 5

30.5

7.9

Benzo [g,h,i] perylene

PHS

34.4

25.0

30.3

35.4

8.5

Naphthalene

PS

165

10.5

30.7

12.5

13.7

Fluoranthene

PS

109

50.7

73.2

37.7

18.3

837

423

480

425

147

Total 16 US EPA PAHs

Interestingly, the results showed that Tata Steel Plant A exhibited much lower annual mass
releases of PAHs (21 to 71 kg / annum) than Tata Steel Plant B (423 to 480 kg / annum). The
reasons for these differences are linked to the fact that much higher concentrations of suspended
solids were measured in the treated effluent of BET plant B (ca. 400 mg / L) compared to BET
plant A (ca. 20 to 50 mg / L). During this investigation, it was found that treatment stability at BET
Plant A was much better than BET plant B. In particular, thiocyanate treatment was relatively poor
at BET plant B owing to very strong coke oven liquor which may inhibit the treatment of
thiocyanate. Also the possibility of unwanted nitrification occurring at that plant could have resulted
in a loss of thiocyanate treatment. When the treatment of thiocyanate was poor, suspended solid
concentrations in the treated effluent increased significantly. Since PAHs are mostly bound to
suspended solids, this resulted in much higher PAH releases at plant B. At BET plant B, in order to
reduce significantly PAH emissions, it would be essential that the factors inhibiting thiocyanate
treatment are tackled first to establish a stable treatment process.
Trace metal annual releases and emission factors at Tata Steel Plant B
The trace metal concentrations data previously reported in Section 3.4.2.1 were used to calculate
annual mass releases for the integrated steelworks. No sample was analysed for Hg between 2011
and 2013 since Hg was only found at very low concentrations in 2010. As a result, the mean
concentration for the samples collected in 2010 (0.09 g / L) was used for calculation. The results
are presented in Table 30.

64

Table 30: Annual mass releases (expressed in kg / annum) and emission factors (expressed in mg
/ tonne liquid steel) of trace metals from the main effluent discharge point (long sea outfall) at
Tata Steel Plant B during 2010 - 2013.

Dissolved

Total

Trace metals
Cd
Pb
Ni
Cr
Fe
Cu
Zn
As
Cd
Pb
Ni
Hg
Cr
Fe
Cu
Zn
As

Mean emission
factors
(mg/tonne LS)

Annual mass releases

(kg/annum)
2013

2012

2011

2010

Between 2010 and 2013

PHS

19.9

22.3

21.2

25.1

6.2

PS

1094

1232

1097

1196

323

PS

174

172

223

157

50.5

SP

201

190

423

145

66.4

SP

141499

157275

195591

125702

43438

SP

334

359

326

221

87.7

SP

38520

48513

56434

43498

13114

SP

77.9

70

77

49.9

19.2

PHS

0.51

1.2

0.94

2.6

0.35

PS

15.0

17.6

13.8

78.6

8.2

PS

96.2

111

136

79.7

29.8

1.5

1.6

1.5

1.3

0.43

8.5

76.1

60.9

7.7

11.9

SP

1616

1126

3967

651

505

SP

129

132

89.9

87.3

31.1

SP

6039

8859

14894

19094

3319

SP

38.3

30.9

27.2

19.2

8.1

PHS
SP

Analysis of the data presented in Table 30 showed that:

2.4.4

The most significant total metal releases from the effluent discharge at Tata Steel Plant B
were Fe (within the range of 126 -196 tonnes / annum) and Zn (ranged from 38 tonnes /
annum to 56 tonnes / annum).
Amongst the metals identified as PHS and PS in the WFD, Pb exhibited the most significant
total annual mass release (ranged from 1094kg to 1232 kg / annum), followed by Ni
(within the range of 157 to 223 kg / annum) and Cd (from 19.9 kg/annum to 25.1
kg/annum).
Hg emissions were very low for the integrated steelworks (ca. < 1.6 kg / annum).
There was no significant difference for total Cd and Pb releases between 2010 and 2013,
however the dissolved Cd and Pb releases were much higher in 2010 compared with the
releases between 2011 and 2013.
Both total and dissolved annual mass releases for Ni in 2011 were approximately 30%
higher than the annual mass releases obtained in 2010, 2012 and 2013. However, it was
difficult to find an explanation for this result at this stage of the study.
Overall, amongst the metals identified as SPs in the WFD, there was no significant
difference for the annual releases of Fe, Cu, Zn and As between 2010 and 2013.

Task 2.4: Identification of priority areas for improving the treatment

and detoxification of wastewater from coke making operations

Analysis of the mass inventory data and examination of the results obtained for the distribution of
pollutants between suspended solids / dissolved fraction led to identify several priority areas for
reducing emissions of the WFD substances from integrated steelworks.
For PAHs, the main areas of concerns were as follows:

PAH emissions are predominantly originating from coke oven operation (>90% from BET
plants).

65

The efficiency of BET plants for degrading low-molecular weight PAHs (ie. naphthalene) was
very high (>99%), however it was significantly lower (ca. 60 to 80%) for middle-molecular
weight (ie. fluoranthene) and high molecular weight PAHs (ie. B[a]P).
PAHs are mostly particle-bound in coke oven effluents, therefore it is likely that only
improved filtration techniques which would reduce suspended solid emissions would also
result in lower PAH emissions.
It was found that the stability of the BET process was critical to control PAH emissions.
When the treatment of key species such as thiocyanate or phenols is severely inhibited,
this resulted in a significant increase in the suspended solids emissions, and therefore in
PAH emissions. As an example, it was observed that PAH emissions from Tata Steel BET
plant B were ca. 10-fold higher than at BET plant A because of instability in thiocyanate
treatment at BET plant B. This constitutes a key area for improvement in the future.

For trace metals, the main areas of concerns were as follows:

The three most significant trace metals emitted in terms of volume from an integrated
steelworks were Fe, Zn and Pb. Ironmaking (BF) and steelmaking (BOS) operations were
the main source of Fe (ca. 70%) and Pb (ca. 90%). Fe, Pb and Zn were almost exclusively
particle-bound. Zn was originating from bothironmaking / steelmaking (ca. 60%) and
cokemaking (ca. 40%) activities.
With regard to Cd and Ni, Ni was emitted in greater quantities than Cd. The main sources
of nickel were ironmaking / steelmaking (ca. 60%), cokemaking (ca. 10%) and the rolling
mills (ca. 10%). In all effluents, Ni was present in the dissolved phase (ca. 60 to 90%) and
therefore it is thought that only adsoption abatement techniques could be used to reduce
Ni emissions. Cadmium sources were mostly from ironmaking / steelmaking (ca. 74%) and
continuous casting (ca. 15%). In both of these these streams, Cd was mostly found in the
dissolved phase.
Arsenic was emitted from two major sources: ironmaking / steelmaking (ca. 50%) and
cokemaking BET plants (ca. 30%). Arsenic was found exclusively in the dissolved phase in
both type of effluents (ca. >90%). Accordingly, treatment options for As would also consist
in adsorption rather than filtration.

2.5

Work Package 3: Impact studies

2.5.1

Task 3.1: Impact studies of coke oven effluents on the chemical

quality of local river basins

2.5.1.1

Impact studies carried out at Tata Steel Plant A

In 2011, Tata Steel undertook a major sampling campaign to characterise the quality of rivers and
water streams in the vicinity of Tata Steel Integrated Works A and to determine the impact of coke
oven and steel making effluents on the water quality. Sampling took place at five locations on the
steelworks and two locations on a local river where effluents get discharged (Fig. 44). Within the
boundary of the Works, there are two main water streams (Beck 1 and Beck 2) where most of the
steelmaking and rolling mills operation waste water discharges occur. In total, 7 sampling locations
were selected. Five of these locations (1 to 5) were situated within the Works boundary on Beck 1,
Beck 2 and at an old quarry pond (See Fig. 44A). The remaining two locations (6 and 7) were
situated on a major local river upstream and downstream from the BET discharge point (See Fig.
44B).
Beck 1 enters the site from the north, near Location 4, whereas Beck 2 enters the site near
Location 5. Therefore, locations 4 and 5 are situated upstream from any Tata Steel wastewater
discharges (background sites). On the other hand, location 2 is situated at the exit of the Works
after the junction between Beck 1 and 2, and after all steelmaking and rolling mills operation
wastewater discharges. This final effluent run through towards the main river situated about 10
miles away before being discharged upstream from location 7. Effluents from the coke plants are
not discharged in Beck 1 and 2, but they are treated in the BET plant situated 6 miles from the
Steelworks before being discharged downstream from location 7 (upstream from location 6).
Details of the sampling and analytical procedures have been summarised in Appendix 4.

66

67

(A)

North
(B)

River
flow

Beck

Fig. 44: Map of passive sampling locations at Tata Steel Plant A showing the sampling locations on (A) the steelworks, and (B) the river.

Trace metal concentrations

In this section, the results obtained for DGTs and the spot samples are reported. As EQS for trace
metals in the WFD are defined for the dissolved phase, only the dissolved phase data obtained for
the spot samples (dissolved) and the results obtained with the DGTs are compared in Fig. 46.
As may be seen from the error bars on Fig. 46 (representing one standard deviation across the four
sampling periods), some elements appeared to present little variation between the four sampling
periods [ie. nickel, cadmium (except at location 1), zinc, arsenic, chromium and copper]. On the
other hand, significant variations were observed for lead and iron.
As expected, concentrations of most trace metals were higher on the steelworks (locations 1 to 5)
than on the river (location 6 and 7). This was particularly evident at locations 1 and 2, which are
situated at the exit of the works after all steelmaking discharges. For instance, for nickel, lead,
cadmium, iron, zinc, chromium and copper, the concentrations found at locations 1 and 2 (both
spot samples and DGTs) were higher than at locations 6 and 7. However, for arsenic, much higher
concentrations were observed on the river than on the Works, showing that the steelworks was not
a predominant source of arsenic in the environment. For all elements, the concentrations observed
at location 6 (downstream of the BET plant discharge) and location 7 (upstream of the BET plant
discharge) were almost identical suggesting that the BET plant effluents had no significant impact
on the concentrations of these pollutants in the river.
During the sampling campaign, it appeared that the concentrations of PHS and PS determined
using the DGTs were below the EQS (ie. for cadmium, lead and nickel) even at the steelworks
location situated downstream from most of the steelmaking discharges (ie. locations 1 and 2).
Overall, the DGT results were typically slightly lower than the results obtained using the spot
sampling techniques and the analysis of the dissolved fraction. This was expected since only weakly
bound metal complexes in solution can be sampled using DGTs. Larger complexes, which can be
predominant when humic levels are high, would not be sampled by DGTs but would be detected in
spot sampling. In terms of EQS, whilst DGT samples did not show any exceedances, cadmium in
some spot samples were found to largely exceed the EQS at locations 1 and 2 and also slightly
exceeded at Location 6 and 7 in the river.

68

Fig. 46: Average concentrations of PS, PHS and specific pollutants in water bodies around Tata
Steelworks A with respect to the Environmental Quality Standard (EQS) between 10th August - 2nd
November 2011. The error bars represent the standard deviation between the four consecutive
sampling periods.
Cd DGT
Ni DGT

Ni dissolved

Cd dissolved

EQS Cd

EQS Ni

2.0

20

1.8
14

1.6

12

1.4
1.2
ug/L

ug/L

10
8

1.0
0.8

0.6

0.4

0.2
0.0

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Fe DGT
Pb DGT

Pb dis solved

Fe dissolved

EQS Pb

80

70

60

6
50
ug/L

ug/L

5
4

40

30

20

10

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

As DGT

Zn dissolved

60

12

50

10

40

8
ug/L

ug/L

Zn DGT

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

30

As dissolved

20

10

2
0

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Cr DGT

Cr dissolved

Cu DGT

3.5

Cu dissolved

12

3.0

10

2.5
ug/L

ug/L

2.0
1.5

6
4

1.0

0.5
0.0

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

PAH concentrations
In this section, the results obtained for both the SPMDs and spot samples are reported. Since EQS
for PAHs are defined for the total phase (dissolved + particulate), both the dissolved and the total
concentrations are reported with regard to the spot samples. The results are summarised in Fig.
47.
As may be seen from the error bars on Fig. 47 (representing one standard deviation across the four
sampling periods), the variability in PAH concentrations across the four sampling periods was
relatively small for most PAH compounds. This applied to results obtained by SPMDs or by

69

analysing the dissolved fraction of the spot samples. However, important variability in the data was
observed when looking at the total PAH results obtained from the spot samples (dissolved +
particulate). This was particularly observed at locations 5, 6, and 7 where relatively high amounts
of suspended solids were collected.
In most cases apart from naphthalene, SPMD results were in excellent agreement with the results
obtained from the analysis of the dissolved fraction (spot sample). In general, it appeared that
lower limits of detection could be reached using the SPMD sampling method for heavy molecular
weight PAHs such as benzo[a]pyrene. SPMD results were systematically much lower than the total
spot samples (dissolved + particulates). This was particularly apparent with HMW PAHs where most
PAHs are present in the particulate phase.
In terms of EQS (reported for total phase PAHs), SPMD results cannot be directly used as they only
represent the bio-available fraction, which is close to the dissolved fraction. Therefore, only spot
sample results are discussed. Naphthalene and anthracene concentrations were much lower than
the EQS at all locations. The EQSs for fluoranthene, benzo[a]pyrene and benzo [b] +
benzo[k]fluoranthene were exceeded at locations 5 (Works), 6 and 7 (river). The EQS for indeno
[1,2,3-c,d]pyrene + benzo[g,h,i]perylene was largely exceeded at all locations. However, the
likelihood of being able to reach concentrations below EQS in water bodies for these compounds
appears difficult considering that the EQS defined in the WFD are extremely low, and below typical
limits of detection for the analysis of PAH compounds using modern analytical techniques. For
instance, the LoD defined for indeno[1,2,3-c,d]pyrene + benzo[g,h,i]perylene using the analytical
procedure developed by Tata Steel is 16 ng / L (See Table 10), but the EQS defined in the WFD is
2 ng / L.
Overall, PAH concentrations measured at locations 6 (downstream of the BET plant discharge) and
7 (upstream of the BET plant discharge) were very similar. This suggested that the coke oven BET
effluents did not have a significant impact on the PAH concentrations in the river.

70

71

Concentration ug/L

Concentration ug/L

0.00

0.05

0.10

0.15

0.20

0.25

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Location 1 Location 2 Location 3 Location 4 Location 5 Location 6 Location 7

EQS 0.05ug/l

Spot sample_Dissolved
Spot sample_Total
SPMD_NLS

B[a]pyr

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Spot sample_Dissolved
Spot sample_Total
SPMD_ NLS

EQS 2.4 ug/l

C o n cen tratio n u g /L

Naph

Concentration ug/L

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

EQS 0.1 ug/l

Location 1 Location 2 Location 3 Location 4 Location 5 Location 6 Location 7

EQS 0.03ug/l

Spot sample_Dissolved
Spot sample_Total
SPMD_NLS

B [b] Flant + B [k] Flant

Location Location Location Location Location Location Location

1
2
3
4
5
6
7

Spot sample_Dissolved
Spot sample_Total
SPMD_NLS

Ant

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Location 1 Location 2 Location 3 Location 4 Location 5 Location 6 Location 7

EQS 0.002ug/l

Spot sample_Dissolved
Spot sample_Total
SPMD_NLS

IDP + B [g,h,i] Per

Location 1 Location 2 Location 3 Location 4 Location 5 Location 6 Location 7

EQS 0.1 ug/l

Spot sample_Dissolved
Spot sample_Total
SPMD_NLS

Flant

Fig. 47: Mean concentrations of PAHs in water bodies around Tata Steelworks A, with respect to the Environmental Quality Standard (EQS), between the
10th August and the 2nd November 2011. The error bars represent the standard deviation between the four consecutive sampling periods.

Concentration ug/L

C o n cen tra tio n u g /L

2.5.1.2

Impact studies carried out near a coke oven BET plant in Italy

Over the period 2012 - 2013, CSM carried out sampling campaigns to characterise local sea and
sediment samples situated 2.5 to 3.0 km away from the effluent discharge point of a major coke
oven plant in Italy (Fig. 48).
Fig. 48: Wastewater discharge points (green) and sampling points (yellow)
near a coke oven plant in Italy.

The samples were analysed for PAHs, phenols and heavy metals. The mean data are reported in
Table 31 and Figs. 49-50. Analysis of the data showed that light molecular weight PAHs were found
almost exclusively in the water whereas high molecular weight PAHs which exhibit low water
solubilities were detected predominantly in the sediment samples (Fig . 49). Although very little
differences were observed with regard to the trace metal concentrations of samples between each
sampling campaign, it was found that phenol and PAH concentrations were significantly lower in
April 2013 compared to August 2012.
Table 31: Seawater and sediment characterization at sampling position
Parameter

Seawater
Aug- 12
g/l

Sediment
Aug- 12
mg/kg

Sediment
Apr-13
mg/kg

Sediment
Jul-13
mg/kg

Ni

25

23.2

21

Cr

25

22.6

24

Fe

34

10300

11800

9870

Cd

<0.05

<0.1

0.105

0.1

Hg

<0.5

<0.5

0,603

0.54

Zn

17

118

49.5

57

As

15

7,3

5.1

9.6

22

25,3

19.7

mg/l
Phenols

7.05

0.45

0.31

COD

2469

72

Fig. 49: PAHs in seawater

Fig. 50: PAHs in sediments

Although very little differences were observed with regard to the trace metal concentrations of
samples between each sampling campaign (Table 32), it was found that phenol and PAH
concentrations were significantly lower in April 2013 compared to August 2012. This was also
evident from a significant change in the overall aspect of the sediments collected (Fig. 51).
Fig. 51: Picture of the sediment samples collected in August 2012 (left) and April 2013 (right).

Those changes were attributed to the fact that significant improvements were carried out in 2012 at
the coke oven BET plant which resulted in reduced PAH and phenol emissions from the plant. In
particular, the filtering system at the exit of coke oven was upgraded, a new buffer tank was build to
be used in case of emergency and finally maintenance of activated carbon filters was optimised.
PAHs in aquatic environments tend to become associated to the particulate matter as a consequence
of their hydrophobic nature. Therefore, sediments represent the most important reservoir of PAHs in
the marine environment. PAHs accumulation in coastal sediments is both due to anthropogenic and
natural emissions. Among anthropogenic factors, petrogenic and pyrolytic sources are the most
important: whereas pyrolytic sources include combustion processes, the petrogenic input is closely
related to petroleum products. Furthermore, PAHs from pyrolytic and petrogenic sources exhibit
different chemical behaviours and distribution in marine sediments. In particular, PAHs from

73

pyrolytic processes are more strongly associated to sediments and are much more resistant to
microbial degradation than PAHs of petrogenic origin since they are characterized by the dominance
of high molecular mass (4-6 rings) PAHs, over those with low (2-3 rings) molecular mass [35]. In
Fig. 52, PAHs were grouped into different classes depending on the number of aromatic rings
present in their structure. As may be seen from Fig. 52, the PAHs with 4, 5 and 6-rings found in
sediments constituted more than 90% of the total suggesting their predominantly pyrolytic nature.
Fig. 52: Group profile of PAHs in the sediments. 1, 2 and 3 represent respectively the data obtained
in August 2012, April 2013 and July 2013.

A further source analysis was carried out by analysing the phenantrene/anthracene and
fluoranthene/pyrene ratio of the sediment samples. This analysis was carried out only for the
samples taken in August 2012. Phenanthrene and anthracene are structural isomers: phenanthrene
is more thermodynamically stable than anthracene. Therefore in petrogenic PAH pollution, the
Phe/Ant ratio is very high. High temperatures during the combustion process help the formation of
anthracene which reduces the Phe/Ant ratio. Because of the differences in reactivity and solubility of
these two pairs of isomers, their respective ratios are not expected to remain constant and cannot
provide a complete picture of PAHs origin through environmental transport, to deposition in marine
sediments [35]. Fluoranthene and pyrene have the greatest stability range and are therefore good
as indicators of thermodynamic vs. kinetic (e.g. petroleum vs. combustion) effects. Generally,
Flu/Pyr ratios above 1 indicate a pyrogenic origin, whereas values below 1 are typical of petroleum
hydrocarbons. In particular, the high Flu/Pyr (>1) and low Phe/Ant ratios (<10) are characteristic of
PAHs of pyrolytic origin, while low Flu/Pyr (<1) and high Phe/Ant ratios (>10) indicate PAHs to
possibly derive from petroleum. In Fig. 53, a cross-plot of Phen/Ant versus Flu/Pyr ratios for
sediments sampled in August 2012 depicts a clear pyrolytic source of the PAHs.
Fig. 53: Cross plot of the Phenanthrene/Anthracene versus Fluranthene/Pyrene ratios for source
characterisation of PAHs in marine sediments.

2.5.2

Task 3.2: Impact studies of coke oven effluents on the biological and
ecological quality of local river basins

In this activity, the biosensors developed in Work Package 1 were tested on a series of real samples
to determine their response and applicability to coke oven / steelmaking effluents as well as river
water samples and soils contaminated with hydrocarbons.

74

2.5.2.1

Genotoxicity tests using the PAH biosensor ADPWH_recA_Sal450

After the development of the toxicity biosensor ADPWH_recA_SAL450 which can detect the toxicity
linked to PAHs, tests were carried out to study its response when exposed to discharge effluents
from the coke oven plant and steelmaking plants. In addition, river samples collected near the final
discharge point of Tata Steel Plant A were also tested for genotoxicity. The results are depicted in
Fig. 54 where the bioluminescence (lum) corrected by the cell density (OD) was plotted againt time.
Lum/OD represents a measure of the toxicity.
Fig. 54: Results from genotoxicity tests carried out using the biosensor ADPWH_recA_SAL450 on
discharge effluent samples from cokemaking steelmaking operations and river samples near the final
discharge point of Tata Steel Plant A.

Coke oven wastewater

River sample water
at steelworks discharge

Steel plant discharge sample

Control

1#-water sample from steel plant 2#-river discharge from steel plant 3#-water sample from coke plant

Analysis of the results showed a positive response of the biosensor for the three types of sample
indicating that the three samples exhibited genotoxicity. In particular, the coke oven effluent
samples was characterised with the highest Lum/OD value with approximately a 50% increase
compared to the control sample. This showed that the coke oven effluent sample exhibited the
highest genotoxicity amongst all the samples tested, and confrmed the potential of the biosensor
developed in the project for detection of PAHs in wastewater.

2.5.2.2

Tests on environmental samples

Acinetobacter baylyi ADPWH_alk

using

the

n-alkane

biosensor

A series of tests were carried out using the Acinetobacter baylyi ADPWH_alk biosensor to test its
response when exposed to different types of crude oil products including mineral, Brent, Chestnut
and Sirri crude oils. For these tests the concentration range of the crude oil was 0.1 100 mg/l. In
addition, the experiments were carried out using both pure water and sea water. The results of the
experiments are depicted in Fig. 55.

75

Fig. 55: Acinetobacter baylyi ADPWH_alk response to different concentrations of mineral oil and
Brent, Chestnut and Sirri crude oils in pure water and seawater.

The ADPWH_alk biosensor response time for oil quantitative estimation was 200240 min. The three
crudes tested were typical oils, and Brent crude was a standard crude oil used by world oil trade.
Although the mineral oil and the crude oils both exhibited a mixture of alkanes and alkenes with
different compositions, the ADPWH_alk induction patterns to the different oils were similar (Fig. 55),
which suggested that a single general calibration curve was sufficient to estimate the oil
concentration in water samples independently from the type of oil from which the contamination
originated. The ADPWH_alk induction in seawater and in pure water was similar, indicating that
seawater had little impact on the bioreporters performance and that ADPWH_alk could be applied to
detect oil spill in seawater.
Additional tests were carried out using contaminated soils with increasing concentraions to calibrate
the response of the biosensor. A curve showing the ADPWH_alk induction ratio against the crude oil
contents in soils is shown in Fig. 56.
Fig. 56: Calibration curve for ADPWH_alkresponse to crude oil in soils.

The results showed that the induction ratio increased with higher concentrations of crude oil in soils.
The calibration curve obtained can be used to measure the contamination of soils by oil using the
ADPWH_alk biosensor for future applications.

76

In conclusion, bioreporters employ living cells (usually genetically engineered bacteria) as detection
elements and they can be a complementary tool to chemical analysis. Bioreporters present several
advantages: rapid, simple, cheap, in situ and importantly providing information of bioavailability.
Bioavailability is one of main concerns of environmental risk management since these two factors
directly link environmental contamination to human health risk. Most environmental samples are
mixtures of complex contaminants. The additive, antagonistic and synergistic effects caused by
complex physical or chemical interactions would make the risk assessment unpredictable if only
chemical analysis (e.g. GC-MS or HPLC) was applied to the estimation of bioavailability of
contaminated samples. Chemical analysis of contaminated soil and water samples usually requires
sample pre-treatment and extraction, which makes inert or active portions of contaminants
indistinguishable, while risk assessment is concerned to the active portion (bioavailability). In
contrast, bioreporters can detect the active or bioavailable fraction of contaminants, and it directly
links contamination to biological effects and make bioavailability assessment more relevant to
human health risk.

2.3.5.3

Task 3.3: Assess the impact of cokemaking effluents with respect to

transitional areas of exceedances

2.5.3.1

Definition of mixing zones

Article 4 of the Environmental Quality Standards Directive (2008/105/EC) introduces the concept of
mixing zones [2]. By definition, a mixing zone is an area of surface water adjacent to the point of
discharge within which the Competent Authority is prepared to accept EQS exceedance for one or
more substances, provided that it does not affect the compliance of the rest of the water body.
Compliance with environmental quality standards is an essential part of the Water Framework
Directive Strategy. Effluent discharge control regimes are normally designed to ensure that
concentrations of polluting substances in the receiving water do not exceed the EQS. However if the
concentration of a contaminant of concern in the effluent is greater than the EQS value at the point
of discharge there will be a zone of EQS exceedance in the vicinity of the point of discharge. The
Directive 2008/105/EC allows Member States to permit such zones of exceedance in water bodies
when a number of criteria are met. Understanding these criteria is important as it enables the
competent authority first to identify whether this level of exceedance is acceptable for a candidate
mixing zone and then to identify the appropriate location for monitoring points. This guidance has
been designed to assist member states to complete this process using a tiered approach. This
guidance must be used in the determination of mixing zones for substances listed in Annex 1A of
Directive 2008/105/EC.
The purpose of the guidance on mixing zones is to establish where a mixing zone is required and
then to determine its size and acceptability using a tiered approach. The tiered approach may be
summarised as follows:
Tier
Tier
Tier
Tier
Tier

0
1
2
3
4

Is the contaminant of concern present ?

Initial screening
Simple approximation
Detailed assessment
Investigative study / validation of the models
(optional)

Tier 0:
Tier 0 assessment consists of identifying those discharges with the potential to cause EQS
exceedence, (i.e. where [PHS or PS] > EQS)
Tier 1:
Tier 1 assessment consists of an initial screening of the discharges identified in Tier 0 to determine
whether they can be considered trivial. The approach is different for rivers and coastal waters as
described below:

For discharges to river the significance test is based upon the impact of the
discharge after complete mixing. The process contribution (PC) is calculated as
follows:
PC = [PHS or PS] / dilution factor.

For small water bodies (< 100 m3/s), the discharge can be considered insignificant if
PC < 4%EQS.

For discharges to coastal waters the significance test is based on an

approximation of the overall volume of the mixing zone. The test is applicable to

77

buoyant, covered discharges, which after initial dilution form a well defined surface
layer.

The size of the mixing zone depends upon the effective volume flux (EVF) which is
defined as:
EVF = Effluent discharge rate x ([PHS or PS]/EQS).

If EVF < 5 m3/s then the discharge can be considered insignificant.

Tier 2:
The purpose of Tier 2 assessment is to eliminate those discharges that are clearly either acceptable
or unacceptable on the basis of a simple case-specific assessment, using an initial indicative
assessment of the size of the extent of EQS exceedence. Part of the guidance document on mixing
zones, a discharge test is provided that may be used to carry out the Tier 2 assessment. The
discharge test is provided as an MS Excel Workbook, and was originally designed as a tool to assess
discharges made to freshwater bodies, but it has now been revised to address a broader range of
water body types. It provides a mechanism for the simple approximation of the dimensions of the
mixing zone, based on the input of discharge data (flow and total concentration of the pollutant in
the effluent), and water body data (flow, dimensions, bed-roughness and the upstreamconcentration of the pollutant in water body). For these types of studies, a number of commercial
tools are also available such as CORMIX, a US EPA mixing zone model and decision support system
for environmental impact assessment of regulatory mixing zones resulting from continuous point
source discharges.
Tier 3:
In complex cases, a more detailed assessment may be required. Tier 3 provides this, often involving
the use of computer-based modelling techniques, to consider the individual circumstances for the
discharge concerned. In this tier, the approach required may be more sophisticated than that
applied at Tier 2, with detailed consideration of the spatial and temporal variation in extent of EQS
exceedence.
Tier 4:
If after assessment there is still uncertainty it may be appropriate to conduct investigative studies to
validate the outputs, refine the approach taken or to characterise the actual impacts occurring within
extents of EQS exceedence.

2.5.3.2

Impact Assessment on the main effluent discharge points at Tata

Steel Plant A

The tiered approach (Tiers 0-2) was used to assess discharges of Priority Substances and Priority
Hazardous Substances from Tata Steel Plant A for several discharge points including DP1, DP2, DP3,
DP4, DP6, DP9, DP10, DP11 and DP12. Details and nature of each discharge points have been
presented previously in Section 2.4.2.1 (Table 16). The discharge points are impacting three distinct
rivers (A, B and C) situated in the vicinity of the Steelworks. Table 32 summarises the flow rates of
each impacted river and the flow rates of each effluent discharges.
Table 32: Details of the effluent discharges assessed.

Discharge
point

River
impacted

Effluent flow rate

(m3 /s)

River flow rate

(m3 /s)

DP1

0.192

0.049

DP2

0.0003

0.112

DP3

0.011

0.113

DP4

0.001

0.124

DP6

0.007

0.131

DP9

0.012

0.062

DP10

0.025

89.9

DP11

0.021

0.0875

DP12

0.062

0.002

78

For the assessment, all the substances that were of potential relevance to Tata Steel operations
were included. These were the PAHs, trace metals and benzene identified in the WFD. The list of
compounds assessed and their respective Environmental Quality Standards are listed in Tables 33
and 34.
Table 33: List of Priority Hazardous Substances included in the Tiered approach.
Substance
Anthracene
Cadmium and its
compounds
Mercury and its compounds
Benzo(a)pyrene
Benzo(b)fluoranthene +
Benzo(k)flouranthene

AA-EQS
Inland surface
waters (g/l)
0.1
0.08 0.25*
(dissolved)
0.05 (dissolved)
0.05

AA-EQS
Other surface waters
(g/l)
0.1
0.2
(dissolved)
0.05 (dissolved)
0.05

0.03

0.03

0.002

0.002

Benzo(g,h,i)perylene +
Indeno(1,2,3-cd)pyrene

Table 34: List of Priority Substances included in the Tiered approach.

Substance

EQS
Other surface waters
(g/l)
8
0.1
7.2 (dissolved)
1.2
20 (dissolved)

EQS
Inland surface
waters(g/l)
10
0.1
7.2 (dissolved)
2.4
20 (dissolved)

Benzene
Fluoranthene
Lead and its compounds
Naphthalene
Nickel and its compounds

For the assessment, analytical data obtained in 2010 were used in the mixing zone assessment. The
data used were the mean value of a number of samples taken during the two years of monitoring.
These results are summarised in Tables 35 and 36, for trace metals and organic contaminants,
respectively.
Table 35: Concentrations of trace metals used in the Tiered approach.
Dissolved (D) and Total (T) concentrations of trace metals
for each discharge point (g / L)
Discharg
e point

Cd
(D)

Cd
(T)

Pb
(D)

Pb
(T)

Hg
(D)

Ni
(D)

Ni
(T)

DP1

0.16

0.21

5.3

37.9

0.06

7.6

8.0

DP2

0.05

0.05

0.52

0.72

0.1

0.1

0.1

DP3

0.01

0.05

0.79

5.3

0.1

0.32

1.6

DP4

2.5

2.6

0.27

1.3

0.1

7.9

9.2

DP6

0.65

0.99

0.13

74.3

0.06

5.2

16.3

DP9

0.04

0.09

0.3

4.2

0.1

1.5

33

DP10

0.03

0.03

2.3

3.3

0.09

5.7

14.9

DP11

0.01

0.01

0.06

0.19

0.1

0.9

1.8

DP12

0.01

0.02

0.08

3.0

0.07

3.2

3.2

79

Table 36: Concentrations of organic contaminants (PAHs and benzene) used in the Tiered approach.
Discharge
point

Ant

Ben

Flant

Naph

B[a]P

B[b]Flant

B[k]Flant

B[ghi]Per

IDP

DP1

0.003

0.1

0.046

0.073

0.016

0.043

0.011

0.02

0.014

DP2

0.002

0.1

0.012

0.047

0.002

0.008

0.003

0.006

0.005

DP3

0.013

0.1

0.129

0.027

0.083

0.148

0.046

0.062

0.05

DP4

0.002

0.1

0.024

0.045

0.001

0.004

0.002

0.007

0.003

DP6

0.018

0.1

0.246

0.105

0.126

0.249

0.071

0.13

0.094

DP9

0.01

0.1

0.146

0.052

0.071

0.145

0.047

0.052

0.043

DP10

0.18

0.1

7.32

0.15

9.86

15.1

3.93

8.24

9.39

DP11

0.001

0.14

0.01

0.032

0.001

0.002

0.001

0.003

0.002

DP12

0.128

0.1

1.11

0.359

0.5

1.03

0.274

0.312

0.252

Ant: Anthracene; Ben: Benzene; Flant: Fluoranthene; Naph: Naphthalene; B[a]P: Benzo[a]pyrene;
B[b]Flant: Benzo[b]fluoranthene; B[k]Flant: Benzo[k]fluoranthene; B[ghi]Per: Benzo[g,h,i]perylene;
IDP: Indeno[1,2,3-cd]pyrene

The trace metal, PAH and benzene concentrations have been compared with the EQS values. It
should be noted that for the metals (Cd, Pb, Ni and Hg) the assessment was originally undertaken
using the dissolved concentrations, which is consistent with the EQS Directive. However as it was
not clear how these metals partition between the solid and aqueous phase in the receiving water,
the assessment has been applied using total metal concentrations where possible, therefore giving a
worst case scenario.
Results from the Tier 0 and 1 assessment:
The results of the mixing zone Tier 0 and 1 assessment for both Priority Hazardous Substances and
Priority Substances are presented in Tables 37 and 38, respectively. The grey shading indicates that
the discharge of a particular substance was considered as trivial (Tier 0). Substances assessed at
Tier 1 were shaded either red or green. The green shading indicates that the discharge was
insignificant after dilution, whereas the red shading indicated that the discharge may not be
insignificant after dilution and therefore may require a mixing zone.
Table 37: Results from Tier 0 and 1 assessment Priority Hazardous Substances.
Is a mixing zone required?
Discharge
point

Ant

DP1

Cd
(T)

Hg
(D)

YES

YES

DP2

B[a]P

B[b+k]Flant

B[ghi]Per + IDP

YES

YES

NO

DP3

NO

YES

YES

YES

YES

DP4

YES

NO

DP6

YES

YES

YES

YES

YES

YES

YES

YES

YES

NO

YES

YES

YES

DP9
DP10

NO

DP11
DP12

NO

YES
YES

YES

YES

YES

80

YES

YES

Table 38: Results from Tier 0 and 1 assessment Priority Substances.

Is a mixing zone required?
Discharge point

Ben

Flant

DP1

Pb
(T)

Naph

Ni
(T)

YES

DP2
DP3

YES

DP4
DP6

YES

DP9

YES

DP10

NO

YES
YES

DP11
DP12
At Tata Steel Plant A, the results from the Tier 0 and 1 assessment suggested that a mixing zone
may be required for all discharges from operations on the site, with the exception of DP2. In all
cases, the substances giving greatest concern, i.e. highest ratio of concentration/EQS were
benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene.
Results from the Tier 2 assessment:
For Tier 2 assessment, a discharge tests tool was used to estimate the extent of the mixing zone,
based upon the characteristics of the discharge and the receiving water body. The tool has been
used to assess four of the discharges identified in Tier 1 (DP1, DP3, DP6 and DP9).
A site visit was undertaken to obtain various parameters relating to receiving water/discharge
characteristics, i.e. effluent pipe diameter, width of receiving water body, and depth of receiving
water. The Tier 2 assessment was normally completed for the substances where the ratio of
concentration/EQS is greatest. In the case of DP1, DP3, DP6 and DP9 the combined concentration of
benzo(g,h,i)perlyene and indeno(1,2,3-cd)pyrene was used. The input data used for this assessment
are summarised in Table 39.
Table 39: Input parameters for the discharge test carried out in Tier 2 assessment.
Parameter
Receiving water flow (m3/s)
Depth of receiving water (m)
Width of Receiving water (m)
EQS for Benzo(g,h,i)perylene and
Indeno(1,2,3-cd)pyrene (g/l)
Benzo(g,h,i)perylene and
Indeno(1,2,3-cd)pyrene (g/l)
Discharge flow (m3/h)
Pipe diameter (m)

DP1
0.138
0.3
3.9
0.002

DP3
0.113
0.5
4.3
0.002

DP6
0.131
0.3
3.9
0.002

DP9
0.062
0.1
2.4
0.002

0.034

0.112

0.224

0.095

594
2.0

39.2
1.0

23.63
2.0

44.2
0.17

The results of this assessment for DP1, DP3, DP6 and DP9 suggested that the extent of the mixing
zone was unlimited, i.e. the concentration of the relevant pollutants cannot be reduced by mixing
to a concentration below the EQS. The implications of an unlimited mixing zone are not yet fully
understood. It is recommened that further work should be carried out in the future to measure
actual concentrations in river B for a number of PAHs, including benzo(g,h,i)perylene and
indeno(1,2,3-cd)pyrene.

2.6

Work Package 4: Enhance PAH degradation in coke making BET plants

using state-of-the-art molecular biology techniques

2.6.1

Background on the molecular biology techniques used in the

ECOWATER project

The development of advanced molecular biology techniques has revolutionised the field of
environmental microbiology over the past few decades, allowing the study of microorganisms in a

81

culture independent manner [36]. This new field of molecular microbial ecology has opened up new
possibilities for studying microbial communities at the genetic level leading to a far greater
appreciation of their diversity in nature. Key technical developments in the field such as 16s rRNA
sequencing, denaturing gradient gel electrophoresis (DGGE) and stable isotope probing (SIP) have
become common methods used to study and quantify the microbial diversity [37]. More recently,
there has been a lot of interest on the use of magnetic nanoparticles (MNPs Magnetic
Nanoparticles) in molecular biology [38]. MNPs are used to functionalise living cells enabling to
remotely control and selectively manipulate cells in vivo. As a result, MNP provides a powerful tool
to investigate cell-chemical interactions [18]. In this section, an introduction to the key molecular
biology methods that will be used / developed in the ECOWATER project to study the microbial
communities involved in the degradation of organic contaminants in coke oven biosludges is
presented.
16s rRNA analysis / denaturing gradient gel electrophoresis (DGGE)
Ribosomes consist of protein and RNA. Because of its highly important role in protein synthesis
(translation), the structures of ribosomes are highly conserved across species. These proteins and
RNAs are encoded in the genome of the bacteria (i.e. in the DNA as genes). Ribosomal proteins are
produced via transcription of the gene into mRNA then translated into the protein (See Fig. 57).
rRNAs are also encoded in the genome but after transcription, there is no translation. In particular,
the 16S ribosomal RNA (or 16S rRNA), which is a component of the 30S small subunit of prokaryotic
ribosomes (Fig. 57), is often used for phylogenetic studies as it is highly conserved between
different species of bacteria and archaea. In addition to highly conserved primer binding sites, 16S
rRNA gene sequences contain hypervariable regions that can provide species-specific signature
sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become
prevalent in environmental microbiology as a rapid and accurate method of bacterial identification.
Fig. 57: Protein synthesis in ribosomes and role of 16S rRNA.

The analysis of 16S rRNA in environmental samples involves the following steps (See Fig. 58):

extraction of nucleic acids (DNA and RNA);

amplification of 16s rRNA fragments by Polymerase Chain Reaction (PCR);
analysis of PCR 16s rDNA PCR products by DGGE (Denaturing Gradient Gel Electrophoresis);
Sequencing of fragments and development of clone libraries for bacterial identification.

This combination of nucleic acid extraction followed by PCR amplification of 16s rRNA genes,
construction of clone libraries, sequencing and comparative sequence analysis have now become
essential techniques for the phylogenetic analysis of microbial communities [36].

82

Fig. 58: Typical molecular biology workflow to carry out a phylogenetic analysis
of an environmental sample using the 16s rRNA.

Originally developed to detect point mutations in human genetics, DGGE is now applied as a tool in
environmental microbiology to separate double stranded 16S-rDNA PCR products [39, 40]. Briefly, a
polyacrylamide gel containing a formamide/urea denaturing gradient is cast, with the denaturant
concentration increasing from top to bottom. As each double stranded PCR product migrates through
the gel, it is subjected to an increasing level of denaturation and begins to melt. Migration is then
retarded due to the interactions between the exposed charged nucleotides and the gel matrix.
Sequence variation in the 16S rRNA genes, giving rise to differences in % G/C content cause 16SrDNA PCR products from different species to denature or melt at different points in the gel. Thus
migration distances differ, revealing a characteristic fingerprint or molecular profile for the
community. DGGE can also be applied in tandem with other state-of-the-art methods such as stable
isotope probing. Together this combination offers a powerful method to assess diversity and
function.
Stable isotope probing (SIP)
While molecular profiling techniques are a powerful tool for revealing microbial diversity, they are
severely limited in terms of assigning metabolic functions to a particular organism or microbial
community. The development of Stable Isotope Probing (SIP) enables the direct study of metabolic
functions of microorganisms in the environment via incorporation of stable isotope labelled
compounds (e.g. C) into their DNA during microcosm or in situ incubation experiments [41. 42].
In a SIP experiment, a 13C labelled compound used as carbon source is fed to the activated sludge
(ie. 13C naphthalene). As a result, the bacteria involved in the degradation of that specific compound
(ie. naphthalene) will incorporate the 13C labelled into their DNA whereas the bacteria that are not
involved in the degradation of that compound will remain unlabelled. Therefore, after recovering the
13
C labelled DNA, it is possible to selectively identify the bacteria responsible for the degradation of
the target compound. This is a powerful technique to identify the microorganisms, often uncultivable
using standard microbiology techniques, involved in degradation mechanisms in complex activated
sludge systems. The selective recovery of the 13C heavy labelled DNA can be achieved via
ultracentrifugation as shown in Fig. 59, allowing further analysis with profiling techniques such as
DGGE or PCR amplification of fragments for sequencing and identification. Often, RAMAN microspectroscopy is also used to confirm the incorporation of 13C into the DNA of the cells. Indeed,
several studies have shown that some Raman bands in 13C-labelled cells are shifting to lower
wavenumbers upon the incorporation of 13C [43-46]. SIP is not exclusively a DNA based technique,
as other biomarkers including RNA and phospholipid-derived fatty acids can also be analysed
following stable-isotope incorporation. The effectiveness of RNA-SIP is now well established and has
been recently used alongside DGGE and sequencing to study the functional composition of two
microbial communities in an activated sludge system treating coke making effluents [47]. SIP will be
used in the ECOWATER project to identify phenol and PAHs degrading bacteria in coke oven
biosludges.

83

Fig. 59: Stable Isotope Probing (SIP) for the identification of uncultivable microorganisms involved in
the degradation of organic contaminants in activated sludge systems.

Magnetic nanoparticles (MNPs)

Magnetic nanoparticles (MNPs) are a class of nanoparticles which can be manipulated using a
magnetic field. Ferrite (Fe2O3) nanoparticles are the most explored magnetic nanoparticles to date,
although some nanoparticles with cobalt and nickel have also been synthesized. Magnetic
nanoparticles offer some attractive possibilities in molecular biology. First, they have controllable
sizes ranging from a few nanometres up to tens of nanometres, which places them at dimensions
that are smaller than or comparable to those of a cell (10100 m) or a gene (2 nm wide and 10
100 nm long). As a result, MNPs can interact and/or bind to living bacterial cells thereby providing
means of manipulating and/or sorting them. Indeed, owing to their magnetic properties, MNPs obey
the law of Coulomb and they can be manipulated using an external magnetic field. This action at
distance opens up many applications in molecular biology for isolating, analysing, selecting and / or
sorting living bacterial cells.
For instance, recently, the University of Sheffield developed for the first time a biocompatible and
highly efficient method for functionalisation of the bacterial cell wall with MNPs (Huang et al). In this
study, MNPs based on Fe2O3 were considered owing to their good biocompatibility. Positively charged
superparamagnetic MNPs of approximately 18 nm internal diameter were able to bind to negatively
charged bacterial cells leading to functionalised cells. This was demonstrated using the soil
bacterium Acinetobacter Baylyi ADP1. The functionalisation of MNPs to the bacterial cells was
achieved with a very high efficiency of 99.96%. The nanoscale MNPs offered very strong magnetic
properties to bacterial cells meaning that cells could be separated efficiently and more importantly
without jeopardizing the cellular viability and functionality. To illustrate this, Fig. 60 shows the
picture obtained by Transmission Electron Microscopy (TEM) of the soil bacterium Acinetobacter
Baylyi ADP1 coated with MNPs, and how the bacterial cells could be spatially controlled by an
external magnetic field. In the ECOWATER project, these MNPs will be used to try and isolate, in a
first application of this kind, phenol- and PAH-degrading bacteria from coke oven biosludges.

84

Fig. 60: TEM pictures showing the functionalisation of a living bacterium (ie. Acinetobacter Baylyi
ADP1) using 18 nm Fe2O3 MNPs (A and B, and picture showing how the bacterial cells could be
spatially controlled by an external magnetic field as a result of the magnetic funtionalisation (C).

2.6.2

Outline of the microbiological

ECOWATER project

analytical

methods

used

in

the

All the microbiological procedures used in the project for the characterisation of coke oven
biosludges have been summarised in Appendix 5 where the technical details about analytical
methods such as PCR, DGGE, SIP and MNPs can be found.

2.6.3

Task 4.1: Studies of cultivable PAH-degrading bacterial strains

present in wastewater and biosludges from coke oven BET plants

The incubation of 100l of biosludge from each of the three plants (Tata Steel Plants A, B and C),
spread onto minimal media agar (MMA) plates, showed colony formation for each of the carbon
sources tested including anthracene, naphthalene and phenol. This result confirmed that cultivable
degrading species were present in each of the coke oven biosludge samples tested. Some examples
are provided in the next paragraphs.
Isolation of phenol degraders
Phenol degraders showed the most rapid and substantial bacterial growth with samples from all sites
forming confluent bacterial lawns across the plates. Where possible, single colonies were picked and
re-streaked on to fresh MMA plates in order to purify them. As may be seen from Fig. 61, control
plates contained no phenol. Plates 1, 2 and 3 were inoculated with phenol and showed substantial
bacterial growth.
Fig. 61: Culture of 100L of the biosludge from Tata Steel BET plant C on minimal media agar
supplemented with phenol as a carbon source. Control plate contained no phenol.

Control

In order to purify a phenol-degrading colony, a single colony was picked and re-streaked onto a
fresh agar plate supplemented with phenol. Fig. 62 shows the growth of a purified colony after being
re-streaked onto a fresh agar plate. These colonies (obtained for the three plants) have been
sequenced and analysed for identification.

85

Fig. 62: Phenol-degrading bacterial colonies in pure culture after re-streaking a single colony from
the plates in Fig. 61. The control plate contained no phenol.

Control

Isolation of naphthalene degraders

Bacterial growth on MMA plates supplemented with naphthalene was observed for all samples
tested. Naphthalene degrading species were then readily isolated from the biosludge samples. As
growth was observed for all samples, an example re-streak plate is shown in Fig. 63 for isolates
from Tata Steel Plant A.
Fig. 63: Naphthalene-degrading bacterial colonies from Tata Steel Plant A in pure culture on minimal
media agar plates. The control plate contained no naphthalene.

Control

Isolation of anthracene degraders

Initially, no colonies appeared to grow on plates supplemented with anthracene. However after a
longer period of incubation, re-inspection of some of the anthracene plates showed substantial
bacterial growth as depicted in Fig. 64.
Fig. 64: Growth of anthracene-degrading bacteria on MMA plates supplemented with anthracene
from a culture realised using an activated biosludge from Tata Steel Plant A.

86

Identification othe main cultivable phenol and PAH degraders at Tata Steel Plant A
For Tata Steel Plant A, a complete identification of the cultivable microbial species isolated and
involved in the degradation of phenols, naphthalene and anthracene was carried out. From the
isolates, DNA extractions were undertaken and the key degraders were identified through 16S rRNA
PCR and sequencing. The results are summarised in Table 40.
Table 40: Identification of the key cultivable degraders isolated from
Tata Steel coke oven BET plant A.
Carbon source
Phenol

Naphthalene

Anthracene

Species

Number

Pseudomonas

Acinetobacter
Unclassified
Total
Commonas
Unclassified
Total
Pseudomonas
Commonas
Unclassified
Total

3
6
12
10
3
13
2
2
3
7

As may be seen from Table 40, of all the 12 isolated phenol degrading strains, 3 were Pseudomonas
and 3 were Acinetobacter. The main naphthalene degraders were Commonas (10 out of 13 species
identified). Pseudomonas and Commonas were also found to be the key degrader of anthracene,
indicating that the main cultivable bacteria that can utilise PAHs in the coke oven biosludge could be
Pseudomonas and Commonas.
In summary, the cultivation-dependent approaches focused on the potential of the isolates to
degrade phenols and a range of PAH substrates such as anthracene and naphthalene. Degraders for
all compounds were isolated from the various treatment plant sludge samples. A wide variety of
bacteria and fungi are known to degrade PAHs and the rate of degradation is related to the number
of rings in the molecule. Generally, low-molecular weight PAHs such as naphthalene are degraded
more rapidly than high-molecular weight PAHs [55]. Naphthalene and phenol degraders were readily
isolated on MMA for all sites, probably owing to the ease with which these two compounds can be
biodegraded, having just two and one benzene rings in their structures, respectively. This correlates
well with the high level of efficiency with which these two compounds are degraded in the BET plant.
Anthracene, however, containing three aromatic rings proved a much more difficult substrate for the
isolation of degraders on MMA. Colony formation on these plates was only apparent after about
three weeks and colonies were small in comparison to phenol and naphthalene colonies.
Identification of all the isolates was carried out, and species including Pseudomonas and Commonas
were identified. However, the cultivation approach showed its limitations because it was very
difficult to quantify the exact contribution of the cultivable bacteria isolated towards the degradation
of phenols and PAHs. The cultivation approach provided useful information about the cultivable
species that were actually present in the sludge however it was very difficult to obtain quantitative
data from this type of analysis, and therefore it was not possible to ascertain which of the bacteria
isolated were predominantly involved in the degradation of phenols or PAHs. In addition, it is also
possible that uncultivable bacteria could actually be the most predominant species involved in the
degradation of phenols and PAHs, but these specied would not be isolated using standard cultivation
techniques. Indeed, It is thought that typically >90% of the bacteria present in an activated sludge
are actually uncultivable. Therefore, although simple cultivation techniques could provide useful
qualitative data between activated sludges, it was found that there were significant limitations to
this type of approaches.

2.6.4

Task 4.2: Studies of non-cultivable PAH-degrading bacterial strains

present in wastewater and biosludges from coke oven BET plants

2.6.4.1

Qualitative analysis of microbial diversity in coke oven activated

sludges using DGGE analysis of 16s rRNA

During the first phase of the project, work focused on the development of validated microbiology
analytical tools to characterise microbial diversity in activated sludges from coke oven effluent BET
plants. After successful development of the techniques, several biosludges collected at BET plants
operated by Tata Steel and/or external cokemaking companies were characterised.

87

For this work, sludge samples were collected and total DNA extractions were carried out at Sheffield
University within 24-h of sample collection. The 16S rRNA fraction of the total DNA extract was
amplified using PCR and analysed by DGGE. The 16S rRNA fraction was selected for phylogenetic
analysis because:

16s rRNA is highly conserved due to its essential role in protein synthesis;
16S rRNA contains highly variable regions allowing differentiation between species;
16S rRNA are present in high numbers in bacterial cells.

Results from DNA extractions

DNA extractions on several biosludge samples collected at Tata Steel BET plants were carried out
during the project. The extractions were very successful with a high yield of high molecular weight
DNA as well as the presence of 16s and 23s rRNA, that are indicative of the active microbial
community in the sludge. To illustrate the results obtained from a typical biosludge DNA extraction,
Fig. 65 shows the presence of genomic DNA (containing DNA from living and dead cells) and 16s /
23s rRNA fragments originating from active microorganisms in a sample collected at Tata Steel BET
Plant A.
Fig. 65: Results from a DNA extraction of a biosludge from Tata Steel BET plant A. Lane 1: 5 l
hypperladder used to determine the size of DNA fragments; Lanes 5-8: Replicate DNA extractions of
the activated sludge.

5 6

Results from 16S-rDNA PCR

The 16S rRNA fractions were isolated and submitted to amplification by PCR using primers GC338F
and 530R. The amplification step is required to obtain sufficient DNA products for DGGE analysis.
Following the optimised method described in Section 2.6.2, PCR amplification was successful. An
intense band was typically seen at ~220 bp indicative of the presence of double-stranded 16s rRNA
PCR products that could be further analysed by DGGE. As an example, this is illustrated in Fig. 66
where the results from a PCR amplification of 16s rRNA samples from Tata Steel BET plants B and C
are shown.
Fig. 66: Results from 16S-rDNA PCR of total DNA extracted from coke oven activated sludges. Lane
1: 5 l hypperladder used to determine the size of DNA fragments; Lane 2: negative control; Lanes
3-5: Tata Steel BET plant B; Lanes 6-8: Tata Steel BET plant C.

220 bp

88

Results from DGGE analysis

The 16s rDNA double-stranded PCR products were separated for analysis by DGGE following the
optimised procedure described in Section 2.6.2. DGGE provides a profile of the microbial diversity in
the biosludge and the relative abundance of the bacterial species in the biosludge. Several sludge
samples were collected from Tata Steel BET plants A, B and C. In addition, sludge samples were also
collected from BET plant D (formerly Tata Steel) and BET plant E (separate cokemaking company
operating in South Yorkshire, UK). Finally, a sludge sample from a sanitary wastewater treatment
plant (CAS) in operation at Tata Steel Plant C was also analysed for comparison purposes. The
results from the DGGE analysis are presented in Figs. 67 and 68.
Analysis of the profiles obtained revealed clear differences between the microbial community profiles
of all the BET plants investigated as well as differences with the sanitary wastewater treatment
plant. Each sample was analysed in triplicate. Clearly, each BET plant exhibited a distinct and unique
bacterial community fingerprint. From these profiles, it was clear that the coke oven biosludges of
each site were characterised by a great diversity in bacterial species including some predominant
species (darker bands observed in Figs 67 / 68). Interestingly, the analysis of several of these
biosludges was repeated after several months and the same fingerprint was obtained indicating the
stability of the bacterial communities at each site. In the case of the former Tata Steel BET plant D,
two separate and independent activated sludge streams were in operation (ie. West and East), but
the DGGE analysis revealed that both streams had actually identical microbial community
fingerprints (See Fig. 68 / lines 1, 2, 3 and 5, 6, 7).
Fig. 67: DGGE analysis of 16s rDNA products from biosludge samples of Tata Steel BET Plant A
(lanes 1, 2 and 3), Tata Steel BET Plant C (Lanes 5, 6 and 7), CAS plant (lanes 8, 9 and 10) and
Tata Steel BET Plant B (lanes 12, 13 and 14).

89

Fig. 68: DGGE analysis of biosludges from Tata Steel BET plants A and D. Lanes 1-3: Former Tata
Steel BET plant D (West stream); Lane 4: Blank; Lanes 5-7: Former Tata Steel BET plant D (East
stream); Lane 9: Blank; Lanes 10-13: Tata Steel BET plant A.

At that stage of the study, it was thought that further isolation of the fragments separated by DGGE
could be carried out for identification purposes. Accordingly, the most intense bands identified in Fig.
67 were assigned a band ID number and were cut-out (excised) from the DGGE gel. After reamplification using PCR, the PCR products were sent out for sequencing to try and identify the
bacterial species. The aim of this study was to possibly identify within the bacterial community
profile which bands corresponded to which degraders (ie. phenol-, thiocyanate, PAH-degraders,
etc.). This process was obviously extremely time-consuming. Unfortunately, the results obtained
from sequencing were very inconclusive and it was not possible to carry out any identification of the
fragments. This was due to the fact that each band did not correspond entirely to a single
microorganism but possibly to several species. Also, it is thought that cross-contamination with
neighbouring bands did occurr during the excision process therefore preventing clear identification of
species. Much more resolving power would be needed to prevent this issue; however this is not
possible with the current DGGE instrumentation commercially available. This showed the limitations
of DGGE analysis for identification of bacterial species in biosludges.
On the other hand, DGGE analysis proved interesting when the bacterial community fingerprints of
sludge samples collected at a BET plant experiencing a loss of treatment (ie. thiocyanate treatment)
were compared with the fingerprints obtained at the same plant under normal operating conditions.
The results are depicted in Fig. 69 showing the 16s rDNA PCR products fingerprints of the former
Tata Steel BET plant D under normal operating conditions (lanes 4 and 6) and during an incident
with loss of thiocyanate treatment (lanes 3 and 5). During this event, biosludge from an external
cokemaking plant (BET plant E) operating in South Yorkshire were transported by tanker to BET
plant D in order to help with plant recovery. Accordingly, an analysis of the 16s rDNA PCR products
for this plant was also carried out (lanes 1 and 2).

90

Fig. 69: DGGE analysis of biosludges from the former Tata Steel BET plant D. Lane 3: West stream
during loss of thiocyanate treatment; Lane 4: West stream before loss of treatment; Lane 5: East
stream during loss of thiocyanate treatment; Lane 6: East stream after loss of treatment. Lanes 1
and 2: BET plant E (External cokemaking plant operating in South Yorkshire).

Interestingly, the results showed clear differences in the fingerprints of the biosludge at the former
Tata Steel BET plant D before and after the loss of thiocyanate treatment. It appeared that
significant bacterial community changes had occurred. For the samples collected during plant failure,
the number of bands observed on the DGGE profile was reduced suggesting a loss of bacterial
diversity. For instance, the most predominant bands observed when the plant was operating
normally have been circled in red in Fig. 69, and it is clear that the intensity of these bands have
significantly decreased. Other bands which were not predominant and/or present before the incident
also became more important. The decrease in the total number of detectable bands in the DGGE
profiles suggests a significant reduction in total bacterial diversity which would result in limited
metabolic potential and thus loss of degradation efficiency. At this stage of the study, it was unclear
what the main cause for the loss of thiocyanate treatment at the BET plant was. However, it was
observed that instability and sometimes loss of thiocyanate treatment at BET plant D and A was
often linked with a small increase in nitrite concentrations found in the aeration cells signalling that
small amounts of unwanted nitrification was taking place. Indeed, both plants are not set up to carry
out nitrification for ammonia treatment. This phenomenon would require more work to be fully
understood however further studies at BET plant D were halted because of the sale by Tata Steel of
the integrated Works and cokemaking plant to an external company at the time.
In summary, molecular biology methods such as PCR and DGGE are suitable to assess and study the
diversity of activated sludge bacterial communities such as those in coke oven effluent BETPs. The
relatively high biomass gave rise to sufficient quantities of 16s rRNA for qualitative analysis by
DGGE. It is clear from the DGGE profiles that each BET plant exhibited a distinct and unique profile
showing that each plant was characterised by different bacterial communities. It was found,
however, that these profiles did not change significantly overtime, unless a loss of treatment was
taking place. DGGE analysis showed that many different species were present in each treatment
plant including some apparently dominant species (darker bands). This, however, did not shed any
light on the identity of the degrading species separated by DGGE because the experiments carried
out to excise the 16s rRNA from the DGGE gels for amplification by PCR and sequencing for
identification were unsuccessful. More selective and powerful techniques which will be developed in
the following sections are needed to achieve the identification of degrading species in coke oven

91

biosludges. It remains, however, that DGGE analysis can provide useful information about the status
and potential time evolution of the bacterial communities in coke oven effluent treatment plants by
fingerprint analysis of the 16s rRNA fragments.

2.6.4.2

Analysis of microbial diversity in coke oven activated sludges using

Stable Isotope Probing and Magnetic Nano Particles (MNPs)

Stable Isotope Probing is a powerful and well-established molecular biology technique capable of
identifying degraders by incorporation of specific stable isotope (13C) labelled substrates. This
technique is usually applied on simple substrates for which fully 13C labelled compounds are
commercially available (ie. phenol, naphthalene). However, this can be very expensive considering
that the costs of fully 13C labelled compounds are important as it is the case for 4- and 5-ring PAHs
such as pyrene of B[a]P. Another drawback of SIP is that it can not recover live cells of uncultured
bacteria that could be later used to study physiology and catabolic repression of degraders.
Alternatively, a technique for isolation of uncultivable bacteria was recently developed using
biocompatible Magnetic Nanoparticles (MNPs) [54]. The power of this novel technique is that
uncultivable bacteria have effectively been cultured and live cells can actually be isolated for further
analysis which is totally impossible with SIP. In this section, the results obtained when applying both
techniques on coke oven biosludges to isolate and identify phenol- and naphthalene-degrading
organisms will be described.
Results from the MNP experiments carried out to isolate phenol-degrading bacteria in
biosludges from Tata Steel BET plant A
On separate occasions, two good-performing biosludges (G-BS) and one poor-performing biosludge
(P-BS) were taken from a coke oven biological wastewater treatment plant operated by Tata Steel
(BET plant A). P-BS was considered as a poor performing biosludge because at the time of sample
collection, severe unwanted nitrification was taking place with loss of thiocyanate treatment
occurring the day before the sludge sample was collected. The unwanted nitrification taking place
was confirmed by the presence of relatively high nitrite concentrations in the aeration cells (within 5
to 10 mg / L). For G-BS, no nitrification was observed and no nitrite was detected in the aeration
cells at the time of sampling, the treatment was working very well for all important species such as
phenols / thiocyanates.
After synthesis of the MNPs (See Section 2.6.2), the two biosludges were functionalised with MNPs.
The principle of these experiments is summarised in Fig. 70. This approach is a completely new
application for MNPs in microbiology developed specifically in the project to study coke oven
biosludges. The idea is to use MNPs to selectively isolate phenol- and naphthalene-degrading
microorganisms, and to recover live bacterial cells in order to carry out additional studies such as
inhibition tests. The technique developed is called MNP-mediated isolation (MMI).
MMI starts by functionalising all the cells from the biosludge with biocompatible MNPs of 183 nm
[54]. The efficiency of MNP functionalisation was greater than 99.9% [54], which ensured that all
cells were initially coated with MNPs. In a second step, the MNP-functionalised cells were reintroduced into a filter-sterilised wastewater in which 250 mg/L phenol (both 13C- and 12C-phenol)
were added as the sole carbon source. In presence of phenol, the active phenol degraders present in
the sludge will start dividing. As a result, the newly divided cells will not be coated with MNPs. In
fact, after several hours, the only cells without MNPs will be the cells actively involved in the
biodegradation of phenols. At that stage, using a permanent magnet, it is therefore possible to
isolate selectively the active degraders which are freely suspended in the water phase. These cells
are designated as MNP-free cells. On the other hand, the rest of the cells (non-divisive or inactive
cells) were attracted and immobilised by the magnet. This approach enables the separation of live
bacterial cells responsible for phenol degradation in-situ from unactive cells in a complex activated
sludge.

92

Fig.70: Principle of the MNP-Mediated Isolation (MMI) technique developed in ECOWATER to isolate
selectively phenol- and naphthalene degrading micrororganisms for active biosludges.

The phenol degradation curves obtained for the two biosludge samples (G-BS and P-BS) are shown
in Fig. 71. After a 2-hour lag phase, the phenol in the good performing sludge G-BS was completely
degraded within 7 hours. In contrast, no phenol degradation was observed in P-BS during the initial
18 hours and phenol degradation was completed only after 36 hours. Although at the time of
collection of the sample, only issues with thiocyanate treatment were observed at the main BET
plant, it would appear that the unwanted nitrification could also have affected phenol treatment
even if no loss of treatment was observed at that time. For both sludges, the bacteria degraded the
fully 13C-labelled phenol and the native 12C-phenol in a similar manner. The phenol degradation
curves showed that the MNP-functionalised biosludge performed as well as the raw biosludge,
confirming that the MNP functionalisation was biocompatible and had no detectable impact on
phenol biodegradation.

93

Fig. 71: Phenol degradation curves (13C and 12C) of the raw biosludges (G-BS and P-BS) and the
MNP functionalised biosludges.

After phenol degradation was completed, the MNP-free cells from the G-BS sample were separated
and recovered using the magnet to separate unactive cells. DAPI staining using 4',6-diamidino-2phenylindole indicated that the MNP-free cell population was 1.480.49105 cells/mL. When 250
mg/L phenol was added into the MNP-free cell suspension, the results showed that the degradation
performance observed was similar to that of the original biosludge, as depicted in Fig. 72. This
demonstrated that the active cells isolated using MNPs from the original raw biosludge G-BS were
responsible for phenol degradation in-situ.
Fig. 72: Phenol degradation curves obtained with the MNP-free cells isolated from the G-GS
biosludge and the raw G-BS biosluge indicating that the MNP-free cells exhibited the same phenol
degradation characteristics as the raw biosludge.

To confirm that the MNP-free cells isolated from the biosludge (G-BS) were indeed phenol-degrading
microorganisms, the MNP-free cells recovered from G-BS spiked with 13C and 12C-phenol were
examined by Raman micro-spectroscopy (Fig. 73). Single cell Raman spectra of the MNP-free cells
were acquired to examine 13C-incorporation, based upon the knowledge that Raman bands in 13Clabelled cells should shift to lower wavenumbers upon the incorporation of 13C from 13C-phenol [4346]. The Raman spectra from the biosludge sample spiked with 12C-phenol were used as 12C-cell
controls. Microscopic images indicated that most MNP-free cells were rod shaped and of a similar

94

size, and that their Raman spectral patterns were similar. SCRS from the cells incorporated with 13C
showed significant Raman shifts from 1001.8 cm-1 to 965.7 cm-1 (phenylalanine band) and 1668.6
cm-1 to 1626.1 cm-1 (protein band), respectively. Other Raman bands such as 641, 723, 781, 1121,
1317, and 1576 cm-1 also shifted due to the incorporation of 13C into the cells. Analysis of Raman
spectra from 135 randomly chosen single cells in the 13C MNP-free cells indicated that 79% of them
were fully labelled with 13C. These results confirmed that the cells isolated in the MNP-free cell
fraction were 13C-cells, and therefore confirmed that the MNP-free cells were phenol-degraders.

1530.3

767.3

1668.6

1576.4

1451.8

1317.1
1121.8

723.6

781.9

1001.8
1044.9

709.7

621.1
641.4
400

1626.1

1306.5

cell
cell
965.7

12C-MFC

1044.9
1090.8

13C-MFC

1451.8

Fig. 73: Raman microspectroscopic analysis confirming that 78.7% of cells were 13C-labelled in MFCs
from biosludge with 13C-phenol as carbon source. (From Zhang et al 2014)

600

800

1 000

1 200

1 400

1 600

1 800

Raman shift (cm-1)

To identify the bacteria isolated in the MNP-free cells of the G-BS biosludge, DNA sequencing of 16srRNA PCR products using 63f and 1387r primers was carried out. The results are presented in Table
42. The analysis revealed that only one strain was present and that the isolated bacterium using the
MMI approach was Ralstonia spp. (accession number: KJ174596). The pyrosequencing of 13C-DNA
from G-BS sludge also showed that 64% DNA reading matched with Ralstonia spp. (Table 41). All
these evidence indicated that the key phenol degrader in the biosludge collected at Tata Steel BET
Plant A was an uncultivable Ralstonia spp., which was completely different from the species isolated
using standard cultivation techniques (Table 40 ; See Section 2.6.3). This result confirmed that
standard microbiology techniques based on simple cultivation techniques are inadequate for
identifying key degraders in complex industrial biosludges. In this case, although some phenoldegraders were isolated using cultivation techniques, these species were not the most predominant
phenol-degraders in the sludge. Advanced techniques using MMI led to the identification of an
uncultivable bacterium as the most predominant phenol-degrading in the sludge.
Table 41: Matching Pyrosequencing DNA with Ralstonia spp.
Sample

Total reading

Matched reading

Percentage
(%)

12

C-DNA fraction*

6978

2465

35

13

C-DNA fraction*

3452

2208

64

4084

4026

98

MNP-free cells

* Sample from G-BS after 7 hours phenol degradation

95

Results from the SIP experiments carried out to isolate phenol-degrading bacteria in
biosludges from Tata Steel BET plant A
To confirm the results obtained using the MNP approach, SIP experiments were carried out for
comparison purposes. Accordingly, a 16s-rRNA polygenetic pyrosequencing analysis was also applied
to the raw biosludge DNA, in particular both the 12C-DNA and 13C-DNA fractions recovered after SIP.
The results are summarised in Fig. 74.
Fig. 74: 16s-rRNA polygenetic pyrosequencing analysis on the 12-C and 13C fractions recovered
after SIP carried out on the biosludge (G-BS) sample.

The results showed that the microbial structure changed during phenol degradation (Fig. 74). After
the phenol was completely degraded at 7 hours, the microbial structure changed significantly in both
the 12C-DNA and 13C-DNA fractions and Burkholderiales was dominant (65%) in the 13C-DNA fraction
(Fig. 74). Ralstonia spp. is a member of the Burkholderiales order, which contains over 200 species,
hence SIP pyrosequencing results from the 13C-DNA fraction were consistent with the sequencing
data from the MNP-free cells. Initially, for example, at 2.5 hours of incubation, the structure of the
microbial community in the 13C-DNA fraction was similar to the biosludge, which might be due to
cross-smearing of the DNA.
Diversity of functional genes for phenol degradation
A key functional gene for phenol degradation is phenol hydroxylase, which converts phenol into
catechol before entering into a TCA cycle. Phenol hydroxylase can be recovered by PCR using
degenerate primers (See Table 66; Appendix 5). Additional studies were carried out to identify the
key functional genes present in the bacteria isolated by standard cultivation techniques, the
Ralstonia spp. isolated using the MMI technique and the functional genes found in the raw biosludge.
The data are presented in Table 42.

96

Table 42: The various types of phenol hydroxylase genes found in (i) the MNPs-free and 13C
fraction cells isolated using MMI, (iii) the original raw biosludge and (iii) the bacteria isolated though
cultivation techniques.
Sample

phe1

pheL

pheMH

pheH

N.A.

N.A.

N.A.

N.A.

Chryseobacterium sp. XY3

P. pseudoalcaligenes XY4

P. putida XY5

P. plecoglossicida XY6

SIP

MNP-free cells (MFC)

Biosludge (G-BS)
Cultured bacteria
Rhodococcus sp. XY2

Uncultured bacteria

N.A.: not analysed.

Amongst the cultivable bacteria, Rhodococcus sp. XY2, Chryseobacterium sp. XY3 and three
different Pseudomonas spp. XY4, 5, 6 were isolated from the biosludge samples. These cultured
degraders were able to grow on minimal medium agar plates with phenol as a sole carbon source.
The 16S-rRNA sequences of Pseudomonas sp. XY4 and XY5 are identical to that of Pseudomonas
pseudoalcaligenes and Pseudomonas putida KT2440 respectively, while 16S-rRNA gene sequence of
Pseudomonas sp. XY6 is 99.6% similar to that of Pseudomonas plecoglossicida. Chryseobacterium
sp. XY3 has an identical pheL phenol hydroxylase subunit gene to Comamonas sp. J5-66 [56]. The
isolated P. pseudoalcaligenes XY4 and P. plecoglossicida XY6 have two domains of LmPH (phe1 and
pheMH) (accession number KJ174598 and KJ174600), which were both identical to the functional
gene in Pseudomonas sp. [57]. P. putida XY5 has different phe1 and pheMH (accession number
KJ174599) that have not been previously reported.
In the original biosludge, all of the four types of LmPH, phe1, pheMH, pheL and pheH, were found.
Pseudomonas has phe1-, pheMH- and pheH- type LmPH. whereas Burkholderia has only phe1- and
pheL- type. The phe1 and pheL of Ralstonia sp. (accession number KJ174604 and KJ174605) in the
MFC are identical to Cupriavidus metallidurans CH34 (formerly Ralstonia metallidurans) (Janssen et
al 2010), which are, however, different to phe1 and pheL in the five cultivable isolates from the
same biosludge. Besides, the sequence of phe1- and pheL- LmPH, fragments shared 98% similarity
with the LmPH genes in Burkholderia [58]. This confirmed that the phenol degrader in-situ was
Ralstonia spp. which was recovered by the MMI method.
Results from the MNP experiments carried out to isolate PAH-degrading bacteria in
biosludges from Tata Steel BET plant A
In order to isolate PAH-degrading bacteria from the biosludge, a series of experiments were carried
out by functionalising the biosludge with MNP. Naphathalene was chose for these experiments and
was added to the MNP functionalised sludge to isolate specifically naphthalene-degrading bacteria.
The following six cultures were set up and incubated at room temperature with agitation at 150
rpm:
S1) 4ml MNP-biosludge + 40l naphthalene + 4ml D2O (70 atom %)
S2) 4ml MNP-biosludge + 40l naphthalene + 4ml H2O
S3) 4ml MNP-biosludge + 40l DMSO
S4) 4ml biosludge + 40l DMSO + 4ml H2O
S5) 4ml biosludge + 40l naphthalene + 4ml H2O
S6) 4ml filtered sludge + 40l naphthalene + 4ml H2O

97

In cultures 1, 2, 5 and 6, a solution of naphthalene (40 g/l) was spiked into the cultures to reach a
final naphthalene concentration of 200 mg/l, representative of the concentrations found typically in
raw coke oven effluents. Samples were taken after 24h (t1) and 48h (t2), and the MNP-free fraction
was separated to recover naphthalene-degraders. To verify naphthalene degradation, the MNP-free
fraction was subjected to analysis using a biosensor specific to salicylate [17]. Indeed, in the case of
naphthalene degradation, salicylate is produced in significant quantities as metabolite. The results
are depicted in Fig. 75.
Fig. 75: Use of a salicylate biosensor to demonstrate naphthalene-degrading activities in a series of
experiments carried out with MNP to isolate naphthalene degraders and without MNP (control).

Results showed that salicylate was produced in significant quantities only in experiment (1, 2 and 5)
for which naphthalene was fed to the cultures. In particular, this confirmed that the bacteria isolated
in the MNP-free fraction (experiments 1 and 2) were naphthalene degraders. The MNP-free fractions
were examined microscopically and, in both samples 1 and 2, numerous uniform rod-shaped cells
were observed. These cells were not observed in the MNP-free fraction of experiment 3 (negative
control).
A PCR amplification of the 16S rDNA PCR was performed on the MNP-free cells recovered in the
second experiment sample 2. After sequencing, the bacteria isolated were Pseudomonas sp. showing
that the dominant active degrader was Pseudomonas. High similarity was actually found with
Pseudomonas plecoglossicida, which was previously isolated from the biosludge as a cultivable
phenol degrader. Some of the MNP-free fraction was also spread onto agar plates supplemented
with a naphthalene crystal as sole carbon source. 16S PCR carried out on this new culture led to the
identification of a colony as Rhodanobacter sp., showing that although the Pseudomonas was the
main active degrader, there were other bacteria present capable of degrading naphthalene.
Further PCR was performed using primers for Comamonas-type (COM1) and Pseudomonas-type
(PSE1) naphthalene 1,2-dioxygenases according to Moser and Stahl 2001. The results show that the
MNP-free cells were positive for Comamonas type (Fig. 76), but since the 16S PCR identified them
as Pseudomonas earlier, this strongly suggested that natural horizontal gene transfer could have
occurred inbetween. In otherwords, it is possible that the Peudomonas took up plasmids from a
Comamonas to enable them to degrade naphthalene.

98

Fig. 76: PCR products observed for Comamonas-type naphthalene 1,2-dioxygenase in the MNP-free
cells from cultures S1 and S2 taken at time point t2.

2.6.5

Task 4.3: Revealing controlling factors affecting PAH biodegradation

in coke oven waste water

2.6.5.1

Physiological testing of MNP-free cells using

throughput phenotypic assay for carbon sources

the

Biolog

high-

A remarkable advantage of MMI was that it was able to isolate live bacteria for eco-physiological
analysis. These analyses were carried out using Biolog high-throughput microarrays for phenotyping
of the MNP-free fraction. It served two purposes: the characterization of phenotypes and the
identification of key factors affecting the performance of phenol degradation. The Biolog PM1 plate
allows the testing of 95 different carbon sources. Effects of each carbon source on cell growth was
categorized into three groups: carbon sources promoting cell growth (Red), carbon sources not
supporting cell growth (Blue), and carbon sources inhibiting cell activities (Grey). The results
obtained are depicted in Fig. 77. To compare, Fig. 78 identifies the carbon sources tested using the
Biolog. It showed that for the Ralstonia sp. isolated using MMI and responsible for phenol
degradation in Tata Steel biosludge MFC, the carbon sources promoting cell growth were D-alanine,
-D-glucose, Tyramine and L-glutamine, whereas the carbon sources inhibiting cell activities were
D-galactonic acid--lactone, and L-galactonic acid--lactone.

99

Fig. 77: Phenotypic microarray (Biolog PM1) of carbon utilisation of MNP-isolated cells (from Zhang
et al 2014) Highlighting: grey compounds inhibiting bacterial growth; red compounds promoting
bacterial growth; blue compounds not supporting bacterial growth.

Fig. 78: Biolog PM1 MicroPlateTM Carbon Sources

2.6.5.2

Physiological testing of MNP-free cells using the Biolog highthroughput phenotypic assay for nitrogen sources

The effects of several nitrogen sources on phenol degradation was also studied using a Biolog PM3B
nitrogen test. Details of the nitrogen sources tested are shown in Fig. 79. The results obtained with
Ralstonia sp. are depicted in Fig. 80.

100

Fig. 79: Biolog PM3B MicroPlateTM Nitrogen Sources

Fig. 80: Phenotypic microarray (Biolog PM3B) showing the effects of various nitrogen sources on
Ralstonia sp. isolated using MMI.

Examination of the results showed that some nitrogen-compounds promoted phenol degradation by
Ralstonia. These included amino-acids such as L-arginine, L-asparagin and L-glutamine. Analysis of
the results showed also that nitrite (Position A3) did not inhibit phenol degradation by the MNP-freecells. This was in contrast with the findings reported in Section 2.6.4.2 where it was observed that
both phenol / thiocyanate degradation could be considerably affected in the biosludge when residual
nitrite concentrations were present in the aeration cells. On the other hand, results from the Biology
analysis showed that hydroxylamine (Position D4) was strongly inhibiting phenol degradation in the

101

MNP-free cells. Considering that hydroxylamine is an intermediate in the chemical reactions taking
place during nitrification denitrification, this suggested that hydroxylamine could act as a potential
inhibitor of phenol / thiocyanate degradation in the BET process. This hypothesis was further tested
in Task 4.4.

2.6.6

Task 4.4: Enhancement of PAH biodegradation in coke oven BET

plants

In order to study further the potential role of hydroxylamine as an inhibitor of phenol degradation in
coke oven biosludges, a series of tests were carried out to identify whether hydroxylamine could be
the main inhibitor for phenol degradation rather than nitrite. For these tests, ammonium chloride,
sodium nitrite, sodium nitrate and hydroxylamine were added into filter-sterilised wastewater as
nitrogen sources. Phenol was present in all treatments at a final concentration of 250 mg/L. The
final concentrations of NH3 as N, NO2- and NO3- were 100 mg/L, 50 mg/L, and 100 mg/L,
respectively. The final concentrations of NH2OH were 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10 mg/L.
Samples were added into a 96-well microplate: each well contained 160 L of filter-sterilised
wastewater, 20 L of appropriate phenol with nitrogen compounds (or water), and 20 L of cell
suspension (or water). The microplate was incubated at 25C in the microplate reader and OD600
was read every 15 minutes. At the end of incubation, the pH values in the 96-well microplate were
measured using indicator strips. Final phenol concentration was measured according to the method
described above, and the percentage of phenol degradation was calculated by final concentration
relative to three cell-free controls on the plate.
Ammonia and its metabolic intermediate compounds NH2OH, NO2- and NO3- were added into the
filter-sterilised wastewater along with the MNP free cells that were enriched in Ralstonia sp. In the
first 10 hours, cell growth in all treatments was similar to the phenol-free control. After 10 hours,
cell growth in the treatment with NH2OH >5 mg/L was inhibited whilst cells with 100 mg/L NH3 as N,
50 mg/L NO2-, 100 mg/L NO3- and <2 mg/L NH2OH continued to grow. Phenol concentration
remained unchanged after 20 hours incubation when NH2OH was greater than 5 mg/L, whilst phenol
had been completely degraded in all other treatment conditions. This result showed that when
NH2OH concentrations were greater than 5 mg/L, severe inhibition of phenol degradation was
observed for Ralstonia sp.. But, phenol degradation was not inhibited at concentrations of 50 mg / L
nitrite, 100 mg/L nitrate and 100 mg/L ammonia (Fig. 82).
Fig. 81: Growth curve of MNP-free cells in presence of hydroxylamine, ammonia, nitrite and nitrate.

102

Fig. 82: Phenol concentrations after 20-h incubation of Ralstonia sp. with hydroxylamine, nitrite,
ammonia and nitrate showing severe phenol inhibition with hydroxylamine concentrations > 5 mg /
L.

The coke plant wastewater treatment facility has reported on several occasions that a sudden
increase of NO2- in wastewater was often associated with a sudden loss of thiocante treatment and
sometimes phenol treatment. In particular, it was observed that the treatment often failed when
NO2- concentration was greater than a threshold of 15 mg/L. It was therefore concluded that nitrite
could act as an inhibitor of phenol / thiocyanate degradation. However, the BET plant does not
monitor the concentrations of hydroxylamine in the aretaion cells. Ammonia-oxidizing archaea and
ammonia-oxidizing bacteria first oxidise NH3 to NH2OH [59-60], which is then oxidised to nitrite
(NO2-) and finally to nitrate (NO3-). The experimental data clearly indicates that it was NH2OH, but
not NO2-, NO3- or NH3, that inhibited phenol degradation in the coking wastewater. The threshold of
this NH2OH inhibition effect was between 2 and 5 mg/L. It is therefore possible that when high
concentration of NO2- are found in the treatment cells of the BET plant, NH2OH accumulation also
takes place and that this key compound could ultimately lead to the BET plant failure. NH2OH is a
volatile and unstable compound in fact, it was observed that when its concentration decreased
below the threshold, phenol biodegradation resumed. It is thought that the accumulation of
hydroxylamine is likely caused by the inefficient conversion of hydroxylamine and nitrite by
oxidoreductase. Therefore, this work showed that by recovering live yet uncultured degraders using
the MMI technique, it was possible to study in more details the parameters that could affect BET
plant efficiency. In this case, hydroxylamine was identified as possible key controlling factor for
phenol biodegradation although more work would be required to confirm this.

2.7

Work Package 5: Novel abatement solutions Industrial application

and transferability

Four water treatment applications have been investigated at the laboratory and pilot scale in the
project ECOWATER to improve the treatment of coke oven effluents. These are summarised below:

Photocatalysis using anatase (Titanium dioxide: TiO2): Tasks 5.1 and 5.2
Zeolite filtration (Aluminosilicate minerals): Tasks 5.1 and 5.2
Powdered Activated lignite treatment: Task 5.3
Fuzzy filter filtration: Task 5.4

This work package describes the results from these investigations and provides cost-benefit analysis
for the best performing abatement technologies.

2.7.1

Task 5.1: Study into the use of heterogeneous photocatalysis using

Anatase (TiO2) for the biodegradation of coke oven waste water

2.7.1.1

Background on photocatalysis using Anatase

Photocatalytic chemistry of titanium dioxide has been studied over the past 25 years for the removal
of organic and inorganic compounds from contaminated water (and air) and for the partial oxidation

103

of organic compounds [61-68]. Photocatalytic activity (PCA) is the ability of a material to catalyse
oxidation/reduction reactions after activation by irradiation using a light of suitable wavelength. Most
of the semiconducting oxides exhibit this property. Three polymorphs of titanium dioxide occur in
nature: rutile, anatase and brookite. Among these, the anatase form of titanium dioxide exhibits
very high photocatalytic activity. PCA strongly depends on the surface redox potential, the band-gap
and the lifetime of photogenerated electron-hole pairs. Anatase, which has a larger band gap (3.2
eV) than rutile, for example, tends to increase the surface redox potentials and prolong the carrier
lifetime.
The reactions involved in the PCA action of anatase are summarised in Table 43 and depicted in Fig.
83.
When anatase is activated by photons, negative electrons and positive holes are formed:
TiO2+ hv TiO2 (ecb + holevb+)

(1)

The most common reaction is the recombination of electrons and holes on the surface, which
reduces the quantum efficiency of the process.
TiO2 (ecb + holevb+) TiO2 + heat

(2)

However, electrons also react with reducible species such as oxygen molecules, ions or organic
compounds, while positives holes can oxidise water leading to the formation of the hydroxyl radical
(.OH), which is a very powerful oxidising agent for a wide range of organic pollutants.
Table 43: Process involved in the photocatatytic activity of anatase (TiO2)

104

Fig. 83: Schematic of the reactions involved in the photocatatytic activity of anatase (TiO2).

Titanium dioxide is the most frequently used photocatalyst in water purification processes since it is
relatively cheap, non-toxic, insoluble in water and very resistant to most chemicals.
Photodegradation can be a very effective method for the destruction of a range of wastewater
organic pollutants such as aromatic compounds, dyes, pesticides, biorecalcitrant polyphenolic or
humic acids [64, 69]. The efficiency of the PCA degradation process depends strongly upon the
experimental conditions including the quantity of catalyst, pollutant concentrations, pH, light
intensity and wastewater inflow rate. The effect of H2O2 or UV irradiation or use of ozone on the
reaction rate has been ascertained by different studies [66, 70-71].
In practice, titanium dioxide can be suspended in the form of a fine powder in the water, however
this leads to the requirement for an additional separation step after PCA treatment to recover the
titanium dioxide. To avoid this problem several solutions have been proposed which can enable the
photocatalyst to be immobilised. For instance, the deposition of fine TiO2 powder on glass surfaces is
the most common technique. This is typically accomplished by coating a glass surface with an
aqueous suspension of fine particles of TiO2 and drying. The prepared film adheres strongly to the
glass because of the electrostatic charge of TiO2 (the glass surface being negatively charged).
Titanium dioxide also shows very good adhesive properties on other material surfaces such as glass
fibres, plastics or even metals. Metal doping was adopted to modify TiO2 and enhance the
photocatalytic degradation of harmful cyanides in aqueous solution. In this respect, Ni-, Cu-, Co-,
and Ag-doped TiO2 were found to be active photocatalysts for UV light induced degradation of
aqueous cyanides generating nitrate and ammonia as main nitrogen containing products [61, 70].
The combination of heterogeneous photocatalysis with chemical and physical operations appears to
be a promising tool for wastewater treatment applications. However, review of the literature shows
that most of the work undertaken in this field has been carried out using artificial systems to study
the photodegradation of single chemical compounds. In the project ECOWATER, it was proposed to
study the benefits of photocatalysis using anatase for the treatment of a complex mixture of
compounds (i.e. coke oven effluent). The use of sunlight should be the final objective for this
application, however to achieve this goal, bench scale investigations using artificial light were carried
out to gain a better knowledge of the influence of physicochemical and operational parameters on
photoreactivity.

2.7.1.2

Background on zeolites

Zeolites are naturally occurring hydrated microporous, aluminosilicate minerals widely used in the
industry for water purification (Fig. 84). They belong to a class of minerals known as tectosilicates.
The structures of zeolites consist of three-dimensional frameworks of SiO4 and AlO4 tetrahedra. The
aluminum ion is small enough to occupy the position in the centre of the tetrahedron of four oxygen
atoms, and the isomorphous replacement of Si4+ by Al3+ produces a negative charge in the lattice.
The net negative charge is balanced by an exchangeable cation (sodium, potassium or calcium).
These cations are exchangeable with certain cations in solutions such as lead, cadmium, zinc,
ammonium and manganese.

105

Fig. 84: Photograph of zeolites.

Owing to their high cation-exchange ability as well as their molecular sieve properties, zeolites have
been widely used for producing different types of industrial product including absorbers, reaction
activators, ion exchangers and hardness reducers. Recently, zeolites have been used in pollution
control, in particular for effluents polluted with heavy metals [72-78]. Zeolites can constitute a costeffective solution for the removal of heavy metals in contaminated wastewater because of their
relatively low price and the fact that their exchangeable cations are not toxic. Some zeolites have
also shown adsorption properties for selected anions and organic compounds in aqueous solution.
Natural zeolites can be modified using acid treatment, ion exchange or surfactant functionalities to
increase their adsorption capacity for organic compounds and anions. Synthetic zeolites can also act
as molecular sieves, i.e. they can selectively sort molecules based primarily on a size exclusion
process. Interestingly, when zeolites are exhausted, they can be thermally regenerated, with high
efficiency without destroying their morphology and maintaining high efficiencies.

2.7.1.3

Laboratory tests results using photocatalytic treatment using

anatase

A series of laboratory scale tests were performed to study the photo-oxidation and the
mineralisation of recalcitrant organic pollutants using two types of Anatase (iCarlo Erba and aeroxide
Anatase), in combination with other advanced oxidation techniques (ie. hydrogen peroxide). In
parallel, laboratory tests were also carried out to study the benefits of zeolite filtration. Finally,
combined laboratory tests using both zeolite filtration and photo-oxidation using anatase were
carried out.
Selection and chemical characterisation of coke oven test samples
At first, the coke-oven wastewater treatment plant has been examined and historic data have been
analysed. Then, samples of different wastewater were collected and chemically characterised. The
wastewater treatment plant selected for this study had a maximum flow rate of 15 m3/h. The
wastewater samples originated from the coke-oven gas treatment system and coke quenching (Fig.
85). These streams are sent to the ammonia stripping tower before entering the biological treatment
section. Afterwards, the coke oven wastewater flows into a tertiary treatment and finally is released
to the sea.

106

Fig. 85: Schematic of the coke-oven water fluxes and sampling points.

Three samples of coke oven wastewater were collected and chemically analysed (Fig. 86) The data
are shown in Table 44. Significant differences were found between each sample particularly with
regard to the COD, suspended solid concentrations, NH4+, H2S and phenol concentrations. Only EQ2
and ICD3 have been considered for photo-oxidation since ICS1 exhibited very high concentration of
H2S.
Fig. 86: Picture of the three cokery wastewater samples.

Table 44: Characteristic of three samples of coke oven wastewater.

Parameters
COD

ICS1
(mg/l)
5697

EQ2
(mg/l)
2123

ICD3
(mg/l)
7700

DOC

5201

2001

7350

BOD5

299

417

577

pH

9.6

9.4

9.6

Suspended solids

2.4

23.6

12.8

<0.05

89

205

SO42-

7.1

30.4

29.5

Cl-

1.5

1077

2857

NO3-

0.12

0.18

0.34

3-

140

1.53

180

5400

54.6

4780

2237

532

1118

n.r.

n.r.

n.r.

PO4

NH4

Total phenols
PAHs

107

H2S

15800

0.11

1430

Cd

<0.01

<0.01

<0.01

Hg

0.017

0,015

0.015

Pb

<0.01

<0.01

0.04

<0.01

<0.01

<0.01

As

<0.01

0,09

0.06

Fe

3.0

6.77

11.2

Zn

0.21

0.49

0.43

Ni

0.01

0.05

0.09

Cr

0.24

0.19

0.08

The PAH analysis results for the sample EQ2 are reported in Table 45.
Table 45: PAH concentrations in EQ2 coke oven wastewater sample used for both photo-oxidation
and zeolites tests
PAHs
Naphthalene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Indeno(123-cd)pyrene
Dibenzo(ah)anthracene
Benzo(ghi)perylene
Acenaphthylene
Acenaphthene
Fluorene
Phenantrene
Anthracene
Fluoranthene
Pyrene
Benzo(a)anthracene

Value
(g/L)
420
75
67
93
82
n.d.
n.d.
21
178
n.d.
89
270
320
203
176
98

Types of anatase used in the ECOWATER project

In the project, two types of anatase were used. The first type tested was designated as Carlo Erba
Anatase whereas the second type of Anatase was designated as Aeroxide. The characteristics of
both TiO2 products are summarised in Table 46.
Table 46: Main characteristics of Carlo Erba and Aeroxide anatase selected for the ECOWATER
project.
Characteristics

TiO2
Carlo Erba
white
powder
23
100 A
99.0-100
113
<0.5
3.7-4.5

Colour
Aspect
Average granulometry (nm)
Crystalline structure
TiO2 (w%)
BET surface area (m2/g)
Loss ignition (%)
pH (in 4% dispersion)

108

TiO2
Aeroxide
white
powder
21
80 A, 20 R
> 95.0
5015
<2
3.5-4.5

Laboratory experiments carried out with Carlo Erba Anatase

CSM started the investigation of the heterogeneous photocatalysis using anatase (TiO2 - powder
from Carlo Erba) in combination with sunlight, H2O2 and UV. During the trials, parameters such as
COD, phenols and BOD5 were analysed. In addition, considering the relatively high concentrations of
hydrogen sulphide and ammonia found in the samples, nitrates and sulphates, were also monitored
as oxidation products. For these tests, the Anatase powder was suspended in solution under
constant agitation.
Each test was performed using 200 ml of EQ2 wastewater with 0, 0.1 and 1.0 g/l of anatase,
(samples were named respectively EQ0, EQ20, EQ200 see Fig. 87, where the opaque solution
corresponds to the highest anatase addition rate). Wastewater samples were irradiated with sunlight
during the whole duration of the experiments (5 h). However, UV lamps were turned on in a
discontinuous manner. In particular, UV lamps were turned on for the first 30 min of reaction, then
switched off for the following 30 min and finally turned on again for further 30 min. For the
experiments with H2O2, 4 ml of hydrogen peroxide solution at 50% was used.
Fig. 87: Trials with EQ2 sample in presence of TiO2 and sunlight (at start reaction time).

COD and BOD5 trends during the photo-oxidation trials are depicted in Fig. 88. As it can be
observed, the activator effect of sunlight was negligible in comparison to H2O2 or UV effects. The
combination of anatase with H2O2 or UV led to a COD reduction slightly greater than the COD
reduction observed without anatase, and a BOD5 reduction about twice the BOD5 reduction observed
without TiO2.
Fig. 88: COD and BOD5 reduction observed during photo-oxidation trials (EQ2 sample).

The phenols trend during photo-oxidation trials is shown in Fig. 89. As may be seen from Fig. 89,
H2O2 had a significant effect upon phenol degradation. However, there were no significant
differences observed with and without anatase.

109

Fig. 89: Phenols trend during photo-oxidation trials (EQ2 sample).

The increase in nitrate and sulphate concentrations, shown in Fig. 90, was mostly due to NH3 and
H2S oxidation. The effects of hydrogen peroxide were very significant. Therefore, during the
oxidation process, there were two inorganic species (ammonia and hydrogen sulphide) interacting
with the organic compounds oxidation.
Fig. 90: Nitrates and sulphates trend during photo-oxidation trials (EQ2 sample).

The pH value decreased from 9.4 to respectively 8.3, 7.4 and 7.0 in EQ0, EQ20, EQ200 samples.
Besides, the photo-oxidation of aromatic organic compounds that decreases water alkalinity (C6H5O
+ 3,5O2 + 4OH CO2 + 3 HCOO + OOCCH=CHCOO + 3H2O), the variation of the pH can be
explained, on the basis of NH4+ and HS-/S2- ions oxidation mechanism (i.e. photo-oxidation of
sulphide: HS +2O2 + OH SO42+ H2O) [7-8].
The ammonia and hydrogen sulphide oxidation was particularly important in ICD3 cokery
wastewater samples, where both species were found in high concentrations (See Table 6). A series
of experiments was also carried out using ICD3 to verify this effect. The same analytical procedure
used for the EQ2 sample was applied in this case (samples were named ICD0, ICD20, ICD200).
During the reaction time, a significant change in the colour of the solution was observed, from light
yellow to green and finally brown (Fig. 91).
Fig. 91: Evolution of the colour of the test solutions during photo-oxidation trials
(Left after 1 h of reaction; Right after 5 h of reaction)

110

In comparison with EQ2 sample, a lower phenol reduction but a higher COD reduction was observed
for sample ICD3, as shown in Fig. 92.
Fig. 92: Phenols and COD reduction during photo-oxidation trials with ICD3 sample.

However, there was a significant effect on NH4+ and HS-/S2- ions, for which concentrations in
wastewater were respectively reduced by more than 70% and 99% (Table 47). In addition, the pH
value decreased from 9.6 to 8.4-8.5, in each test, as previously observed in the case of EQ2 sample.
Table 47: Ammonia and sulphide concentrations at start and end reaction time.

HS-/S2t= 0
t= 5h
NH4+
t= 0
t= 5h

ICD0 (mg/l)

ICD20 (mg/l)

ICD200 (mg/l)

1430
4.1

1430
4.2

1430
4.0

4780
1025

4780
1275

4780
1425

On the basis of these results, it was decided that only EQ2 wastewater samples would be used for
further tests. The photo-oxidation of organic compounds is affected, in a minor way, by the presence
of other oxidable compounds. Therefore, a new set of tests was defined focusing on the combined
effects between anatase and H2O2. This testing was set up using a DOE approach.
The parameters tested were:

TiO2: 0.1 and 1 g/l

H2O2: 1 and 10%v/v
Treatment time: 2 and 4 h.

As already observed in previous tests, with respect to the untreated wastewater, the results
indicated an increase of both nitrate and sulphate concentration, respectively from 0.1 mg/l to 9.2
mg/l and from 71.4 mg/l to 581 mg/l, and a decrease of both phenols and ammonia values,
respectively from 338 mg/l to 62.8 mg/l and from 43.4 mg/l to 23.2 mg/l. With regard to the COD,
results showed that it was strongly affected by the presence of unreacted hydrogen peroxide in
solution (in fact the COD data obtained were higher than the initial sample). For these reasons, in
order to evaluate the effect of the main parameters on the photo-oxidation process, the phenol
reduction was considered. There was a negligible effect of the treatment time.
Trials were performed, using 1 g/l of TiO2, at different starting pH value: respectively 8.8 and 7.4.
The pH value decrease was more evident at a basic pH than at a neutral pH (Fig. 93). A neutral pH
resulted in a significant increase in sulphate concentrations from about 75 mg/l to 250 mg/l. COD
and phenols reduction was more efficient in alkaline than neutral environment (Fig. 94).

111

Fig. 93: pH trend during the photo-oxidation tests.

Fig. 94: COD and phenols reduction.

Experimental data showed that photocatalysis with anatase powder, in combination with H2O2, was
preferable with respect to photocatalysis using a fixed bed and to all other combinations (with
sunlight, UV exposition, without activator). Additional tests were also performed in order to
determine the optimum TiO2 quantity to treat 200 ml of EQ2 wastewater. In each test, a percentage
of 1% of H2O2 was used. Although ammonia was reduced more than 90% (and sulphate
concentrations increased from 30 mg/l until 500 mg/l, due to the oxidation of H2S), the reduction of
phenols was lower than expected (on average, below 50%). This result was explained with the low
reactivity of the selected anatase powder.
Laboratory experiments carried out with TiO2 Aeroxide Anatase
Another type of anatase, named TiO2 aeroxide, was used and new set of trials were performed. As
shown in Fig. 95, there was a great difference between the aeroxide and the TiO2 powder efficiencies
with much improved abatement efficiencies obtained with the aeroxide powder.
Fig. 95: Comparison between TiO2 aeroxide and TiO2 powder (Carlo Erba) abatement efficiency.

Further tests were performed in order to determine the percentage of hydrogen peroxide to be used
in the pilot plant scale experimentation with aeroxide powder (Fig. 96). As may be seen from Fig.
96, the best results were obtained when combining 0.1 g/l of anatase and 1% of H2O2.

112

Fig. 96: Tests with Aeroxide powder and hydrogen peroxide.

Photocatalytic laboratory experiments with a fixed bed

Concerning the possibility to use TiO2 in a fixed support, a specific paint was selected for coating
different type of supports. This photo-catalytic paint, named Fotometal, is provided by an Italian
company (Global Engineering S.p.A.) based in Milan. For the tests, a stainless steel support was
coated with Fotometal paint (Figs. 97-98); the coating had 0.25 m of thickness. This plate was
used in the trials, simulating a fixed bed reactor.
Fig. 97: Stainless steel support coated by Fotometal paint.

Fig. 98: Test with TiO2 in a fixed support.

Different experimental conditions were tested, varying the time of UV exposition (between 0.5h and
3h), the thickness of the wastewater on the support (between 1 cm and 5 cm) and the reaction
temperatures (between 25C and 40C). The time of UV exposition appeared to have little effect on
COD degradation, with respect to the wastewater thickness. In particular, trials have been
performed with 1.5 and 2.0 cm of water thickness over the TiO2 fixed bed (Fig. 99).

113

Fig. 99: COD and phenol reduction in function of water thickness over the TiO2 fixed bed.

2.7.1.4

Laboratory tests results using zeolite adsorption

Owing to their properties, zeolites were used to reduce the pollutant concentrations in the coke oven
wastewater. For this study, the zeolites used in the project were supplied by Tosoh Corporation as
extruded cylinders. The type of the zeolites was mordenite (naturally occurring zeolites). The typical
particle size distribution was more than 80% in the range 1.682.83 mm, and more than 95% in
the range 1.41 2.83 mm). The specific surface was 31.2 m2/g. The bed volume of zeolite column
is 930 ml (the used column has a diameter of 6 cm and an height of 36 cm, packet height 32 cm),
BV/h = 1.2*10-3.
The wastewater utilised for zeolite tests was the same wastewater used for the photo-oxidation tests
(EQ2) and its characterisation is repored in Tables 44-45. Different tests were performed by
changing the water flow rates (i.e. 0.4 l / h and 8.5 l/h) that leached from the column containing
zeolites (Fig. 100). Other trials were made but only the limit situations have been reported in Fig.
102. The intention was to show how important is the flow-rate and the corresponding effect on COD
adsorption. In case of high flow-rate, preferential ways are followed by the wastewater. Two
different solutions were used having different COD values; a trial with 8.5l/h had a diluted
wastewater, and a trial with 0.4l/h had the real wastewater sample. In effect, results from Table 49
are 1.27 mg COD / g zeolites instead of 1.67 mg COD / g zeolites and 0.67 mg phenols / g zeolites
instead of 0.27mg phenols / g zeolites.
Fig. 101 shows the colour variation of the wastewater solution at the exit of the zeolite column. This
visible effect corresponds to an increase in the COD concentrations step by step when the column
becomes saturated and the coke oven wastewater is leached without being treated (Fig. 102).
Higher flow rate corresponds to reduced contact time between water and zeolites, so the saturation
level seems to be lower. In particular, during the experimental trials, the asymptotic COD level was
about 600 mg/l with a flow rate of 0.4 l/h whereas it was about 400 mg/l with a flow rate of 8.5 l/h.
different trials have been made but only limit situations have been reported in Fig. 102. The
intention was to show how important is the flow-rate and the corresponding effect on COD
adsorption. In case of high flow-rate, preferential ways are followed by the wastewater. Two
different solution were used having different COD (trial with 8.5l/h had a diluted wastewater, trial
with 0.4l/h had the real wastewater sample).
Moreover, phenols, ammonia and iron concentrations have been analysed in some samples collected
at the exit of the zeolite column, for the two trials. The results are shown in Fig. 103. As it can be
observed, the effects on iron were insignificant, whereas both ammonia and phenols were well
captured by the zeolites.

114

Fig. 100: Zeolite column.

Fig. 101: Picture of coke oven wastewater samples at the exit of the zeolite column
(on the left first samples, on the right final samples).

Fig. 102: COD concentrations at the exit of the zeolite column.

Fig. 103: Phenols, ammonia and iron concentrations at the exit of the zeolite column.

Table 48 shows the calculated amounts of COD, phenol and ammonia absorbed by the zeolites,
expressed in mg / g zeolites, based on the results of the laboratory experiments.

115

Table 48: Adsorption capacity of zeolites

trial

Flow-rate (l/h)

mgCOD/gzeolites

mgphenols/gzeolites

mgNH4+/gzeolites

8,5

1,27

0,41

0,020

0,4

1,60

0,67

0,028

The laboratory trials led to very encouraging results, therefore additional tests were carried out to
study the regeneration of zeolites by thermal treatment and the abatement efficiency of zeolites
after thermal regeneration. A diagram showing the gas emissions obtained during the regeneration
thermal treatment of zeolite (zeolite quantity = 1 kg) is shown in Fig. 104. As may be seen from Fig.
104, a peak of total hydrocarbon (THC) was observed as soon as the temperatures reached above
200C, indicating the regeneration process. The thermal treatment experiments were conducted in a
rotary kiln to achieve temperature homogeneity and a suitable degree of mixing.
Fig. 104: THC emissions during zeolite regeneration.

Figs. 105-106 show the phenols, COD and NH4+ concentrations at the inlet and outlet of a column of
regenerated zeolites. Residual COD and NH4+ concentrations were observed owing to the high flowrate used which may have caused breakthrough in the zeolite column. Nevertheless, the results
confirmed the possibility to efficiently thermal regenerate the zeolites and use it for further water
treatment. Indeed, the COD, BOD5 and phenols reduction rates obtained with regenerated zeolites
were 60%, 65% and >95%, respectively.

116

Fig. 105: COD, phenols and ammonia inlet / outlet concentrations during a laboratory trial with
regenerated zeolites.

To complete the laboratory trials, a series of experiments were carried out to study the effects of
zeolites upon PAH emissions. For these tests, it was decided to carry out in parallel photocatalytic
tests using both types of anatase (Carlo Erba and Aeroxide). Also, combined tests using first zeolite
filtration followed by photocatalytic treatment using TiO2 aeroxide were carried out. The results are
summarised in Table 49.
Table 49: PAHs abatement efficiency in laboratory tests carried out using zeolites, anatase
photocatalytic treatment and combined zeolite / anatase treatment.
PAHs

H2 O 2

TiO2 Carlo
Erba +
H2 O2

%
Naphthalene
Chrysene
Benzo(b)fluoranthene

Zeolites
only

TiO2
aeroxide
+ H2O2

TiO2
Carlo
Erba +
UV
%

1. Zeolites
2. TiO2
aeroxide +
H2 O2
%

92.9
62.4
68.1

43.6
50.7
47.8

48.3
44.0
35.8

91.2
89.9
84.3

96.6
79.5
81.3

99.8
92.3
90.6

Benzo(k)fluoranthene

82.0

71.0

28.0

78.7

84,1

88.2

Benzo(a)pyrene

57.5

48.8

40.2

83.9

65.5

87.1

Benzo(ghi)perylene

29.2

14.3

38.1

89.0

47.1

94.5

Acenaphthylene
Fluorene
Phenantrene

89.5
91.3
84.5

69.7
74.2
67.8

43.3
38.2
39.3

76.7
81.5
82.0

90.9
79.6
89.0

97.8
86.6
95.1

Anthracene

78.7

60.3

38.8

73.7

84.5

88.6

Fluoranthene

82.0

70.9

37.4

88.7

86.0

92.4

Pyrene

65.9

67.6

44.9

79.4

70.9

83.5

Benzo(a)anthracene

56.3

50.0

30.6

84.2

64.7

90.4

Analysis of the results showed that the best abatement efficiency for PAHs was obtained when
combined tests using zeolites and TiO2 aeroxide were carried out. Overall, the second best
abatement efficiencies were obtained using zeolites filtration only. The abatement efficiency

117

obtained using the anatase Carlo Erba was significantly lower than with the aeroxide powder. As
conclusion of this part of the study, it was therefore decided to perform pilot plant scale tests
combining both adsorption on zeolites and photo-oxidation using the aeroxide anatase.

2.7.2

Task 5.2 Scaling up of the anatase (TiO2) photocatalysis process and

cost benefit analysis

Laboratory tests both with anatase and zeolite have proved that, in order to detoxify coke oven
wastewater, the best solution was to use in combination a zeolite column followed by a mixed bed in
which photo-oxidation reactions take place. Accordingly, a combined zeolite / photooxidation pilot
plant was built for this study. The schematic of the pilot plant is depicted in Fig. 106.
Fig. 106: Schematic of the zeolite / photooxidation pilot plant.

2.7.2.1

Design of the photocatalytic reactor - Fluid-dynamic simulations

In order to identify the best agitation system configuration for the photo-oxidation reactor, fluiddynamic simulations with Computational Fluid Dynamics (CFD) code Fluent were carried out. The
simulation was done with CFD code Ansys-Fluent running on Processors Dual Intel Xeon 3.0 Ghz32GB RAM [79]. The main model features were: body-fitted coordinates with about 200,000 hybrid
cells; unsteady Reynolds Averaged-Navier-Stokes equations; standard wall functions; simple
scheme with spatial discretisation with momentum-2nd order upwind; turbulence described via the
- model.
The simulation was done considering a reactor size of 15x30x8 cm and an agitation palette of 2x1
cm. The different conditions investigated were the following:

reference case: laboratory conditions with one stirrer at fixed position and one paddle
angular velocity = 1000 rpm;
check 1: as the reference but with an angular velocity at = 2000 rpm;
check 2: 2 impellers on central axis with = 2000 rpm;
check 3: as check 2 but the impellers were positioned in diagonal. Indeed, the literature
suggests that mixing in a reactor is usually enhanced when stirring configurations are
asymmetric [80].

First, the dead zones were calculated using the CFD model by considering the volume where the
local velocity was lower than half the average velocity [81]. The simulation results are shown in
Figs. 107 to 110.

118

Fig. 107: Computed velocity map in the reactor (m/s). Reference case.

Fig. 108: Computed velocity map in the reactor (m/s).Check 1

Fig. 109: Computed velocity map in the reactor (m/s).Check 2

119

Fig. 110: Computed velocity map in the reactor (m/s).Check 3

A comparison between the dead zones calculated with CFD modelling was carried out for each
condition. The calculations were realised by considering that the dead volume was equal to 1-tave,
calculated /t ave, nominal where t ave, nominal = Volumetric Flow rate / reactorVolume. The results are presented
in Fig. 111.
Fig. 111: Comparison between different scenarios dead zoned.

From the CFD simulations it was concluded that:

an improved stirring was achieved with two stirrers and high angular velocity
the diagonal double stirrers positioning seems to reduce possible stagnant zones between
the stirrers.

In conclusion, the diagonal configuration was recommended for the pilot scale tests. However, with
regard to the angular velocity, the typical (1000 rpm) value was preferred because higher with
several stirrers would lead to very high energy consumption for stirring. Fig. 112 depicts the pilot
plant composed of a zeolite filtration column with a photocatalytic reactor in series equipped with an
agitation system comprising two stirrers in diagonal.

120

120

Fig. 112: Pilot plant composed of a zeolite column connected in series to a photocatalytic reactor,
with diagonal configuration of the agitation system.

2.7.2.2

Results from the pilot plant tests

After commissioning of the pilot plant, a series of pilot scale tests were carried out with the pilot
plant in the combined zeolite / photocatalysis configuration using the Aeroxide anatase. For
comparison purposes, tests were carried out using only the photocatalytic reactor and the zeolite
column separately. Finally, additional tests were carried out using two zeolite columns connected in
series. Details of the pilot scale tests are summarised below:

Photocatalytic reactor with anatase only: 12-h treatment;

Photocatalytic reactor with anatase only: 24-h treatment;
Zeolite filtration + photocatalytic reactor with anatase: 24-h treatment;
Zeolite filtration only;
2 zeolite columns in series.

The pilot plant tests used a column of 10.5 cm diameter and 45 cm of height, (packed height was
40cm). This column was filled with about 2.7 kg of zeolite. The flow rate in absolute values was 4.0
l/h, in BV/h = 1.15*10-3. The breakthrough point of the column was estimated to correspond to
about 3 l of wastewater. The quality of the coke oven wastewater is shown in Table 46 in terms of
PAH concentrations, and wastewater quality remained constant during the trial test with a maximum
variability within 5% for each parameter. Tests were performed at room temperature. Light/UV
radiation was not used since very low yield of pollution removal was achieved during laboratory
tests, (in the laboratory study one 150 Watt lamp was used).
Fig. 113 shows the colour of the coke oven wastewater effluents after treatment as received, after
zeolite filtration and after zeolite + photocatalytic treatment showing a significant improvement in
the effluent.

121

121

Fig. 113: Comparison between the colour of coke oven effluents with and without zeolite /
photocatalytic treatment.

Table 50 summarises the percentage reduction of COD, phenols and BOD5 obtained with the pilot
plant tests. Figures 114 and 115 show the trend of COD and phenols during the trials carried out
with the zeolite column and the corresponding yield are illustrated.
Table 50: Percent reduction of COD, phenols and BOD5 obtained during the pilot scale tests realised
at CSM.
Time

COD (%)

Phenols (%)

BOD5 (%)

12h anatase

12.1

46.7

8.9

24h anatase

13.1

48.0

11.6

24h anatase + zeolite

71.7

91.7

65.6

1x zeolite

67.0

91.9

62.3

2x zeolite

78.8

99.0

69.8

Fig. 114: Trend of COD and phenols during trials with zeolite column.

Fig. 115: COD and phenols yield during trials with zeolite column.

122

From a technical point of view, the combined tests using zeolite and photocatalytic degradation
proved difficult to conduct owing to the respective residence times for both processes. Indeed,
several hours were required for the percolation of the coke oven effluent through the zeolite column
and a residence time of 24-h was needed in the photocatalytic reactor. In practise, it proved very
difficult to synchronise both processes. As a result, both steps were carried out separately in
succession. Therefore, in order to scale up such an application to the industrial scale, further work
would be needed to allow both steps to be synchronised. Analysis of the results showed that the use
of anatase only did not lead to the best abatement efficiencies for COD, phenols and BOD5 (Table
50). The best abatement efficiencies were obtained when two zeolite columns in series were used
with abatement efficiencies of 99%, 70% and 79% for phenols, BOD5 and COD, respectively. The
tests carried out with only one zeolite column and in combination with anatase led to marginally
lower abatement efficiencies in comparison, but still very high abatement efficiencies (ca. 92%, 65%
and 70% for phenols, BOD5 and COD). These results were used to calculate the zeolite adsorption
capacity for COD. For the pilot scale tests, the zeolite adsorption capacity was 2.2 mgCOD/gzeolite
which was slightly higher than the one calculated during the laboratory tests (ca 1.6 mgCOD/gzeolite).
Table 51 shows the percentage reduction for PAHs for the tests carried out using two zeolite
columns in series and combined tests using zeolite and photocatalysis. Similar ranges for emission
reduction were observed under both conditions, however higher PAH percent reduction ranging from
76% to 95% were obtained using two zeolite columns connected in series indicating that this option
may be preferable to the combined zeolite / photocatalys option for the treatment of PAHs.
Tests were also carried out to study the effects of zeolites upon the adsorption of trace metals.
These tests were carried out by CSM using two zeolite columns connected in series. All the samples
were analysed by Tata Steel using ICP/MS. The results are summarised in Table 52 showing the inlet
and outlet trace metal concentrations, and the abatement efficiency. The inlet and outlet
concentrations are also depicted in Fig 116. For most metals, very good abatement efficiencies were
obtained using two zeolite columns for treatment of the coke oven wastewater. In particular, very
high adsorption was observed for lead (ca. 99%) and cadmium (ca. 96%). However, zeolite
adsorption was relatively low for zinc (ca. 25%), manganese (ca. 42%) and chromium (ca. 25%).
Table 51: Percent reduction of PAHs obtained during pilot scale tests realised using two zeolite
columns in series and combined zeolite / photocatalytic treatment.

Naphthalene

95.3

Combined zeolite /
photocatalytic tests using
TiO2
(Abatement - %)
90.2

Chrysene

88.7

86.1

Benzo(b)fluoranthene

87.6

82.5

Benzo(k)fluoranthene

83.5

74.1

Benzo(a)pyrene

86.2

80.1

Benzo(ghi)perylene

87.6

80.4

Acenaphthylene

84.3

71.8

Fluorene

81.6

75.4

Phenantrene

83.7

77.3

Anthracene

75.9

67.7

Fluoranthene

85.5

82.7

Pyrene

78.4

73.8

Benzo(a)anthracene

85.6

78.8

PAHs

Two zeolite columns in

series
(Abatement - %)

123

Table 52: Trace metal concentrations and abatement efficiency in inlet / outlet coke oven effluent
samples during pilot scale tests using two zeolite columns in series.
Untreated
Wastewater
(g / L)

Two zeolite
columns in series
(g / L)

Two zeolite columns

in series
(Abatement - %)

Al

139

24.9

82.1

18

0.66

96.3

Cr

1350

1010

25.2

Mn

157

92

41.7

Fe

7290

87.9

98.8

Co

40

5.12

87.2

Ni

123

41.8

66.0

Cu

110

12.6

88.6

Zn

65

48.8

25.0

As

19

3.78

80.1

Cd

4.5

0.17

96.3

Pb

30

0.19

99.4

Trace
metals

Fig. 116: Inlet / outlet trace metal concentrations obtained during the treatment of coke oven
effluents using two zeolite columns in series.

124

Several studies have been carried out to determine the cation exchange selectivity of synthetic and
natural zeolites for heavy metals. One of the most significant studies presents a summary of the
results for more than 27 natural and synthetic zeolites [82]. In this work, results are reported for a
natural mordenite zeolite similar to the one used during the pilot scale tests which exhibited very
high selectivity for lead and much lower selectivity for zinc, in good agreement with the results
obtained during this study.

2.7.2.3

Cost-benefit analysis

In general, an adsorption process involves identical columns, whose number depends on flowrate
and pollutant concentration. Some of them are in adsorption mode, others in the regeneration
mode. For the cost-benefit analysis, it was decided to focus on different types of column size as
described in Table 53. The zeolite cost was fixed at 0,2 / kgzeolite. For PAHs, an adsorption capacity
of the zeolites equal to 5.5 mg PAHs / kgzeol was used which would lead to an abatement of PAHs >
80%. The zeolite treatment plant would be positioned post-BET plant after the aeration cell and the
clarifiers.
Table 53: Data on columns geometrical features and zeolite costs.
Height
(m)
8

Diameter
(m)
1.24

Volume
3
(m )
10

Mass of zeolite per

column (kg)
11120

Zeolites cost per column

()
1460

10

1.24

12

13344

1810

10

1.6

20

22400

3038

15

1.6

30

33360

4529

20

1.6

40

44480

6039

For this exercise, cost calculations were carried out using Tata Steel BET plant A as an example. For
BET plant A, the following parameters were taken into consideration for the calculations:

Daily flow rate: 2138 m3 / day;

Average PAH concentrations (post-treatment): 40 g / L;
Amount of PAHs to be treated each day: 85.5 g.

Using these values, it was estimated that approximately 13500 kg of zeolites would be needed per
column to treat the effluents at BET plant A. Accordingly; the installation of 2 treatment streams
each containing two zeolite columns in series with capacity of 12 m3 per column would be suitable
for treating PAHs in effluents from the BET plant. These two streams could then be operated
alternatively, with one stream in adsorption mode and the other stream in regeneration mode. For
this configuration, cost calculations were carried out. They are summarised in Table 54.

125

Table 54: Cost-benefit analysis results for the treatment of coke oven effluents using zeolites.
Zeolite capacity
(mgPAHs/g
zeolites)

Number of
column per
stream

Cost abatement first

year + installation
(/g PAHs)

Annual abatement
costs after first
year (/g PAHs)

PAHs
abatement
(%)

5.5

6.45

0.53

>80

2.7.3

Task 5.3. Study into the use of improved filtration sorbents for the removal
of organic and inorganic pollutants in wastewater from coke making
operations

In this task, work focused on the building, commissioning and optimisation of a pilot scale BET plant
by Tata Steel in order to carry out studies into the potential benefits of using powdered activated
lignite as a solution for the removal of trace metals and PAhs from coke oven effluents.

2.7.3.1

Background on Powdered Activated Carbon Treatment (PACT)

Activated sludge processes that are supplemented with powdered activated carbon (PAC) into the
aeration tank are known as powdered activated carbon treatment (PACT) process [83, 84]. In this
type of system, aerobic microbial activity is deliberately promoted using PAC. PACT processes show
stable performance because PAC added into the aeration tank of the activated sludge processes
remove toxic substances by adsorption and also increase the biodegradation capabilities of the
biosludge. Effectively, PACT combines two processes, sorption and biodegradation. The combination
of these two processes reduces problems such as shock loading of toxic substances, improve solid
liquid separation, reduce the effluent suspended solid concentration, and reduces foaming. In
addition, PACT has low capital and operating costs and typically exhibits stable treatment
performance.
The mechanisms of PAC enhancement of the activated sludge process are not entirely clear. A view
of biological activity and carbon adsorption acting independently is very likely too simple. First, the
use of PAC in BET plants results in a bioregeneration process whereby renewal of the adsorptive
capacity of activated carbon occur through the action of microorganisms [84]. In addition, it is
thought that the activated carbon can also act as a 'sink', where adsorbed pollutants can be
metabolised in the bacterial biofilm by slowly diffusing out of the carbon structure. This mechanism
is particularly relevant for slowly degradable, absorbable compounds for which the contact time with
the biomass is important. Initially this class of compounds is removed by adsorption. However, given
sufficient contact time, the biomass can by some means degrade these compounds, partially
renewing the carbon surface. In a conventional BET plant, these slowly degradable compounds are
in contact with the biomass for a length of time equal to the hydraulic retention time (several
hours). In a PACT system, once adsorbed, these compounds are in contact with the biomass for a
length of time equal to the sludge age, up to 50 days [84].
The final discharges from coke oven BET plants are invariably coloured, which may vary from pale
yellow to deep brown dependent upon the coal blend and the characteristics of the carbonisation
process. The colour derives from the presence of polyhydric organic substances with molecular
weights in the same range (1,000 to 8,000 Daltons) as those of naturally-occurring humic and fulvic
acids. The substances found in coke oven effluents are thought to consist of polymerised phenols
and, in addition to colour, they are largely responsible for the residual COD of treated effluents.
These compounds may also inhibit the biological treatment process, thus necessitating the addition
of dilution water to overcome their inhibitory effect in some cases. The inhibitory behaviour of these
compounds generally precludes the possibility of using treated coke oven effluent as dilution water
in the BET process. A previous study has shown that PAC could be used to decolourise treated coke
oven effluents [85].
Most of the work into the advantages of PACT has used target compounds, which are readily
biodegradable, and of low molecular weight, such as organic acids or phenols which are also readily
absorbable. There has been little work carried out to investigate the environmental benefits of PAC
for the removal of heavy metals. In the ECOWATER project, the potential benefits of using PAC for
the removal of recalcitrant high molecular weight PAHs such as B[a]P and the removal of selected
trace metals was therefore studied. The type of carbon selected for the study was activated lignite.

126

2.7.3.2

Commissioning and optimisation of a pilot scale BET plant by Tata

Steel

In the project, work focused on the building, commissioning and optimisation of a BET pilot scale
plant at Tata Steel Plant A. The pilot plant was built during the first 6 months of the year and
commissioned in July 2012. Optimisation tests were carried out between August and November
2012.
The pilot scale plant is composed of:
Inlet storage tank for raw coke oven effluent (0.50 m3);
Peristaltic pump for transfer of raw coke oven effluent to the aeration tank;
Aeration tank (0.72 m3);
Clarifier system for sludge settling;
Peristaltic pump for return of settled sludge to the aeration tank;
Vitox oxygen injection system (maintaining dissolved oxygen concentrations ranging from 2
to 4 mg / L);
Mono pump for internal recirculation of the mixed liquor suspended solid in the aeration
basin;
Heaters to maintain a temperature of 28C in the aeration cell;
Dissolved oxygen, pH and temperature probes.

A schematic of the pilot plant is depicted in Fig. 117 including pictures detailing the different parts of
the pilot plant.

Fig. 117: Schematic (A) and pictures (B, C, D) of the pilot scale BET plant commissioned
at Tata Steel Plant A in July 2012.

127

Fig. 117: (Continued). Schematic (A) and pictures (B, C, D) of the pilot scale BET plant
commissioned at Tata Steel Plant A in July 2012.

Inlet storage
tank
(raw coke oven
effluent)

Clarifier

Aeration Cell

Mono pump
(Aeration cell
recirculation)

Oxygen injection
system
(VITOX)

Inlet / return sludge

pumps

Fig. 117: (Continued). Schematic (A) and pictures (B, C, D) of the pilot scale BET plant
commissioned at Tata Steel Plant A in July 2012.

Dissolved oxygen
probe

pH probe

Flow from inlet

reservoir to
aeration cell

Heat control

Flow from
aeration cell
(Start-up)

Return sludge flow

128

Fig. 117: (Continued). Schematic (A) and pictures (B, C, D) of the pilot scale BET plant
commissioned at Tata Steel Plant A in July 2012.

Clarifier
Waste sludge
valve

Outlet
Treated
effluent
Feed from
aeration cell
to clarifier
After commissioning of the BET plant in July 2012, a programme of work was set out to optimise it.
The primary objectives were to determine the best operating procedure for the pilot plant in order to
achieve almost complete abatement for key compounds such as thiocyanates and phenols. At Tata
Steel Plant A, the main plant is currently not designed for ammonia treatment via nitrification /
denitrification. However, the plant is typically operated with an extended sludge age (ca. 140 days)
to minimise the costs associated with sludge wasting and processing. Theoretically, nitrification of
ammonia can take place with sludge age > 40 days. As a result, the extended sludge age means
that partial unwanted nitrification of ammonia sometimes take place in the main treatment plant
resulting in a sudden increase in nitrite concentrations in the aeration cell. When this phenomenon
occurs, thiocyanate and/or phenol treatment can be inhibited. To prevent this occuring at the pilot
plant and to investigate further this issue during the optimisation trials, it was decided that the
sludge age for the pilot plant would be significantly reduced to below 40 days. Accordingly, a
program over 16 weeks was put in place to progressively reduce the sludge age from 140 days
down to below 40 days, as shown in Fig. 118. In total, the pilot plant operated consistently for 12
weeks, but had to be stopped owing to the mechanical failure of the Mono pump used to recirculate
the mixed liquor suspended solids in the aeration cell. At week 12, a sludge age of approximately 60
days was reached, however, it was not possible to pursue further the optimisation trials.

129

Fig. 118: Sludge age reduction program during the optimisation tests of the BET pilot plant
commissioned by Tata Steel.
Sludge age reduction programme at the pilot scale BET plant
160

Target sludge age

Current sludge age
140

Sludge age (Days)

120

Plant failure
100

80

60

40

20

19th November

29th August

16

15

W
ee
k

14

W
ee
k

13

W
ee
k

12

W
ee
k

11

W
ee
k

10

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

W
ee
k

S
ta
rt

During the twelve weeks of operation, samples of the inlet, outlet, return sludge, and aeration cells
were collected every two days to follow closely plant efficiency. The samples were analysed for a
wide range of species including COD, thiocyanate, suspended solid concentrations, free cyanide,
ammonia and total phenols. In addition, nitrite concentrations were monitored in the aeration cell to
identify the occurrence of unwanted nitrification, if any. The results are depicted in Fig. 119.

130

131

80

60

40

20

Low phenol inputs from 24/10/2012

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

21/11/12

19/11/12

100

21/11/12

120
17/11/12

Total phenols (Outlet - mg / L)

19/11/12

Total phenols (Inlet - mg / L)

15/11/12

160

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

140

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

21/11/12

19/11/12

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

02/09/12

50

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

31/08/12

m
g/ L

21/11/12

19/11/12

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

02/09/12

31/08/12

2000

04/09/12

02/09/12

31/08/12

02/09/12

m
g/ L

31/08/12

mg / L
mg/ L

Fig. 119: Monitoring results for suspended solids, thiocyanate, COD, total phenols, free cyanide
nitrite and ammonia in inlet / outlet and return sludge / aeration cell samples collected at the pilot
scale BET plant during the 12 weeks optimisation campaign.
14000
Aeration cell suspended solids (mg / L)
Return sludge suspended solids (mg / L)

12000

10000

8000

6000

4000

Leak on the return sludge pump

(6 / 11/ 2012)

300
Thiocyanathe (Inlet - mg / L)
Thiocyanathe (Outlet - mg / L)

250

200

150

100

Loss of treatment
(6/11/2012)

1200
COD (Inlet - mg / L)
COD (Outlet - mg / L)

1000

800

600

400

200

132

21/11/12

19/11/12

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

120

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

02/09/12

21/11/12

19/11/12

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

02/09/12

21/11/12

19/11/12

17/11/12

15/11/12

13/11/12

11/11/12

09/11/12

07/11/12

05/11/12

03/11/12

01/11/12

30/10/12

28/10/12

26/10/12

24/10/12

22/10/12

20/10/12

18/10/12

16/10/12

14/10/12

12/10/12

10/10/12

08/10/12

06/10/12

04/10/12

02/10/12

30/09/12

28/09/12

26/09/12

24/09/12

22/09/12

20/09/12

18/09/12

16/09/12

14/09/12

12/09/12

10/09/12

08/09/12

06/09/12

04/09/12

02/09/12

31/08/12

31/08/12

mg / L
4

31/08/12

mg / L

mg / L

Fig. 119: (Continued) Monitoring results for suspended solids, thiocyanate, COD, total phenols, free
cyanide nitrite and ammonia in inlet / outlet and return sludge / aeration cell samples collected at
the pilot scale BET plant during the 12 weeks optimisation campaign.
12
Cyanide (Inlet - mg / L)
Cyanide (Outlet - mg / L)

10

Total cyanide values

2
Free cyanide values

14
Nitrite Aeration cell (mg / L)

12
Loss of thiocyanathe treatment
(6/11/2012)

10

Nitrification start from 24/10/2012

140

NH3 (Inlet - mg / L)
NH3 (Outlet - mg / L)

100

80

60

40

20

The conclusions that may be drawn from the analytical results obtained during the optimisation tests
are as follows:

The suspended solid concentrations in the aeration cell and the return sludge showed a
decrease trend over the twelve weeks of experimentation indicating that the sludge age
reduction program was working. More sludge was wasted in the pilot plant in comparison
with the main plant. At the main treatment plant, it was estimated that the amount of

sludge wasted per m3 of coke oven liquor treated ranged between 4 6 L / m3, whereas 11
L of sludge per m3 of coke oven liquor was wasted at the pilot plant.
Very good and stable operation of the pilot plant was observed over the entire duration of
the optimisation trials with near-full abatement of thiocyanate (ca. 200 mg / L down to
below 2 mg / L ; > 99% removal), total phenols (ca. 80 - 120 mg / L down to below 10 mg /
L ; > 99% removal), COD (ca. 1000 mg / L down to below 200 mg / L ; > 80% removal)
and free-cyanide (ca. 1 mg L down to below 0.09 mg / L ; > 90% removal).
There was an increase of ammonia concentrations after treatment from ca. 60 mg / L to ca.
100 mg / L. This was also observed in the main plant owing to the fact that ammonia was
produced from the degradation of thiocyanate during treatment.
Two periods of partial unwanted nitrification were observed during the twelve weeks
experimentation. In the first period, nitrite concentrations in the aeration cell as high as 6
mg / L were measured however this did not result in any loss of treatment for other key
species (ie. phenols, thiocyanates). In the second period, which started on the 24th October
2012, much more severe nitrification was observed with nitrite concentrations in the
aeration cell reaching 12 mg / L at which point the treatment of thiocyanate was lost for
more than a day. It is unclear what parameters triggered the sudden increase in nitrification
during the second period after a relatively long period without any nitrite present in the
aeration cell. However, it must be noted that this coincided with a period of low phenol
inputs to the pilot plant. In addition, the sludge age was also well above 40 days at that
time meaning that unwanted nitrification could still occur since the sludge age was still
relatively long.

Overall, the results obtained during the optimisation tests showed that the pilot BET plant operated
very satisfactorily for future research in the project. Since the optimisation tests had to be stopped
prematurely, it was not possible to fully assess the possible effects of reducing the sludge age below
40 as a solution to prevent unwanted nitrification occuring during the treatment process.

2.7.3.3

Activated lignite injection laboratory tests undertaken by Tata Steel

The two PAC adsorbents selected by Tata Steel for the project ECOWATER are activated lignite
products originating from the Rhenish region in Germany. These lignite products are extracted,
produced and supplied by Rheinbraun Brennstoff GmBH [86]. These are designated as HOK
products and are activated in a rotary hearth furnace under specific conditions. The two products
selected differ mainly by their particle size. The HOK Super activated lignite is the finest activated
lignite product supplied by Rheinbraun with particle size typically below 0.125 mm. The HOK
Pulverised is a coarser product with particle size below 0.400 mm. A picture of the two adsorbents
selected in the ECOWATER project is provided in Fig. 120. In addition, Table 55 summarises the
main physical characteristics of the two HOK products.
Fig. 120: Pictures of the two HOK activated lignite adsorbents selected for testing in the
ECOWATER project.

HOK super
0 to 0.125 mm

HOK pulverised
0 to 0.400 mm

133

Table 55: Physical characteristics of the two activated lignite products (HOK Super and Pulverised)
tested for PACT in the ECOWATER project.

Grain size

Bulk density
Specific surface

HOK Pulverised

HOK Super

> 0.315 mm
: 5% wt
0.200 - 0.315 mm : 8% wt
0.125 0.200 mm : 12% wt
0.090 0.125 mm : 15% wt
0.063 0.090 mm : 10% wt
< 0.063 mm
: 50% wt

< 63 m (minimum 80%)

0.55 g / cm3

0.44 g / cm3

300 +/- 30 m2 / g

300 +/- 30 m2 / g

The two products were tested initially in laboratory conditions to identify the best adsorbent for
future pilot scale trials. For each product, a series of adsorption tests was carried out by adding
increasing amounts of activated lignite, with addition rates ranging from 250 mg / L to 4000 mg / L,
to coke oven effluent samples collected at Tata Steel Plant A (after biological treatment). After
three hours of agitation at 28C under laboratory conditions, the samples were filtered through a
glass fibre 0.45 m filter and analysed by spectrophotometry at 470 nm to determine the residual
colour of the effluent. The results obtained by spectrophotometry are summarised in Table 56 and
depicted in Fig. 121.
Table 56: Results from laboratory tests realised to study the effects of HOK Pulverised and Super
for coke oven effluent treatment with regard to colour removal efficiency.
Addition rate (mg / L)

Absorbance
(470 nm)

% colour
removal

HOK
Pulverised

4000
3000
2000
1000
500
500
500
250
0
(Blank with 4000 mg /
L HOK Pulverised)

0.025
0.033
0.050
0.115
0.236
0.222
0.240
0.369
0.533
0.007

97
95
92
80
57
59
56
32
-

HOK Super

4000
3000
2000
1000
500
500
500
250
0
(Blank with 4000 mg /
L HOK Super)

0.025
0.039
0.067
0.170
0.310
0.299
0.323
0.425
0.560
0.012

98
95
90
72
43
45
44
26
-

134

Fig. 121: Graph showing the percent colour removal from treated coke oven effluent during
laboratory experiments using activated lignite products (HOK Pulverised and Super).
1.0

% Colour Removal

0.9
0.8
0.7

HOK Super < 0.1 mm

0.6
HOK Pulverised < 0.4 mm

0.5
0.4
0.3
0.2
0.1
0.0
0

1000

2000

3000

4000

HOK Concentration mg/l

Analysis of the results showed that > 90% of the coke oven effluent colour was removed by both
activated lignite products at addition rates > 2000 mg / L. At addition rates below 2000 mg / L, both
products exhibited similar colour removal efficiency although the coarser HOK Pulverised material
did provide slightly better results than HOK Super. For instance, at 500 mg / L addition rate, ca.
60% colour removal was obtained with HOK Pulverised, whereas ca. 45% was obtained with HOK
Super.
Tests were also realised to determine the effects of activated lignite upon the concentrations of
dissolved trace metals including Ni, Cd, As and Pb. Tests were realised using a water sample from
Tata BET plant A (DP10 : discharge point 10). The results are summarised in Fig. 122. As may be
seen from Fig. 122, the most significant reduction was observed with nickel. With 4000 mg / L of
HOK super and/or pulverised, Ni was almost completely depleted from the solution as a result of
the HOK adsorption. At about 500 mg / L, almost 50% of nickel was depleted from the solution.
The highest reduction was obtained with HOK Super. For As, only very small reductions were
observed when HOK was added in quantities higher than 1000 mg / L. For Cd and Pb, small
reductions were observed and the results showed that increasing the amounts of HOK did not lead
to more reduction of the dissolved concentrations.
Fig.122: Graphs showing the concentrations of Ni, As, Cd and Pb in the dissolved phase with
increasing amounts of HOK Super or Pulverised added.
12.0

Pb (With HOK Pulverised)

10

Pb (With HOK Super)

Ni (With HOK Pulverised)

Ni (With HOK Super)

10.0

ug / L

ug / L

8.0
6

6.0

4.0

2.0

0.0

0
DP10

250 mg / L

500 mg / L

1000 mg / L

2000 mg / L

3000 mg / L

DP10

4000 mg / L

250 mg / L

500 mg / L

1000 mg / L

0.16

Cd (With HOK Pulverised)

Cd (With HOK Super)

0.14

2000 mg / L

3000 mg / L

4000 mg / L

As (With HOK Pulverised)

7.0

As (With HOK Super)

6.0
5.0

0.1

ug / L

ug / L

0.12

0.08
0.06

4.0
3.0

0.04

2.0

0.02

1.0

0
DP10

250 mg / L

500 mg / L

1000 mg / L

2000 mg / L

3000 mg / L

0.0

4000 mg / L

DP10

250 mg / L

500 mg / L

1000 mg / L

2000 mg / L

3000 mg / L

4000 mg / L

The results obtained during the laboratory scale tests were promising especially for the possible
reduction of the colour of the final effluent and the nickel dissolved concentrations. However, the
tests were carried out using a coke oven effluent sample collected after treatment. To confirm this,

135

tests carried out with HOK added directly into the aeration cells had to be carried out at the pilot
scale.

2.7.3.4

Activated lignite injection pilot scale tests undertaken by Tata Steel

Tests were realised at the pilot scale by adding controlled amounts of activated lignite into the
aeration cell to determine possible improvements in terms of abatement efficiencies. For these tests,
only HOK Super was tested. The pilot plant was operated for duration of 20 days without activated
lignite. This was followed by a period of 30 days when HOK Super was added at a rate of 400 mg /
L. Simultaneous inlet / outlet samples were collected to measure the concentrations of suspended
solids, PAHs, trace metals and the colour of the final effluent. Fig. 123 depicts the tests carried out
using the pilot plant commissioned by Tata Steel.
Fig. 123: Schematic describing the activated lignite tests carried out at the pilot
scale BET plant of Tata Steel Plant A.
HOK
addition

Oxygen
Injection
Clarifier

Influent

Treated
effluent

Return sludge

Sludge wasting

Without activated lignite, the suspended solid concentration in the final effluent was 25 17 mg / L,
whereas with activated lignite the suspended solid concentration was 20 3 mg / L. Overall, there
was significantly less variability in the suspended solid concentrations and a small reduction of
suspended solid concentrations in the final effluent was also observed. This was due to the fact that
the addition of HOK Super at 400 mg / L did improve slightly the settleability of the sludge in the
clarifier.
With regard to the colour of the final effluent, the tests realised at the pilot scale with HOK super
were disappointing. The mean absorbance of the final effluent measured by spectrophotometry at
470 nm without lignite was 0.309. When using HOK Super at 400 mg/L, the mean absorbance of
the final effluents was only reduced to 0.295, corresponding to less than 5% of colour removal.
These results were not in agreement with the laboratory scale data. It appeared that adding HOK
into the aeration cell did not lead to a significant reduction of the colour of the effluent, as observed
when HOK was added to the treated final effluent. Interactions between HOK and the activated
sludge may be responsible for this.
The abatement efficiencies obtained with and without activated lignite have been summarised in
Table 57 for the 6 PAHs considered in the BAT-AEL limit and several trace metals including Cr, Cu,
Ni, Zn and Pb. With regard to PAHs, the results obtained showed only a very small increase in the
abatement efficiencies. Overall, abatement efficiencies were improved by less than 10% for most
PAHs when HOK Super was added at 400 mg / L. With regard to metals, only a significant increase
in the abatement efficiencies of Ni (ca. 20% increase) was observed. For all other metals, no
significant improvements were observed. This result was in good agreement with the laboratory
tests showing significant reduction in the concentrations of nickel, in particular, in the final effluent
with increasing amounts of activated lignite. Nickel is predominantly present in coke oven effluents
in the dissolved phase (>60%) as previously shown in Section 2.4.1.1, therefore it is
understandeable that this particular metal would be adsorbed preferentially by HOK Super.

136

Table 57: Abatement efficiencies of PAHs and trace metals with and without
activated lignite (HOK Super).
Mean abatement efficiency
Without HOK Super

PAHs
Fluoranthene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Indeno[1,2,3-cd]pyrene
Benzo[g,h,i]perylene
6 BAT-AEL PAHs
Trace metals
Cr
Cu
Ni
Zn
Pb
Cd

2.7.3.5

94
64
71
69
67
61
73

With HOK Super

400 mg / L
96
73
78
79
69
67
81

35
65
47
66
57
50

35
63
67
70
62
59

Cost benefit analysis of activated lignite addition

A cost-benefit analysis was carried out for the use of activated lignite (HOK Super at 400 mg / L).
For this analysis, calculations were based on Tata Steel BET plant A. Using the BET plant capacity
and the amounts of sludge wasted per annum to take into account the losses of HOK Super
throughout the year it was possible to estimate the costs of activated lignite addition. In terms of
benefits, possible reductions of suspended solids by ca. 20%, PAHs by ca. 10% and nickel by ca.
20% were considered. The results of the cost-benefit analysis are summarised in Table 58. It
showed that the average costs of reducing PAHs by ca. 10% using activated lignite were about 1.0
Euros / g PAH reduced the first year. For nickel, the costs were 2.5 Euros / g nickel reduced on the
first year with 20% maximum reduction. Finally for suspended solids, the costs of abatement were
0.85 Euros / kg suspended solids reduced the first year with 20% maximum reduction.
Table 58: Cost-benefit analysis results for the treatment of coke oven effluents
using activated lignite.
Plant characteristics
3
Plant capacity (m / day)
Average return slude suspended solid concentrations (mg / L)
HOK addition rate (mg / L)
HOK proportion in return sludge (%)
3
Amounts of return sludge wasted (L / m liquor treated)

2138
15000
400
4
5

Required amounts of HOK Super

Initial addition of HOK Super for start-up (kg)

Addition of HOK Super to compensate losses via sludge wasting

(kg / annum)
Cost of HOK

Super + delivery (Germany UK) in Euros / ton

912
2341

1020

Abatement costs
Nickel (First year) (Euros / g nickel reduced) ca. 20% reduction
Nickel (Second year onwards) (Euros / g nickel reduced) ca. 20% reduction
Estimated Ni abatement per annum( kg / annum)

2.5
1.8
1.3

PAHs (First year) (Euros / g PAHs reduced) < 10% reduction

PAHs (Second year onwards) (Euros / g PAHs reduced) < 10% reduction
Estimated PAH abatement per annum ( kg / annum)

1.0
0.7
3.2

Suspended solids (First year) (Euros / kg SS reduced) ca. 20% reduction

Suspended solids (Second year onwards) (Euros / g SS reduced) ca. 20% reduction
Estimated suspended solids abatement per annum ( kg / annum)

137

0.85
0.61
3902

2.7.4

Task 5.4. Study into the use of Fuzzy filtration for the removal of
organic and inorganic pollutants in wastewater from cokemaking
operation

2.7.4.1

Background on fuzzy filtration technology

The Fuzzy filter is an innovative and cost-effective compressible media filter, suitable for the
removal of suspended solids from waste water. It has been developed and is currently
commercialised worldwide by BOSMAN, a water treatment company based in the Netherlands
[87]. A fuzzy filtration unit is equipped with a compressible filter medium and is a modular system
that can be used for various different applications such as:

Post-treatment of effluent wastewater (tertiary filtration);

Primary clarifier effluent;
Industrial water treatment.

A schematic of a typical fuzzy filtration unit is depicted in Fig. 124. A fuzzy filtration unit consists of
a square housing, in which the filter medium is captured between 2 perforated plates. The filter is
operated in up-flow. A strainer in the inlet pipe prevents relatively big parts to flow into the filter
and clog it. In the lower part of the filter the influent is divided equally over the total filtration area
before it reaches the lower, fixed mounted, perforated plate. The solids are trapped in the filter bed
and the filtered water leaves the filter on the top. Because the upper plate can be adjusted, the
compression and with that the porosity of the filter bed can be altered. This means that the filter can
be fine tuned and adjusted to changed circumstances. The filter is packed with Fuzzy filter balls
made of polyvaniladene, a highly porous synthetic filter medium (Fig. 124). Each ball has a diameter
of approximately 30 mm. Unusual features of the filter are (i) the porosity of the filter bed can be
modified by compressing the filter medium and (ii) the size of the filter is increased mechanically to
backwash the filter. The filter medium also differs from standard filters such as sand or anthracite
filters in that the wastewater to be filtered flows through the medium as opposed to around the
filtering medium.
Fig. 124: Schematic of a typical fuzzy filter system and picture showing fuzzy filter balls.

Operation of the fuzzy filter

The filter system works up-flow. A schematic showing the different steps in the filtration process is
provided in Fig. 125. In the distribution room, the influent is distributed equally over the bottom
area of the filter before flowing trough the lower perforated plate. In the filter bed the suspended
solids are removed, and the filtered water leaves the filter at the top. The pressure on the filter is
monitored continuously. The washing cycle will start automatically if the pressure reaches a setpoint, or at a preset time interval. The Fuzzy filter uses air and water to clean the filter media.
During a washing cycle influent flows into the filter, and an external compressor feeds the filter with
air in the bottom section of the filter, in order to bring the filter medium in a turbulent flow. It is not

138

necessary to use filtered water to clean the filter. This way the filter medium, which is between the
two perforated plates, will be cleaned thoroughly. The suspended solids that come out the filter
medium will leave the filter continuously during the washing cycle. After the washing is completed,
the filter medium is compressed again, and the filtration process continues.
Fig. 125: Fuzzy filter operation.

Particle size range for fuzzy filter application

The most important parameter to consider for the fuzzy filter to be efficient is the particle size range
of the suspended solids present in the waste water. Fuzzy filters are highly efficient for the removal
of particles within a very large particle size range (ie. from 4 m to above 500 m) and, as such,
exhibit similar performance to sand filtration for particles size within the range 5 to 30 m (Fig.
126).
Fig. 126: Particle size removal for fuzzy filters in comparison with other wastewater treatment
filtration technologies.

139

When using the fuzzy filters, the finer particles (ie. 4 to 10 m) can be collected by compressing the
fuzzy filter balls to a maximum setting in order to have smaller voids to capture small particles.
Conversely, if the wastewater effluent requiring treatment does not contain large amounts of small
particles, the perforated plate can be lifted up to allow larger voids in the filter as shown in Fig. 127.
Fig. 127: Schematic showing that the compression rate of the fuzzy filter can be adjusted to collect
smaller particles (high compression rate) or larger particles (low compression rate).

2.7.4.2

Pilot scale tests using fuzzy filters realised by Tata Steel

Characterisation of coke oven effluent samples for particle size

Samples of treated coke oven effluent (after clarification) were collected and submitted for particle
size analysis.
The coke oven effluents were collected from Tata Steel BET plant A (after the clarifier N.1) and Tata
Steel Plant B (from sump N.6). The results are summarised in Table 59 where the mean diameter
and the volume distribution of particles are reported. The parameters measured for particle size
analysis were:

D[4,3]: Equivalent volume mean diameter (or De Broncker mean diameter);

D[3,2]: Equivalent surface area mean diameter (or Sauter mean diameter);
D[V,0.5]: The diameter for which 50% of the particles (in volume) are above or below this
value;
D[V,0.9]: The diameter for which 90 % of the particles (in volume) are below this value;
D[V,0.1]: The diameter for which 10 % of the particles (in volume) are below this value.

Table 59: Mean diameters particle size distribution (in volume) of treated coke oven effluents from
Tata Steel BET plants A and B.
Sample
Treated
effluent
Treated
effluent

D[3,2] D[4,3] D[V,0.1] D[V,0.5] D[V,0.9]

coke oven
(Plant A)
coke oven
(Plant B)

9.3

41.3

4.9

37.6

79.7

5.1

28.5

1.8

17.3

71.8

The results from the particle size analysis are also depicted in Fig. 128 where the particle size
distribution (1m interval) of both treated coke oven effluents is shown.

140

Fig. 128: Particle size distribution (in volume) of treated coke oven effluents from
Tata Steel BET plants A (Left) and B (Right).
Volume (%)

Volume (%)

10

100

10

100

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10
0
0.1

0
1.0

10.0

100.0

1000.0

Particle Diameter (m.)

10
0
0.1

0
1.0

10.0

100.0

1000.0

Particle Diameter (m.)

Analysis of the data suggested that particle size in both coke oven effluents were above 4 m with,
in particular Sauter mean diameter of 9.3 m (Plant A) and 5.1 m (Plant B). Based on this analysis,
it was therefore possible that fuzzy filters could improve significantly the coke oven effluent
treatment at both BET plants by polishing the final effluent before discharge.
Details of the pilot scale tests
The pilot scale tests were carried out by Tata Steel using a pilot scale mobile fuzzy filter unit
supplied by BOSMAN for duration of three weeks in November 2012. The mobile unit used for the
tests is depicted in Fig. 129. It is a standard 20 feet container fully equipped with a fuzzy filter tower
with a capacity of approximately 15 - 20 m3 / h. For these tests, the inlet was connected to the main
coke oven effluent outlet tank where post-treatment samples could be pumped in for fuzzy filtration
treatment. During washing of the filter, air was injected into the fuzzy filter tower using a blower of
2.2 kW. The wash water was discarded into a separate tank placed outside the container, and
pumped back into a separate outlet area prior to discharge.

141

Fig. 129: Pilot scale fuzzy filter unit installed at Tata Steel BET plant A for testing (November 2012)

Fig. 129: (Continued) Pilot scale fuzzy filter unit installed at Tata Steel BET plant A for testing
(November 2012)

Outlet
Turbidity
probe
(out)

Blower
(Air supply)
Fuzzy filter
Tower
(filtering mode)

Inlet

Several tests were carried out to determine the best operating conditions for coke oven effluent
filtration. The two main parameters tested were the flow rate and the compression rate.
Flow rates:
For the pilot scale fuzzy filter, flow rates typically range from 5 to 15 m3 / h. For these tests, flow
rates of 7, 10 and 15 m3 / h were used.

142

Compression rates:
The depth of the filter bed determines the pore size of the filtration bed. A smaller bed depth means
a more compressed, denser filter bed in which smaller particles can be captured. Typical values for
filter bed depth are 48 72 cm (equating to 40% - 10% compression rates). The compression rate
is defined as follows:
Compression rate = (Initial bed height actual bed height) / Initial bed height

x 100%

The initial bed height prior starting the tests with the pilot fuzzy filter unit is always 80 cm.
Therefore, as an example, if the actual bed height was 60 cm, then the compression rate would be:
Compression rate = (80 60) / 80 x 100 = 25%
In the tests carried out by Tata Steel, three compression rates were used: 41.25%, 31.25% and
47.5%.
Results of the pilot scale tests
For all the tests carried out, a maximum set pressure of 350 psi was set as maximum pressure for
the fuzzy filter. The first experiments that were carried out aimed at determining how much time
was necessary under different flow and compression rate settings to actually reach the maximum
set pressure, at which point a wash cycle is initiated. Fig. 130 depicts the curves obtained for three
settings (ie. 15 m3 / h and 41.25 % ; 10 m3 / h and 47.5% ; 7 m3 / h and 47.5%).
Fig. 130: Graph indicating the time necessary for the pressure in the filter to reach the maximum
set point of 350 psi as a function of compression and flow rates.
400
Compression : 41.25%
Flow rate

: 15 m / hour

350

Flow rate

Pressure (psi)

Compression : 47.5%

Compression : 47.5%

Flow rate

: 10 m / hour

: 7 m / hour

300
250
200
150
100
50

39

33

27

24

36

40

15
21

11

18

ho
ur
s

Hours

Establishing these curves at the beginning of the optimisation programme was important because
they provided an estimation of the frequency of washing cycles for a particular combination of flow
rate / compression rate. As may be seen from Fig. 130, the results indicated that the lowest
frequency of washing (ie. 40 hours) was obtained with the highest compression rate (ie. 47.5%) and
the lowest flow rate (7 m3 / h), whereas the highest frequency of washing (ie. 11 hours) was
obtained with the highest flow rate (ie. 15 m3 / h) and the smallest compression rate (41.25 m3 /
h). Although the aim would be to minimise the number of washing cycles overall, the results
obtained showed that even with very high flow rates (ie. 15 m3 / h), the frequency of washing cycle
would still be relatively low (ca. 11 hours), therefore this would not constitute a major determinant
factor.
Figure 131 depicts the appearance of the fuzzy filter tower after 2-h operation at a compression rate
of 40% and a flow rate of 10 m3 / h, and this is compared with the appearance of the tower prior
starting the filtration process (ie. after a washing cycle). As is evident from Fig. 132, significant

143

amounts of suspended solids were captured by the fuzzy filter tower. This was confirmed when
samples from the inlet, outlet and wash water were collected and filtered prior to analysis as shown
in Fig. 132. After filtration, the opacity in the outlet samples was definitely lower than in the inlet
sample indicating much lower concentrations of suspended solids in the outlet sample after fuzzy
filtration. This was also evident after filtration of the inlet / outlet samples.
Fig. 131: Picture of the fuzzy filter tower after 2-h operation (40% / 10 m3 / h) and immediately
after a washing cycle.

Fig. 132: Pictures showing the appearance of liquid and suspended solid samples taken at the inlet,
outlet and inside the filtering tower (ie. wash water) during the fuzzy filter tests.

Wash water

Inlet

Inlet

Outlet

Outlet

Wash water

The suspended solid results obtained for the inlet, outlet and wash water samples are summarised in
Table 60. As may be seen from Table 60, suspended solid concentrations in the outlet samples were
always lower than in the inlet samples indicating that the fuzzy filtration process was efficient at
capturing particles in coke oven effluents. Amongst the parameters tested, the best operating
conditions were 7 m3 / h and 47.5% compression rate which led to 60% abatement in suspended
solids. At higher flow rates and lower compression rates, the percent abatement was not as high
but typically about 40%.

144

Table 60: Suspended solids concentrations in inlet, outlet and wash water coke oven effluent
samples during pilot scale fuzzy filter tests at Tata Steel Plant A.
Date

29/10/2012
31/10/2012
02/11/2012
05/11/2012
07/11/2012
09/11/2012

Inlet
Suspended
solids
(mg / L)

Outlet
Suspended
solids
(mg / L)

17.4
13.8
30.9
25.0
50.7
52.9

11.2
8.7
18.6
16.2
30.0
21.1

Wash
water
Suspended
solids
(mg / L)
704
1110
1250
986
1260
1890

Suspended
solids
reduction
(%)

Fuzzy filter
compression
rate
(%)

Flow rate

35
36
40
35
41
60

41.25
41.25
41.25
31.25
47.5
47.5

15
15
15
15
10
7

(m3 / h)

This result was confirmed by the data obtained with the continuous turbidity analyzers measuring
simultaneously the turbidity of inlet and outlet samples. Fig. 133 shows the data recorded by the
turbidity analyzer probes during the pilot scale tests. The results indicated that under most
operating conditions a minimum of 50% reduction in the turbidity of the coke oven effluent was
achieved using fuzzy filters.
Fig. 133: Graph showing the turbidity (NTU units) of the influent and effluent samples and the
percent abatement during the pilot scale fuzzy filtration tests.

Turbidity
100

Influent Turbidity [NTU]

Effluent Turbidity [NTU]

90

90%

Turbidity Percent Removal [%]

70%

80

70
50%

30%
50

Removal

Turbidity [NTU]

60

10%

40

30
-10%
20
-30%
10

-50%

26/10/2012 00:00 28/10/2012 00:00 30/10/2012 00:00 01/11/2012 00:00 03/11/2012 00:00 05/11/2012 00:00 07/11/2012 00:00 09/11/2012 00:00 11/11/2012 00:00 13/11/2012 00:00

Inlet and outlet samples were also analysed for their PAH content. The results obtained are depicted
in Fig. 134. The results showed that relatively poor abatement efficiencies were obtained during the
first tests where high flow rates and low compression rates were used (ie. from 29/11 to 03/11),
however much better abatement efficiencies were obtained with low flow rates and high
compression rates ranging from 50% to 80%. For instance, on the 9/11, the most significant PAH
emissions reduction of ca. 80% was achieved with a flow rate of 7 m3 / h and a compression rate of
47.5%, confirming that these were the best operating conditions. Therefore, as discussed earlier
with the suspended solid emissions reduction, it would appear reasonable to expect at least 50%
PAH emissions reduction using fuzzy filters as post treatment technique.

145

Fig. 134: Graph showing the PAH concentrations (Sum of the 16 US EPA PAHs) of the influent and
effluent samples and percent abatement during the pilot scale fuzzy filtration tests at Tata Steel
plant A.

PAH
250

1
Influent PAH [ug/l]
Effluent PAH [ug/l]

0.9

PAH Percent Removal [%]

200

0.8

PAH concentration (ug/l)

0.7
150

0.6
0.5

100

0.4
0.3

50

0.2
0.1
0

26
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Under the best operation conditions (7 m3 / h ; 47.5% compression rate), tests were also carried
out to measure the abatement efficiencies obtained by fuzzy filtration for several trace metals. The
results are summarized in Table 66. As may be seen from Table 61, the abatement efficiencies for
several trace metals including Al, V, Co, As and Pb were relatively low (ca. <10%). The only
significant abatement of trace metal concentrations was found for Zn (ca. 42%), Ni (ca. 28%), Cu
(ca, 27%) and Cd (ca. 19%).

Table 61: Concentrations of trace metals in coke oven effluents after biological treatment
and after fuzzy filtration and abatement efficiency obtained using fuzzy filters.
Trace metals

Coke oven effluent

After BET plant
(g / L)

Coke oven effluent

After fuzzy filtration
(g / L)

Abatement
efficiency
(%)

Al

568

537

5.5

43

42

0.6

Cr

21

18

13

Mn

86

68

21

Fe

2116

1830

13

Co

2.0

1.8

Ni

14

10

28

Cu

2.0

1.5

27

Zn

15

9.0

42

As

7.3

7.0

2.5

Cd

0.14

0.11

19

Pb

6.3

5.8

5.8

2.7.4.3

Cost benefit analysis of the fuzzy filtration technology

A cost-benefit analysis of the fuzzy filtration technology was carried out. For this exercise, a
quotation was requested from BOSMAN. Calculations were based on the implantation of the

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technology at Tata Steel BET plant A. Based on the plant full capacity (ie. 126 m3 / h), it was
proposed to install two parallel Fuzzy filter units (post treatment) after the existing clarifiers at the
BET plant. The total costs for the installation and commissioning of the Fuzzy filter units was
200kEuros with a very small footprint area of 12 m2. It was estimated that civil costs would be ca.
100kEuros. For energy costs, a distinction had to be made between BET plants with gravity flow
available versus BET plants without. At Tata Steel BET plant A, gravity flow was available so the
lower energy costs associated with this configuration were used. The results of the analysis are
summarised in Table 62 where the costs associated with the abatement of nickel (ca. - 28%), PAHs
(ca. -50%) and suspended solids (ca. -50%) are summarised. As may be seen from Table 63, the
abatement costs for the first year were relatively high for nickel (ca. 180 Euros / g nickel reduced),
(ca. 21 Euros / g PAHs reduced) and (ca. 30 Euros / kg of suspended solids reduced). From the
second year onwards, when considering only the energy and maintenance costs, the abatement
costs using fuzzy filtration were much lower (ca. 13.2 Euros / g nickel reduced), (ca. 1.5 Euros / g
PAHs reduced) and (ca. 2.2 Euros / kg of suspended solids reduced). These costs were higher than
the costs of activated lignite addition, however much better abatement efficiencies could be attained
with fuzzy filtration.
Table 62: Cost-benefit analysis results for the treatment of coke oven effluents using fuzzy filtration
at Tata Steel BET plant A.
Plant characteristics
Plant capacity (m3 / h)
Number of fuzzy filter units required
Footprint area of the plant (m2)

126
2
12

Costs of fuzzy filtration

Initial installation and commissioning of two fuzzy filter units (kEuros)
Energy consumption (Wh / m3) Gravity flow available
Energy consumption (Wh / m3) No gravity flow available
Annual ener gy costs Gravity flow available (kEuros)
Annual ener gy costs No gravity flow available (kEuros)
Installation costs (civils, pumps, pipes) (kEuros)
Annual maintenance costs (Fuzzy balls replacement / cleaning) (kEuros)
Total costs (kEuros - first year)
Total costs (kEuros second year onwards)

200
8
28
1.1
3.9
100
20
323.9
23.9

Abatement costs
Nickel (First year) (Euros / g nickel reduced) ca. 28% reduction
Nickel (Second year onwards) (Euros / g nickel reduced) ca. 28% reduction
Estimated Ni abatement per annum (kg / annum)

180
13.2
1.8

PAHs (First year) (Euros / g PAHs reduced) ca. 50% reduction

PAHs (Second year onwards) (Euros / g PAHs reduced) ca. 50% reduction
Estimated PAH abatement per annum ( kg / annum)

20.9
1.5
15.5

Suspended solids (First year) (Euros / kg SS reduced) ca. 50% reduction

Suspended solids (Second year onwards) (Euros / kg SS reduced) ca. 50% reduction
Estimated suspended solids abatement per annum ( kg / annum)

2.8

Work Package 6 Project management / Transfer of knowledge

2.8.1

Task 6.1 - Project coordination (Tata Steel)

29.6
2.2
10925

Tata Steel compiled the contribution of individual partners to produce the annual, mid-term and final
reports. A mid-term presentation was given in Oviedo in May 2011. Tata Steel provided a summary
slide for the project as requested by the TGS9 project officer. Finally, Tata Steel ensured that the
progranmme of work was going according to plan by regularly updating and exchanging emails with
partners.
2.8.2

Task 6.2 Evaluation of results, meeting, reporting and publications

All partners have contributed to the preparation of the reports and mid-term presentation. Seven
coordination meetings have been held every 6-month to allow data exchange and discussions about
the results. At least one meeting was hosted by each of the partners. The list of the coordination
meeting locations and dates are shown below:

August 2010: Kick-off meeting hosted by Sheffield University (UK)

January 2011: 2nd coordination meeting hosted by Tata Steel (UK)
October 2011: 3rd coordination meeting hosted by CSM (Italy)

147

July 2012: 4th coordination meeting hosted by Tata Steel (UK)

January 2013: 5th coordination meeting hosted by Tata Steel (UK)
September 2013: 6th coordination meeting hosted by CSM (Italy)

Separate meetings have also been taking place between partners to organise sampling trails and
plan the experiments in the project. In total, 10 publications were published over the course of the
project. This will be discussed into more details in Section 4 of the report.

2.8.3

Task 6.3 Dedicated workshop organisation

Several attempts were made to organise a workshop towards the end of the ECOWATER project,
however, owing to the difficult financial climate faced by the steel industry it was not possible to
organise it. Indeed, several potential participants were not able to attend and declimed the offer
owing to strict travel restrictions. The transfer of knowledge will be discussed into more details in
Section 3.

148

3.

Conclusions, exploitation and impact of the research results

3.1

Challenges addressed by the ECOWATER project

The first objective of ECOWATER was to address the challenges faced by the steel industry to fulfil
the requirements of the WFD. With the WFD coming into force, tighter environmental quality
standards were introduced for a range of Priority and Priority Hazardous Substances (PS / PHS)
such as trace metals and PAHs in local river basins, however there was a significant gap in the
knowledge across the steel industry on the emissions of these substances in wastewater effluents
from cokemaking and ironmaking / steelmaking operations. Research was required in this area to
identify the main emission sources, determine effluent toxicity, quantify emissions and measure the
environmental impacts of steelworks.
The second aspect of ECOWATER was concerned with the investigation of the potential of state-ofthe-art molecular biology techniques for understanding further biodegradation processes in coke
oven BET plants. In recent years, there has been an explosion in microbial molecular biology
techniques. The environmental sector is well placed to adapt many of these techniques to gain
efficiencies across a range of biological treatment streams. Novel molecular biology techniques
could not only provide the flexibility and adaptability that is required in treatment but could deliver
significant environmental improvements by enhancing the biodegradation capabilities of bacteria
therefore minimising the use of other less cost-effective abatement solutions. The biological effluent
treatment process is well established as BAT for the treatment of coke oven effluents and generally
operates efficiently. Normally, these plants are capable of destroying a wide range of organic and
inorganic compounds that form the major part of the chemical oxygen demand of the effluent.
However, the current state-of-the-art has evolved in an empirical manner and there is still a lack of
in-depth understanding of the fundamentals of the process, which prevents the full potential of
aerobic biological treatment plants from being attained. In the project ECOWATER, it was proposed,
for the first time, to use a range of novel state-of-the-art molecular biology techniques to identify
unculturable microorganisms that plays a major role in the degradation of organic pollutants such as
phenols and PAHs.
The third important aspect of the ECOWATER project was concerned with the development of
innovative technological solutions for the removal of PS and PHS in wastewater effluents from the
steel industry in order to ensure that integrated steelworks comply with the final discharge consent
in the future. In particular, the potential application of a photocatalytic oxidation process using
anatase (TiO2) was studied in the project. Photocatalytic oxidation is an interesting approach for
water treatment because the process gradually breaks down the contaminant molecules and
therefore no sludge requiring disposal to landfill is produced. In parallel, work was carried out at the
pilot scale to investigate the potential of advanced adsorption techniques using sorbents such as
powdered activated carbon (activated lignite) and zeolites which present high adsorption
capabilities, respectively, for the removal of organic and inorganic pollutants. Finally, pilot scale
tests were also carried out to investigate the potential for coke oven effluent treatment of a new
high efficiency filtration technology (Fuzzy filtration), which has only been used in municipal waste
biological effluent treatment plants.
The impact of the ECOWATER project on the EU steelmaking industry in those three particular areas
will be discussed into more details in the next section.

3.2

Impact of the ECOWATER project on the EU steelmaking industry

Analytical developments for chemical characterisation, toxicity testing and impact

measurements
One of the main objectives of the project was the development of novel sampling and analytical
methods for the characterisation of trace metals and PAHs at trace levels in effluents from the steel
industry, and ambient samples such as local rivers. This was achieved in Work Packages 1 and 3.
Analytical procedures for the analysis of PAHs using GC-MS, trace metals using ICP-MS and mercury
using CVAF were developed and validated in the early phase of the project. These techniques
allowed the determination with very high sensitivity (low ppb range) and very high accuracy of the
WFD priority substances in all types of effluents generated at an integrated steeworks. EU
steelmaking companies will be able to benefit directly from the analytical development work carried
out and described in the ECOWATER project.
To assess the toxicity of cokemaking / steelmaking effluents, two approaches were selected. The
first approach consisted in the use of Direct Toxicity Assessment (DTA) tests using living organisms
for the measurement of the EC50. The second approach was concerned with the development and
validation of very sensitive biosensors for the detection of PAHs / n-alkanes constructed using

149

advanced molecular biology techniques. For the DTA approach, the most suitable ecotoxicity tests
were selected for freshwater and seawater (Oyster Embryo-Larval development; Marine algae
inhibition of growth) and applied to cokemaking / steelmaking effluents. Although the DTA tests
provided a good estimation of the EC50 value, they were very expensive and proved extremely
difficult to organise / set-up owing to a range of technical limitations including the need for a
minimum of 3-week period for preparation and growth of the organisms and the necessity of
analysing the samples within 12-h of collection. In contrast, two biosensors were developed for the
detection of PAHs and n-alkanes (ie. oils) in effluents from the steel industry. The PAH biosensor
was constructed by cloning the human cytochrome P450 (CYP) in a soil bacterium (Acinetobacter
baylyi). All PAH compounds are oxidised by CYP which metabolically converts PAHs into carcinogens.
Therefore, the advantage of using CYP was to develop a biosensor that could respond to all PAHs
(including PAH mixtures) rather than having to develop a biosensor for each individual PAH
compound. The alkane bioreporter developed in the project was also constructed through cloning of
the soil bacterium Acinetobacter baylyi. The resulting biosensor showed a unique ability to adhere to
an interface of oil and water and to emulsify mineral and crude oils into oil droplets at the
micrometre level. It also was able to respond to alkanes with various chain lengths from C7 to C36,
the first biosensor reported in environmental microbiology exhibiting such a wide range of detection.
The two biosensors were tested on a range of effluents from the steel industry and showed a very
good response. As opposed to the DTA approach, both biosensors were very easy to use, exhibited
good sensitivity and had the potential for direct in situ measurements with further development. In
the steel industry, these biosensors could be used as a warning system and this could be
advantageous for commercialisation of the biosensor for online and in situ purposes. It is possible
that Sheffield University will patent the PAH biosensor developed in the ECOWATER project in the
near future. These biosensors could also have several applications outside the steel industry in
particular for the environmental detection of oil spills, for instance.
Another significant development in the ECOWATER project was concerned with the use, for the firsttime in the steel industry, of passive sampling techniques such as DGT (Diffusive gradient in thin
films samplers) and SPMD (Semi-permeable membrane devices) for monitoring the concentrations
of trace metals and PAHs in local rivers near the effluent discharge points. As opposed to standard
spot sampling techniques which only yields an instantaneous measurement of pollutant levels and
require the use of expensive electrically-powered equipment, passive samplers are left unattended
in the environment for periods of approximately three-weeks allowing continuous monitoring of
aqueous contaminant levels. As a result, the problem of potentially significant short-term
concentration variations is addressed since only time-weighted average concentrations are
determined using passive samplers. In addition, they often provide a direct measurement of the bioavailable fraction for the pollutants of concern, which is essential to assess the contamination of
local rivers by trace metals or PAHs. The two types of passive samplers were tested during a major
impact study at a UK integrated Works. This campaign showed that the results obtained using DGTs
and SPMDs were useful in assessing the ambient concentrations of a range of trace metals (ie. Ni,
Cd, Pb, Fe, As, Zn, Cr and Cu) and PAHs (including B[a]P) in respect of the EQS proposed in the
WFD. Passive sampling techniques were much easier to implement and less costly overall than
standard spot sampling techniques. The use of these techniques should be promoted across the
steel industry for impact assessement purposes in the future. These sampling techniques for impact
assessment are generic enough to be directly used by other coke plants in order to ensure that they
will be able to comply with the Environmental Quality Standards defined by the Water Framework
Directive.
Source characterisation
A significant number of measurements over a 3-year period were carried out at two major
integrated steelworks in the UK to characterise and quantify the main emission point sources of
trace metals and PAHs in the steel industry.
This work led to identify without ambiguity that cokemaking activities were responsible for >90% of
PAH emissions in wastewater from the steel industry. Interestingly, comparison of the PAH data at
both integrated sites revealed a very high discrepancy between the emissions of the two sites. The
relatively high PAH emissions observed at one of the site were associated with significant instability /
poor performance of the BET plant for treatment of thiocyanate and phenols, in particular. This
resulted in high suspended solid emissions. Since PAHs were mostly particle-bound, high suspended
solid emissions did result in high PAH emissions at this site. It highlighted a priority area for the
improvement of PAH emissions wich would require to keep a perfect control of BET plants for
thiocyanate and phenol treatment in order to maintain low PAH and suspended solid emissions. BET
plants are adopted by the vast majority of EU steel and coke producers, so that there is considerable
investment in this technology throughout Europe. The findings of the Ecowater project should help
ensure that these plants are operated more efficiently in the future to control PAH emissions.

150

With regard to trace metal emissions, the work carried out in the project showed that the three most
significant trace metals emitted in terms of volume from an integrated steelworks were Fe, Zn and
Pb. Ironmaking (BF) and steelmaking (BOS) operations were the main source of Fe (ca. 70%) and
Pb (ca. 90%). Fe, Pb and Zn were almost exclusively particle-bound. Zn was originating from
ironmaking / steelmaking (ca. 60%) and cokemaking (ca. 40%) activities. With regard to Cd and Ni,
Ni was emitted in greated quantities than Cd. The main sources of nickel were ironmaking /
steelmaking (ca. 60%), cokemaking (ca. 10%) and the rolling mills (ca. 10%). Cd sources were
mostly from ironmaking / steelmaking (ca. 74%) and continuous casting (ca. 15%). In both of these
streams, cadmium was mostly found in the dissolved phase. Arsenic was emitted from two major
sources: ironmaking / steelmaking (ca. 50%) and cokemaking BET plants (ca. 30%). Finally,
mercury emissions were very low at all discharge points.
Two complete emissions inventory were realised at major integrated steelworks. This work
constitutes the first source of information across the steel industry concerning the emissions of WFD
substances in effluents of the steelmaking / ironmaking and cokemaking processes. The data
reported in the report (ie. generic emission factors / annual mass releases) could be used by other
steelmakers in Europe to carry out preliminary assessment of their releases of PHS / PS. It will also
be useful to other steelmaking companies to compare their current emissions of WFD substances
and establish benchmark performance in the future.
More effort is required in the future to understand the relatively high variations observed in PAH and
trace metal emissions both in the cokemaking and steelmaking / ironmaking effluents. It is thought
that these emissions are greatly influenced by the quality of the raw materials used (ie. coke, iron
ores) as well as process parameters. Investigating the effects of raw material inputs / process
parameters upon the chemical quality of effluents in the steel industry was outside the scope of the
ECOWATER project, but it is clear that more work is required in this area.
Potential of molecular biology for BET plant biosludge characterisation
One of the most innovative aspects of the project was to investigate the potential of state-of-the-art
molecular biology approaches to study and identify the bacterial communities involved in the
degradation of key pollutants (ie. phenols / PAHs) in coke oven BET plants. With the development of
advanced molecular biology techniques in the field of environmental microbiology over the past few
decades, microorganisms in activated sludge processes can now be studied in a culture independent
manner. This new field of molecular microbial ecology has opened up new possibilities for studying
microbial communities at the genetic level leading to a far greater appreciation of their diversity in
nature. In the project ECOWATER, key technical developments in the field such as 16s rRNA
sequencing, denaturing gradient gel electrophoresis (DGGE) and stable isotope probing (SIP) have
been employed to study and quantify the microbial diversity in coke oven biosludges. In addition, a
completely new approach using magnetic nanoparticles (MNP Magnetic Nanoparticles) to
functionalise living cells and isolate selectively degraders has been developed for the first time in
this project.
First, standard cultivation methods were tested to isolate the cultivable species that were actually
present in coke oven biosludges and involved in phenol and PAH degradation. Although several
bacteria were isolated and identified after sequencing of their DNA, it was very difficult to obtain
quantitative data from this type of analysis, and therefore it was not possible to ascertain which of
the bacteria isolated were predominantly involved in the degradation of phenols or PAHs. Indeed, It
is thought that typically >90% of the bacteria present in an activated sludge are actually
uncultivable. Therefore, although simple cultivation techniques could provide useful qualitative data
between activated sludges, it was found that there were significant limitations to this type of
approaches.
Further analyses were carried out to study and compare the profiles obtained by DGGE analysis of
the 16srRNA isolated after a total DNA extraction of sludge samples. This type of analysis revealed
clear differences between the microbial communities profiles of all the BET plants investigated.
Clearly, each BET plant exhibited a distinct and unique bacterial community fingerprint. From these
profiles, it was clear that the coke oven biosludges of each site were characterised by a great
diversity in bacterial species including some predominant species. Interestingly, the analysis of
several of these biosludges was repeated after several months and the same fingerprint was
obtained indicating the stability of the bacterial communities at each site. However, this technique
did not allow identifying with certainty phenol and PAH degraders, therefore only qualitative data
were obtained. The DGGE method, however, enabled to identify changes in the composition of
microbial communities when a loss of treatment occurred at the BET plant where a clear change of
the bacterial community fingerprint was observed.

151

The most successful technique employed for bacterial identification was a method specifically
developed in this project using MNP. The idea was to use MNP to selectively isolate phenol- and
naphthalene-degrading microorganisms, and to recover live bacterial cells in order to carry out
additional studies such as inhibition tests. The technique developed was called MNP-mediated
isolation (MMI). MMI started with functionalising all the cells from the biosludge with biocompatible
MNP of 183 nm. The efficiency of MNP functionalisation was greater than 99.9%, which ensured
that all cells were initially coated with MNP. In a second step, the MNP-functionalised cells were fed
with both 13C- and 12C-phenol and/or naphthalene as the sole carbon source. In presence of phenol,
the active active phenol degraders present in the sludge started dividing. As a result, the newly
divided cells were not coated with MNP. After several hours, the only cells without MNP were the
cells actively involved in the biodegradation of phenols, and it was possible to selectively isolate
them using a permanent magnet to separate them from the rest of the cells (non-divisive or inactive
cells) which were attracted and immobilised by the magnet. This approach enabled the separation of
live bacterial cells in a complex activated sludge.
In the case of Tata Steel BET plant A, the bacteria isolated in the MNP-free cells of the biosludge
were identified by DNA sequencing. It showed that only one strain was present and that the isolated
bacterium using the MMI approach was Ralstonia spp., belonging to the Burkholderiales order, which
contains over 200 species. The phenol-degrading capability of the MNP-free cells was confirmed by
Raman microspectroscopy and by SIP using pyrosequencing analysis of the 13C-DNA recovered from
the sludge after incubation with 13C-phenol. All these evidence indicated that the key phenol
degrader in the biosludge collected at Tata Steel BET Plant A was an uncultivable Ralstonia spp.,
which was actually completely different from the species isolated using standard cultivation
technique. This result confirmed that standard microbiology techniques based on simple cultivation
techniques were inadequate for identifying key degraders in complex industrial biosludges. In this
case, although some phenol-degraders were isolated using cultivation techniques, these species
were not the most predominant phenol-degraders in the sludge. Advanced techniques using MMI led
to the identification of an uncultivable bacterium as the most predominant phenol-degrading in the
sludge. Further studies were carried out with MNP to identify naphthalene-degrading bacteria and a
series of uncultivable Pseudomonas bacteria were recovered and identified with this technique.
A remarkable advantage of MMI was that it was able to isolate live bacterial cells for ecophysiological analysis. Therefore, it was possible to study the effects of potential chemicals which
could inhibit or promote phenol degradation. This was carried to investigate the potential cause for a
sudden loss of treatment at Tata Steel BET plant A for thiocyanate and sometimes phenol. It was
observed that the treatment often failed when NO2- concentration in the aeration cells was greater
than a threshold of 15 mg/L. It was therefore supposed that nitrite could act as an inhibitor of
phenol / thiocyanate degradation. Inhibition tests carried out on the Ralstonia sp. isolated from the
sludge revealed that nitrite did not inhibit phenol degradation. However, it was found that
hydroxylamine (NH2OH) exhibited one of the most significant inhibition effects with threshold
between 2 and 5 mg/L. Ammonia-oxidizing bacteria responsible for nitrification in activated sludge
first oxidise NH3 to NH2OH, which is then oxidised to nitrite (NO2-) and finally to nitrate (NO3-). The
results of this study suggested that the accumulation of hydroxylamine in the aeration cells, likely
caused by the inefficient conversion of hydroxylamine and nitrite, could be responsible for treatment
failure.
The results obtained in the project ECOWATER showed that the application of advanced molecular
biology techniques for identifying key degraders in coke oven activated sludges and potentially
reveal their controlling factors had huge potential. Techniques such as SIP and MNP, in particular,
were the two most promising methods that could unveil the microbial composition and architecture
of the complex communities involved in the treatment process. BET plants are well established as
best available technology (BAT) for coke oven effluent treatment, accordingly the results obtained in
the project will benefit most European steelmakers. Within the time frame of the project, the
significant amount of time spent on adapting DGGE and SIP techniques and developing a completely
new method (MMI) for analysis of coke oven biosludges meant that the work mostly focused on one
particular site and two types of species (phenol- and naphthalene-degrading bacteria). Clearly, there
is now significant opportunities to apply these techniques to other BET plants in Europe as well as to
identify additional species involved in the degradation of other key compounds such as thiocyanate
for instance.
Innovative abatement technologies for coke oven effluent treatment
Four water treatment applications were investigated at the laboratory and pilot scale in the project
ECOWATER to improve the treatment of coke oven effluents. These were photocatalysis using
anatase (TiO2), zeolite filtration, powdered activated lignite adsorption and fuzzy filtration.

152

Photocatalytic tests using Anatase showed that the best abatement efficiencies for phenols and COD
in coke oven effluents were obtained when combining the anatase powder (Aeroxide) with 1% H2O2.
In parallel, very good adsorption test results were obtained using filtration of coke oven effluents
over a column packed with a naturally-occurring type of zeolite (Mordenite). Accordingly, a pilot
scale plant was built and commissioned by CSM to carry out combined pilot scale tests using
photocatalysis and zeolite filtration. In addition, tests using two columns of zeolite in series were
also carried out. Overall, the best abatement efficiencies (> 80%) for PAHs and phenols were
obtained at the pilot scale using both types of configuration. For most metals, very good abatement
efficiencies were obtained using two zeolite columns for treatment of the coke oven wastewater. In
particular, very high adsorption was observed for lead (ca. 99%) and cadmium (ca. 96%). However,
zeolite adsorption did not perform so well for the abatament of zinc (ca. 25%), manganese (ca.
42%) and chromium (ca. 25%). A cost-benefit analysis of the zeolite filtration technology was
carried out. It was proposed that the technology could be applied post-treatment as an effluent
polishing technique. For PAHs, the cost of abatement was estimated to be 6.45 Euros / g PAHs, and
the expected abatement was >80%.
Activated lignite injection tests were carried out by Tata Steel using a pilot BET plant commissioned
and optimised during the project. Although very good results were obtained during laboratory tests
in terms of colour reduction and trace metal abatement, the tests carried out at the pilot scale were
disappointing overall. The use of activated lignite in the aeration cell (HOK Super) at 400 mg / L
did not result in significant reduction of the colour of the final effluent. PAH emission reduction was
only ca. 10%. With regard to the trace metals, there was no effect of lignite upon the emissions of
Cd and Pb. The only trace metal for which a significant abatement improvement was found was
nickel (ca. 20% improvement). The main benefit of adding activated lignite was a slight
improvement in the settleability of the activated sludge which resulted in slightly lower and less
variable suspended solid emissions. In terms of cost, the cost of abatement was estimated to be 1.0
Euros / g PAHs, but the expected abatement was only 10%. For nickel, the cost of abatement was
2.5 Euros / g nickel with 20% abatement.
Another application tested at the pilot scale in the project was fuzzy filtration, an innovative and
cost-effective compressible media filter, suitable for the removal of suspended solids from waste
water. It has been developed and is currently commercialised worldwide by BOSMAN, a water
treatment company based in the Netherlands. A fuzzy filtration unit is equipped with a compressible
filter medium composed of fuzzy filter balls and is a modular system that is typically used posttreatment of effluent wastewater as a polishing technique. It has been used so far principally at
municipal wastewater treatment plant and oil refineries, therefore the work presented in the project
constitute the first application to cokemaking. The results showed that with the highest compression
rate (47.5%) and a relatively low flow rate of 7 m3 / h, this technology led to significant abatement
of suspended solids (>50%) and PAHs (>50%). It is also possible that the abatement efficiency
could be further improved by adding additional fuzzy filter balls in the filtration tower for the same
compression rate; however it was not possible to carry out these tests during the project. A costbenefit analysis of the technology was carried out using Tata Steel BET plant A with total capacity of
126 m3 / h as a basis for calculations. The results showed that the abatement costs for the first year
were relatively high for PAHs (ca. 21 Euros / g PAHs reduced) and suspended solids (ca. 30 Euros /
kg of suspended solids reduced). From the second year onwards, when considering only the energy
and maintenance costs, the abatement costs using fuzzy filtration were much lower (ca. 1.5 Euros /
g PAHs reduced) and (ca. 2.2 Euros / kg of suspended solids reduced). Therefore, the technology
could potentially be beneficial for treatment of coke oven effluents leading to significant reduction of
PAH and suspended solid emissions (ca. both 50%) in the future.
In this project, the two most promising technologies identified were the fuzzy filtration and the
zeolite filtration (alone or in combination with photocatalysis). The fuzzy filtration is currently the
most advanced and the most mature solution and therefore, building on the experience of
BOSMAN in previous applications (municipal wastewater), fuzzy filters could be installed at coke
oven plants relatively easily. Further work would be needed, however, to evaluate the zeolite
filtration technology and refine the cost analysis.

3.3

Dissemination of the results / possible patents

All of the information regarding the development and the implementation of novel treatment
technologies (such as zeolite and fuzzy filter) can be readily transferable to other European coke
making plants. For each technology, a cost-benefit analysis was carried out to ensure that
technologies were fully evaluated and transferable to other cokemaking plants in Europe. It is
expected that the findings will benefit most European coke plants regardless of their age or

153

individual design since the vast majority of European coke plants are currently using biological
effluent treatment plants for water treatment.
Several publications in peer-reviewed journals and/or international conferences have already been
published during the course of the project and additional publications will be presented at the
forthcoming European Steel Environment and Energy Congress in September 2014 at Teesside
University (UK) to disseminate the research carried out in this project. In addition, Sheffield
University may try to patent at least one of the biosensor developed in this project. All the partners
managed to publish at least one paper during the project. The list of publications so far is
summarised below.
a.

Zhang D., Huang W., 2012. Characterization and modelling of transcriptional crossregulation in Acinetobacter baylyi. ACS Synthetic Biology, 274-283.

b.

Zhang D., Huang W., 2011. Whole-cell bacterial bioreporter for actively searching and
sensing of alkanes and oil spills. Microbial Biotechnology, 87-97.

c.

Zhang D., Berry J., Jiang B., Huang W, 2014. Cultivating the uncultured bacteria in-situ: the
application of magnetic-nanoparticle-mediated isolation (MMI) to recover functional yet
uncultured bacteria from complex environmental microbiota. International Society for
Microbial Ecology (ISME), Accepted for publication March 2014. In press.

d.

Aries, E., Chen, J., Collins, P, Hodges, J., Pearson, S., 2011. Characterisation of Priority
Hazardous Substances and Priority Substances in cokemaking and steelmaking effluents
from UK integrated steelworks. In proceedings of the 2nd International Conference and
Exhibition on Clean Technologies in the Steel Industry, 26-28 September, Budapest.

e.

Chen J, Aries E, Collins P, Hodges J, 2012. Emissions inventory of Priority Hazardous

Substances and Priority Substances from the Water Framework Directive in effluents from
integrated steelworks in the UK. In Proceedings of the 30th Journees Siderurgiques
Internationales (JSI), 18-19 December, Paris.

f.

Webster, S., Aries, E., Huang, W., 2011. Molecular biology approaches to assess PAH
degradation in coke oven biological effluent treatment plants. In Proceedings of the 5th
European Bioremediation Conference, July 4-7, Chania, Crete.

g.

Aries E., Chen J, Collins P, 2013. Project ECOWATER (2010-2013): Enhanced tratement of
coke oven plant wastewater. Article published in the 2013 Coke Oven Manager Association
year book, pp 227-262.

h.

Raper E, Chen J, Sutcliffe A, Aries E, Arnott E, Soares A, Stephenson T, 2014. Studies into
the removal of priority pollutants in cokemaking effluents using a pilot scale biological
activated sludge treatment plant. In Proceedings of the European Steel Environment and
Energy Congress. September 15-17, Teesside.

i.

Chen J, Aries E, Collins P, Sutcliffe A, 2014. Pollution emissions in effluents from an

integrated steelworks in the UK. Journal of Environmental Quality. Submitted February
2014.

j.

Chen J, Aries E, Anderson D, Hodges J, 2014. Water Framework Directive Substances in the
Steel Industry. In Proceedings of the European Steel Environment and Energy Congress.
September 15-17, Teesside.

k.

Pistelli, M.I., Mirabile, D., Beone, T., Serra, M., Zanlucchi, S., 2012. Approccio DOE per
acque di cokeria, ICP (Rivista dell'Industria Chimica) N.11, 2012, pp 68-73.

154

4.

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5.

AA

List of abbreviations

Annual average

BAT-AEL

Best available Technology Associated Emission Limit

B[a]P

Benzo [a] pyrene

BAT

Best available technology

BET

Biological effluent treatment

BF

Blast furnace

BOD

Biological oxygen demand

BOD5

Biological oxygen demand in a 5-day period

BOS

Basic oxygen steelmaking

BREF

Best available techniques reference document

CFD

Computational fluid dynamics

CNS

Thiocyanate

COD

Chemical oxygen demand

CVAF

Cold vapour atomic flourescence

CYP

Cytochrome P450

DGGE

Denaturing gradient gel electrophoresis

DGT

Diffusive gradient in thin films

DNA

Deoxyribonucleic acid

DOE

Design of experiment

DP

Discharge point

DTA

Direct toxicity assessment

EC50

Half maximal effective concentration

EPA

Environmental protection agency

EQS

Environmental quality standard

EU

European Union

EVF

Effective flux volume

FEP

Flourinated-ethylene-propylene

FISH

Fluorescent in situ hybridisation

GC/MS

Gas chromatography -mass spectrometry

GFP

Green fluorescent protein

HPLC

High performance liquid chromatography

ICP/MS

Inductively coupled plasma -mass spectrometry

ICP/OES

Inductively coupled plasma -optical emission spectroscopy

ISO

International organisation for standardisation

LoD

Limit of detection

LS

Liquid steel

LSO

Long sea outfall

MAC

Maximum allowable concentration

MMC

Mitomycin C

MMI

Magnetic nanoparticles-mediated isolation

MNP

Magnetic nanoparticles

MPPE

Macro-porous polymer extraction

PAC

Powdered activated carbon

PACT

Powdered activated carbon treatment

PCA

Photocatalytic activity

PCR

Polymerase chain reaction

PHS

Priority hazard substances

PS

Priority substances

160

PTFE

Polytetraflouroethylene

PW

Pure water

q-PCR

Real-time quantitive polymerase chain reaction

rDNA

Recombinant DNA

RNA

Ribonucleic acid

RSD

Relative standard deviation

SD

Standard deviation

SP

Specific pollutant

SIP

Stable isotope probing

SPE

Solid phase extraction

SPMD

Semi-permeable membrane device

SW

Sea water

THC

Total hydrocarbon

TKN

Total Kjeldahl nitrogen

TSS

Total suspended solids

TWA

Time weighted average

UDS

Unscheduled DNA synthesis

US EPA

United States Environmental Protection Agency

UV

Ultra violet

WFD

Water framework directive

161

6.

Appendix 1
Table 63: Details of the experiments carried out by CSM to investigate possible synergetic /
antagonist effects between trace metals and organic compounds upon toxicity (EC50) measured
using Microtox test.

case

1
2
3
4
5
6
7
8
9
10
11
12
A1
A2
A3
A4
A5
A6
A7
A8

phenol
-mg/l20
20
20
20
10
10
10
10
20
20
10
10
5
5
5
5
1
1
1
1

M1
M2
M3
M4

N1
N2
N3
N4

Synthetic solutions
heavy metals -g/lpH
9
7
9
7
9
7
9
7
7
7
7
7
9
7
9
7
9
7
9
7

50000
10000
10000
50000
10000
50000
50000
10000
50
10
50
10
5
1
1
5
1
5
5
1

1
1
1
1

7
7
7
7

5
5
1
1

5
5
1
1

7
7
7
7

1
1
1
1

pH
8.5
7.1
9.2
7.0
7.8
6.6
8.0
6.9
6.6
6.8
7.0
6.8
8.9
7.0
8.9
7.0
8.9
7.0
8.9
6.9
Fe-g/l5
1
5
1
salicilic
acid
-mg/l5
1
5
1

162

Results
EC50
-5 min1.5
4.9
2.8
4.0
1.7
1.8
0.9
3.4
5.0
11.1
4.2
16.9
24.4
18.6
20.3
19.0
17.4
16.2
18.8
16.4

EC50
-30 min1.2
3.1
1.8
2.5
1.8
1.3
0.6
3.2
n.d.
11.6
n.d.
11.3
13.5
15.5
17.0
14.4
16.5
13.5
14.7
15.0

7.0
69
7.0
7.0

22.3
18.8
16.6
17.3

11.0
15.0
16.4
12.4

7.6
7.7
7.6
7.5

21.6
20.0
18.2
20.1

17.5
15.4
13.4
14.4

164

PHS

PHS

PHS

PHS

PHS

PS

PS

Benzo [b + j] fluoranthene

Benzo [k] fluoranthene

Benzo [a] pyrene

Indeno [1,2,3-cd] pyrene

Benzo [g,h,i] perylene

Naphthalene

Fluoranthene

PHS

PHS

PHS

PHS

PHS

PS

PS

Benzo [b + j] fluoranthene

Benzo [k] fluoranthene

Benzo [a] pyrene

Indeno [1,2,3-cd] pyrene

Benzo [g,h,i] perylene

Naphthalene

Fluoranthene

Total 16 US EPA PAHs

PHS

Anthracene

Total 16 US EPA PAHs

PHS

0.61

0.087

0.065

0.029

0.027

0.030

0.022

0.059

0.007

Mean

0.57

0.062

0.087

0.032

0.023

0.019

0.018

0.053

0.006

Mean

0.34-1.4

0.026-0.226

0.048-0.108

0.006-0.091

0.009-0.075

0.011-0.083

0.009-0.061

0.024-0.154

0.005-0.014

0.62

0.084

0.077

0.028

0.024

0.023

0.020

0.054

0.008

Mean

0.21-1.4

0.013-0.213

0.042-0.163

0.006-0.050

0.004-0.047

0.002-0.046

0.003-0.040

0.004-0.116

0.002-0.020

Range

(n = 8)

Range

(n = 9)

0.25-0.65

0.012-0.062

0.047-0.112

0.006-0.034

0.005-0.029

0.002-0.024

0.003-0.032

0.008-0.076

0.002-0.009

DP9

0.35

0.029

0.087

0.013

0.013

0.010

0.012

0.029

0.004

DP7

0.38-0.77

0.04-0.072

0.057-0.149

0.017-0.043

0.011-0.034

0.01-0.025

0.008-0.024

0.022-0.073

0.004-0.009

Range

(n = 8)
Mean

(n = 16)
Range

DP2

DP1

51.8

4.2

0.38

4.7

4.5

6.1

3.4

10.9

0.16

Mean

0.42

0.054

0.063

0.028

0.021

0.021

0.018

0.041

0.005

Mean

34.6-94.4

2.9-7.0

0.22-0.55

3.2-8.6

2.7-8.0

4.5-10.9

2.0-6.1

7.0-20.3

0.12-0.19

Range

(n = 40)

DP10

0.17-1.0

0.013-0.126

0.041-0.091

0.006-0.081

0.004-0.069

0.002-0.074

0.003-0.053

0.004-0.139

0.002-0.011

Range

(n = 8)

DP3

1.9

0.280

0.099

0.081

0.068

0.102

0.068

0.222

0.046

Mean

0.36

0.033

0.135

0.007

0.004

0.005

0.003

0.004

0.003

Mean

0.16-4.1

0.013-0.592

0.038-0.212

0.006-0.183

0.004-0.152

0.002-0.262

0.003-0.160

0.004-0.547

0.002-0.106

Range

(n = 10)

DP11

0.20-0.73

0.013-0.080

0.045-0.312

0.006-0.007

0.003-0.004

0.002-0.016

0.002-0.003

0.001-0.006

Range

(n = 8)

DP4

0.20

0.019

0.043

0.006

0.004

0.003

0.003

0.008

0.002

Mean

0.63

0.079

0.084

0.036

0.027

0.026

0.025

0.059

0.006

Mean

0.11-0.27

0.010-0.039

0.032-0.058

0.003-0.007

0.002-0.004

0.001-0.005

0.001-0.005

0.002-0.016

0.001-0.003

Range

(n = 8)

DP12

0.25-1.6

0.013-0.222

0.059-0.109

0.006-0.114

0.004-0.087

0.002-0.091

0.006-0.078

0.004-0.204

0.002-0.015

Range

(n = 9)

DP6

Table 64: Mean and range of PAH concentrations (expressed in g / l) in effluents from Tata Steel Plant A between 2010 and 2013.
Effluents from the cokemaking process are highlighted in grey.

Appendix 2

Anthracene

PAHs

7.

166

8.

11.1

92.4

0.63

Mn

Co

2.4

50.6

SP

As

41.6

9.8

123

9.6

0.03

11.0

Al

SP

SP

Cr

Zn

PHS

Hg

SP

PS

Ni

Cu

PS

Pb

SP

0.25

PHS

Cd

Fe

0.90

Co

3.9

313

Mn

290

14.6

2.9

119

14.8

2310

12.9

14.3

29.6

0.33

0.32

3.8

115

91.7

1.8

11.5

2.6

117

23.9

0.05

5.6

0.34

0.02

0.39

52.4

121

331

1.8

16.2

2.8

922

36.9

7.9

6.1

0.10

(n = 2)

(n = 4)

Al

SP

As

SP

Fe

SP

SP

Cr

Zn

PS

Ni

SP

PS

Pb

Cu

PHS

Cd

DP2

DP1

2.3

30.9

111

62.9

2.2

4.4

3.2

116

9.3

0.05

6.7

0.27

0.06

2.7

78.1

116

134.

2.4

49.5

10.5

718

50.7

38.2

2.5

0.13

(n = 2)

DP3

0.62

12.2

58.6

32.3

1.8

5.8

5.1

26.1

10.2

0.05

5.6

0.02

2.0

0.67

39.1

61.6

286

1.9

17.9

6.0

287

11.6

10.8

1.4

2.0

(n = 2)

DP4

0.46

32.0

66.0

436

0.79

11.9

1.3

192

6.1

0.02

6.4

0.45

1.1

0.68

15

76.0

1093

1.1

50.4

7.6

2737

8.9

7.5

6.5

1.2

(n =2)

DP6

2.6

703

4.1

10.7

0.62

7.0

0.65

43.7

0.05

0.03

2.8

0.03

0.03

2.6

756

9.2

75.9

1.6

10.2

0.89

619

1.4

2.8

0.63

0.04

(n = 2)

DP7

0.25

39.5

15.0

6.5

1.8

4.5

1.6

15.4

3.2

2.3

0.20

0.02

0.50

243

23.4

641

2.5

21.1

4.5

1764

8.2

3.4

5.6

0.07

(n = 2)

DP9

1.9

51.7

25.6

224

8.0

18.0

1.0

882

16.8

0.17

5.7

4.8

0.04

2.2

86.3

30.2

4345

9.1

816

2.2

1998

23.2

12.0

9.4

0.08

(n =10)

DP10

0.78

0.41

4.4

34.6

1.2

0.37

0.32

43.7

8.3

0.05

6.4

0.23

0.02

0.84

3.2

4.5

41.4

1.2

0.37

0.35

79.2

12.5

9.0

0.02

0.02

(n = 2)

DP11

0.33

17.5

0.30

2.4

0.27

0.37

0.03

38.3

6.1

0.05

0.46

0.02

0.02

0.40

18.3

0.32

5.6

0.23

0.37

0.06

81.6

13.1

7.9

0.22

0.02

(n=2)

DP12

Table 65: Mean concentrations of trace metals (total and dissolved concentrations; g / l) in effluents from Tata Steel Plant A in 2012.

Appendix 3

Trace metals

Total

Dissolved

9.

Appendix 4

Sampling and analytical procedures used in Work Package 3 Impact studies

Sampling methods
The campaign consisted of four consecutive 21-days sampling periods where replicate passive
samplers (DGTs for trace metals and SPMDs for PAHs) were deployed. In addition, spot samples
were also collected for comparison purposes. With regard to the DGTs, two types were used. The
Chelex DGTs contained a resin made of Chelex and were used to monitor for most trace metals
including Ni, Cr, Pb, Cd, Zn, Fe and Cu. The FeO DGTs contained an iron oxide (ferrihydrite) gel
suitable for trapping specifically As, Sb and V [33]. Table 66 summarises the experimental set-up for
the four sampling campaigns while Fig. 45 shows a typical deployment of DGTs and SPMDs at a Tata
Steel site.
Table 66: Sampling details for the passive samplers monitoring trial at Tata Steel Site A.
Period

Start
date

End
date

Type of
samplers
Chelex DGTs

10/08/11

31/08/11

FeO DGTs
SPMDs
Chelex DGTs

31/08/11

21/09/11

FeO DGTs
SPMDs
Chelex DGTs

21/09/11

13/10/11

13/10/11

02/11/11

FeO DGTs
SPMDs
Chelex DGTs
FeO DGTs
SPMDs

Samples collected
No samples at Loc 4 and 5
Triplicates at Loc 1, 2, 3, 6, 7
No samples at Loc 4 and 5
Triplicates at Loc 1, 2, 3, 6, 7
No samples at Loc 4 and 5
Duplicates at Loc 1, 2, 3, 6, 7
No samples at Loc 1 and 4
Triplicates at Loc 2, 3, 5, 6, 7
No samples at Loc 1 and 4
Triplicates at Loc 2, 3, 5, 6, 7
No samples at Loc 1 and 4
Duplicates at Loc 2, 3, 5, 6, 7
Triplicates at Loc 1, 2, 3, 5, 6, 7
and duplicates at Loc 4
Triplicates at Loc 1, 2, 3, 5, 6, 7
and duplicates at Loc 4
Duplicates at all locations
Triplicates at all locations;
Triplicates at all locations;
Duplicates at all locations

Spot samples

None

At all locations on
21/09/11

At all locations on
21/09/11 and
13/11/11
At all locations on
13/11/11 and
02/11/11

Fig. 45: Deployment of passive samplers (ie. DGTs and SPMDs) at Tata Steel Works to monitor for
the concentrations of PAHs and trace metals in local rivers and internal water streams.

(A)

(B)

Deployment:
- DGTs were ordered in bulk from DGT Research (Lancaster, UK) and kept in the fridge
until transport to the site. During transport, and until their deployment, the samplers were
kept in cool boxes.
- SPMDs were prepared by NLS (Llanelli, UK) and delivered the day before sampling and
kept in the fridge until transport. For time of the transport to site, and until their
deployment, the samplers were kept in cool boxes.

168

- Passive samplers were contained in dedicated holders and deployed using ropes. They
were then secured to a fixing point and a weight was used to ensure that the samples
remained under water at all times.
- For spot samples, about 1L of water was collected using a bucket and placed in PTFE
sampling bottles.

Retrieval:
Upon retrieval, samplers were transported back to the laboratories in cool boxes where they were
stored in fridges until (i) in-house recovery and analysis of trace metals on DGTs, (ii) dispatch
to
NLS for the analysis of PAHs on SPMDs, and (iii) in-house analysis of the spot samples for trace
metals and PAHs on both the dissolved and total phases.
Temperature of the sampled water was measured at both the times of deployment and collection.
Analytical methods and calculations

SPMDs

Recovery of the PAHs from the SPMDs was carried out by NLS (Llanelli, UK) and consisted of a
dialytic extraction step into an organic solvent such as n-hexane, followed by an additional clean-up
sample preparation step using gel permeation chromatography, prior to GC/MS analysis. Field
blanks were also collected and analysed following the same procedure.

DGTs

DGT sample preparation and analysis was carried out by Tata Steel following the guidelines of the
DGT manual provided by Lancaster University [34]. It consisted in dismantling the DGT and
collecting the binding resin using a pair of tweezers. The resin was then placed in a 1.5 mL vial to
which 1mL of 1M HNO3 was added. The solution was then left overnight to elute and diluted 10
times in 1% HNO3 prior to ICP-MS analysis. Concentration of an element in the water sampled
(CDGT) was determined using the following set of equations:
CDGT = M g / (D t A)
Where:

M is the mass of metal accumulated on the resin (See Equation 2)

g is the thickness of the diffusive gel and filter (0.93 mm)
D is the diffusion coefficient of the metal in gel
t is the deployment time
A is the exposure area (3.14 cm2)

M = Ce (VHNO3 + Vgel) / fe
Where:

Equation 1

Equation 2

Ce is the metal concentration of the eluate (in 1M HNO3)

VHNO3 is the volume of HNO3 added to the resin (1 mL)
Vgel is the volume of the resin gel (0.15 mL)
fe is the elution factor of the metal (typically 0.8)

Field blanks were also collected and analysed following the same procedure.

Spot samples

PAHs in spot samples were determined using Solid Phase Extraction (SPE) followed by analysis by
Gas Chromatography - Mass Spectrometry (GC-MS) as detailed in WP1 (Task 1.3). Briefly, effluent
samples were filtered to remove the suspended particulate matter. The filtrate was subsequently
extracted using solid phase extraction to extract the PAHs present in the dissolved phase while the
filters and residue from the initial filtration was extracted using accelerated solvent extraction (ASE)
to extract PAHs bound to the suspended particulate matter. At this stage, the filtered phase can be
either analysed directly for the determination of dissolved PAHs, or combined to the ASE extract and
analysed to determine the total PAH concentration in the samples. Quantification was achieved by
GC-MS using a set of surrogate, internal and recovery deuterated PAH standards.
Trace metals in spot samples were determined by inductively coupled plasma mass spectrometry
(ICP-MS) as detailed in WP1 (Task 1.3). Briefly, for determination of the dissolved concentration of
trace metals, samples were filtered through a 0.45 m pore diameter membrane filter, pre-digested
on a hotplate/hotblock using nitric acid at 90C during 30 min, and digested with nitric acid using a
microwave instrument. For determination of the total concentration of trace metals, water samples
were pre-digested without filtration on a hotplate/hotblock using nitric acid at 90C during 30 min.

169

Subsequently, samples were digested with nitric acid using a using a microwave instrument. All
samples were analysed using an Agilent 7500cx ICP-MS equipped with an Octopole Reaction
System.
Results from the dissolved phase spot samples are expected to be relatively close to the passive
sampling techniques since passive samplers only collect the bioavailable / labile species.
10.

Appendix 5

Details of all the molecular microbiological methods used in Work Package 4 - Molecular
biology characterisation of coke oven biosludges
Sample Collection
In the first phase of the project, activated sludge samples were obtained from all four European Tata
Steel sites (A, B, C and D) for qualititative analysis and comparison. In the second phase of the
project, work focused on identifying the microbial composition of Tata Steel BET plant A specifically.
Usually, 1L sample was collected from the active return biosludge, in sterilised containers and stored
at 4C prior to analysis (ie. DNA extraction). Repeat samples were taken on a recurring basis every
3 4 months.
Standard culture of isolates
Activated sludge (100 l) from each site was spread onto MMA (Minimal Media Agar) plates (2.5g
Na2HPO4, 2.5g KH2PO4, 1.0g NH4Cl, 0.1g MgSO4.7H2O, 10l saturated CaCl2 solution, 10l saturated
FeSO4 solution, 1ml Bauchop & Elsden solution and solidified with 1.3% noble agar in 1L H2O. The
various compounds tested including phenol (250 mg / L), naphthalene (single crystal) and
anthracene (100 l of 2.7 mM) were added as sole carbon sources for isolation and identification of
the cultivable organisms. Plates were incubated at 25oC in the dark for about 2 weeks. Single
colonies were picked from positive plates and streaked onto fresh MMA plates using the same carbon
source in order to purify the isolates. In the case of anthracene which was insoluble in water, the
compound was dissolved in n-hexane first at the above concentrations, then spread on the surface
of the agar the plates and left in the fume hood in order for the n-hexane to evaporate.
DNA extractions
Briefly, 30ml of activated sludge was centrifuged at 3000 rpm to separate solid and aqueous phases.
0.5g of sludge was added to a BIO-101 tube (Q-biogene). 1ml DNA extraction buffer (100mM Tris
HCl, 100mM sodium EDTA, 100mM phosphate buffer, 1.5M NaCl and 1% CTAB) was added to each
tube and samples were incubated at 65C for 30 mins. Cells were then lysed using a Fast-prep
beadbeater (30s at 5.5). Samples were cooled on ice before centrifugation at 14 000 rpm. Aqueous
phase was extracted and mixed with an equal volume of chloroform:isoamyl alcohol. After a final
centrifugation, the aqueous phase was extracted and DNA was precipitated by adding isopropanol
and incubating at room temperature for 2 hours. The DNA was then pelleted by centrifugation at
14000 rpm, the supernatant was removed and the pellet was washed twice with 70% ethanol. After
a final centrifugation step, DNA pellets were air dried for 5 minutes and then re-suspended in 50l
nuclease free water. 4l extracted DNA was mixed with 1l 5x loading buffer and electrophoresed
on a 1% agarose gel at 85V for 20 minutes to check concentration and quality.
PCR amplification of 16s rDNA
The V3 region of the 16s rRNA gene was amplified by PCR using the primer pairs GC338F and 530R
as described previously [48]. Each 50l PCR reaction contained 29 l water, 5l Dream Taq buffer
(Fermentas), 5 l dNTPs (Sigma), 5 l GC338F, 5 l 530R, 0.5 l BSA (NEB), 0.5 l Dream Taq
polymerase (Fermentas) and 3 l total community biosludge DNA extract. Samples were amplified in
a BioRad C1000 thermal cycler running the following program: 2 min 95C followed by 35 cycles of
95C 1 min, 60C 1 min, 72C 2 min, then 72C for 30 min. Successful PCR products were detected
on a 1% agarose gel as a 220 bp product.
DGGE analysis
16s rDNA PCR products were separated on 10% (w/v) polyacrylamide gels with a 30-60%
formamide / urea (Severn Biotech) denaturing gradient. Gels were electrophoresed at 60C at a
constant voltage of 85V for 16h overnight. Gels were then stained with Sybr Gold for 30 mins and
washed with UHQ water prior to imaging in a BioRad Versadoc system. See appendix for
polyacrylamide gel recipe. Bands of interest were excised from the gel, dissolved in 10l water
overnight at 4C and re-amplified using the above protocol.

170

Preliminary assay for determining 13C incorporation of phenol prior to SIP experiments
250ml of biosludge in sealed 500ml conical flasks were incubated at 30C with shaking over a 10h
time period. At time 0, each incubation was spiked with 1ml phenol (75mg/ml), giving a working
concentration of 300g/ml. 2ml of biosludge was taken prior to phenol addition and every hour
thereafter. Samples were taken using a 5ml syringe to pierce the suba-seal stoppers, thus
preventing contamination or excessive phenol loss. Each 2ml sludge sample was centrifuged at
13000xg and supernatants were used for the phenol assay. Phenol concentrations were assayed as
described previously [41]. Briefly, supernatants were diluted 1/100 with UHQ water then 100l
aliquots were treated with 2.5l 0.5M NH4 OH, adjusted to pH 7.9 with 22.5l phosphate buffer.
Then, 10l of 14-aminoantipyrine at 100mM was added followed by 10l 250mM potassium
ferricyanide. Samples were left to react for around 10 minutes and the colour change was measured
spectophotometrically at 500nm. Phenol concentrations could then be calculated from standard
calibration curves prepared in parallel.
Stable Isotope Probing experiments
C-DNA was separated by equilibrium density gradient centrifugation using a protocol described by
Neufeld et al. [49]. Briefly, 1 g of extracted DNA was mixed with a CsCl gradient buffer to a
volume of 1.2 mL, combined with 4.6 mL of 7.163 M CsCl solution and inverted gently. The mixture
was then transferred into 5.8-mL ultracentrifuge tube (Beckman) with an average density of 1.729
g/mL. After balancing and sealing, the tubes were spun in an ultracentrifuge (Optima L-80 XP,
Beckman Coulter) at 44,100 rpm (~177,000 g, VTi65.2 rotor, Beckman) for 40 hours. The DNA of
different density was retrieved by gradient collection into 12 fractions of 400 L volume from the
bottom of the ultracentrifuge tube. The injection of deionized water was manipulated from the top
of the ultracentrifuge by a low-flow peristaltic pump (Watson Marlow Ltd.). The DNA fractions were
then purified by glycogen and PEG 6000 solution for 2 hours, washed with 70% ethanol, air-dried
and dissolved in 50 L DNase-free water. The DNA from the 12C phenol control was manipulated
with the same centrifugation, fractionation and purification procedures.
13

Raman micro-spectroscopy procedure

Raman micro-spectroscopy was used to confirm incorporation of 13C into the cells DNA durng SIP
experiments. Briefly, cells were harvested from the biosludge supernatants by centrifugation at 3500
rpm for 10 minutes, and were washed with water three times. Each cell suspension (2~5 L) was
spread onto a calcium fluoride (CaF2) slide and allowed to air dry prior to Raman analysis. Single
cell Raman spectra (SCRS) were acquired using a confocal Raman microscope (LabRAM HR, HORIBA
Scientific, UK). A 100 magnifying dry objective (NA=0.90, Olympus, UK) was used to observe and
acquire Raman signals from single cells. The Raman scattering was excited with a 532-nm Nd:YAG
laser (Torus Laser, Laser Quantum, UK). The laser power on a single cell was about 15 mW. Each
Raman spectrum was acquired between the range 1989 cm-1 and 336 cm-1, with 1021 data points
and resolution of ~1 cm-1. LabSpec software (HORIBA Scientific, UK) was used to control the
Raman system and acquire Raman spectra. Acquisition time was 20 s for each measurement of
single cells.
Nucleotide sequencing and computational analysis
PCR amplicon libraries of the small subunit ribosomal (16S) RNA gene V1-V3 hypervariable region
(Escherichia coli positions 5-534) were generated for each individual sample. PCR was performed
using the forward primer (NNNNNNN-TGGAGAGTTTGATCCTGGCTCAG) and the reverse primer
(NNNNNNN-TACCGCGGCTGCTGGCAC). Unique hepta-nucleotide sequences (seven bases) were
synthesized at the 5 end of each pair of primers as barcodes, which helped to assign sequences to
different samples. Pyrosequencing of the 16S PCR-amplicons was carried out using a Genome
Sequencer FLX Titanium platform (Roche, USA) where, on average, 400 bp-long reads were
produced.
With regard to the quality filtering of the raw data, we discarded sequences < 150 bp or > 1,000 bp,
sequences that had an average quality score < 35 in each 50-bp window rolling along the whole
read and sequences containing primer mismatches, uncorrectable barcodes, ambiguous bases, or
homopolymer runs in excess of 8 bases. The sequences that passed the quality filters were then
analyzed using the MOTHUR software package [50]. Sequences were assigned to operational
taxonomic units with a 97% threshold of pairwise identity, and then classified taxonomically using
the Greengenes16S rDNA reference database and a confidence threshold of 80% [51]. The
Greengenes taxonomies were used to generate summaries of the taxonomic distributions at
different levels. To quantify the content of Ralstonia sp. in the MNP-free suspension, all the 454
sequences were aligned against the full-length 16s rRNA sequence of Ralstonia sp. as the reference
(accession number: KJ174596) using BLASTN (e<10-30, identity>88%). DNA sequences of 16SrRNA and phenol hydroxylase from this study are available in Genbank (accession number from
KJ174591 to KJ174607).

171

PCR amplification of 16S rRNA and phenol hydroxylase genes

The PCR amplification was carried out in a C1000 thermal cycler (Thermo, USA). Each reaction (50
L) contained 0.5 L Dream Taq DNA polymerase (Promega, USA), 5 L deoxynucleoside
triphosphates at a concentration of 10 mM, 2.5 L of each primer, 1 L DNA template and 38.5 L
water. DNA fragments of 16s rRNA genes were amplified by the primer pair of 63f and 1387r, as
listed in Table 67.
Table 67: Primers used for amplification of 16s rRNA and phenol hydroxylase genes.
Primer

Sequence (5-3)

Reference

63f

CAGGCCTAACACATGCAAGTC

[52]

1387r

GGGCGGWGTGTACAAGGC

[52]

phe1f

GA(G/A)GGCATCAA(A/G)AT(C/T)

[53]

phe3r

CAG(C/G)CG(A/G)T(A/T)ACC(G/T)CGCCAGAACC

[53]

pheUf

CCAGG(C/G)(C/G/T)GA(G/A)AA(A/G)GAGA(A/G)G
AA(G/A)CT

[53]

pheLr

GG(A/G/C)A(G/T/C)(A/G)TTG(C/T)CCGGGTC

[53]

pheMHr

GAT(T/C/G)GGCAC(A/G)TTGTCTTC

[53]

pheHr

GTGGCCATGTCGCCATTGA

[53]

After activation at 94C for 10 minutes, 35 cycles were undertaken with 94C for 1 minute, 56C for
1 minute, and 72C for 1 minute, followed by final extension at 72C for 10 minutes. The largest
subunit of multicomponent phenol hydroxylases (LmPH) was amplified with the primer pair of
phe1f/phe3r (Table 1) as follows: 10 minutes activation at 94C; 35 cycles of 94C for 1 minute,
56C for 1 minute, and 72C for 1.5 minutes; final extension at 72C for 10 minutes. The PCR
program for the primer pheUf/pheMHr and pheUf/pheHr was as follows: activation at 94C for 10
minutes; 5 cycles of 94C for 1 minute, 58C for 1 minute, and 72C for 1 minute; 5 cycles of 94C
for 1 minute, 57C for 1 minute, and 72C for 1 minute; 25 cycles of 94C for 1 minute, 56C for 1
minute, and 72C for 1 minute; 72C for 10 minutes as final extension. For the primer set
pheUf/pheLr, the DNA was amplified as: activation at 94C for 10 minutes; 5 cycles of 94C for 1
minute, 55C for 1 minute, and 72C for 1 minute; 5 cycles of 94C for 1 minute, 54C for 1
minute, and 72C for 1 minute; 25 cycles of 94C for 1 minute, 53C for 1 minute, and 72C for 1
minute; 72C for 10 minutes as final extension. The PCR products were checked by electrophoresis
with 1.5% (wt/vol) agarose gel (Sigma) in TBE buffer.
MNPs synthesis and functionalisation
MNPs synthesis was carried out as previously described [54]. Briefly, 1 mL of FeCl2 (1.0 M) and 2
mL of FeCl3 (2.0 M) were mixed, to which 25 mL of NaOH (2.0 M) was slowly added drop by drop,
with constant vortex mixing, for 30 minutes. The synthesized MNPs were harvested by a permanent
magnet and the supernatant was replaced with deionized water of the same volume. This washing
step was repeated 6 times until the pH was neutral. Five mL of MNPs were then mixed with 45 mL of
poly(allylamine hydrochloride) (PAAH) solution (10 mg/mL) and stabilized for 60 minutes in an
ultrasound bath with 40-kHz and an output energy of 75 W. After centrifugation at 10,000 g for 10
minutes, the pellet was re-suspended in 50 mL deionized water and dispersed well by vortex. The
final solution was passed through a 0.2 m filter and was then ready for bacterial functionalisation.
For the experiments carried out with coke oven biosludges, ten mL of biosludge was centrifuged at
3000 rpm for 10 minutes and the bacterial pellet was harvested and resuspended in the same
volume of deionized water. The cell suspension was mixed with 10 mL of PAAH-stabilized MNP
solution. The bacteria-MNP mixture was incubated at room temperature for 20 minutes with 150
rpm shaking. The MNP functionalised bacteria were then separated from the aqueous phase by a
permanent magnet, followed by resuspension in deionized water. The washing step was repeated
three times to remove bacteria that were not functionalised by MNPs. Finally, the MNP functionalised
bacteria were resuspended in 10-ml filter-sterilised wastewater. To prepare filtered-sterilised
wastewater, the sludge was centrifuged at 4000 rpm for 10 minutes and the supernatant was
passed through 0.2 m filters twice to remove cells.

172

MNPs mediated cell isolation and counting

After completion of phenol and / or naphthalene degradation, the inactive bacteria were immobilised
by a permanent magnet, which allowed for the recovery of MNP-free cells in the supernatant. These
MNP-free cells (MFC) should be the active and divided cells in the microbial community that were
involved in phenol / naphthalene degradation. The cells in the suspension were stained by 4,6diamidino-5-phenyl-indole (DAPI) (Kubista et al 1987) and counted under a Zeiss Axioplan 2
epifluorescence microscope. The MFC in the suspension were centrifuged at 3000 rpm for 10
minutes and resuspended in the same volume of phosphate-buffered saline (PBS, 54.44 mg KH2PO4
and 106.8 mg Na2HPO412H2O in 10 mL deionized water). To enumerate the population of the MNPfree cells, 20 L of the cell suspension was diluted to 1 mL with autoclaved UHQ water, buffered with
PBS. This dilution was then incubated in the dark with DAPI stain, corresponding to a working
concentration of 12.5 g/mL, filtered onto a 0.2 mm black polycarbonate filter paper and mounted
onto a glass slide with Fluoroshield mounting medium (Sigma). The resultant slides were analysed
and imaged under fluorescent light using the microscope with a DAPI filter cube. The cells were
detected by using 358-nm UV light for excitation and 461 nm for emission. In order to determine
the cell density of the supernatant, cell counts were performed for 15 randomly chosen fields of
view.
In-situ 13C-phenol degradation
Fully 13C6-labelled phenol was added to 25 mL of both the original and the MNP-functionalised
biosludge samples to a final concentration of 250 mg/L. Normal 12C phenol was added to a sample of
the original biosludge as a control and 13C/12C phenol in deionized water was used as a blank
control. Three replicates were carried out for each treatment. Samples were taken every 30 minutes
from the incubations to determine the residual phenol concentrations. Phenol concentration was
measured by a spectrophotometric method described by the American Public Health Association
(Greenberg et al 2005). Briefly, 100 L of cell-free sample was diluted in 900 L deionized water,
dosed in the following order with 400 L of 2.0 M NH4OH, 200 L of 2% (w/w) aminoantipyrine and
400 L of 2% (w/w) K3Fe(CN)6. The absorbance of the mixture was then measured at 500 nm
wavelength using a microplate reader.

173

List of Figures
Fig. 1: Process flow diagram of Tata Steel BET plant A. ..........................................................................................17
Fig. 2: Picture of the aeration basins at Tata Steel BET plant A during oxygen injection in the treatment cells using
the Vitox system. ......................................................................................................................................................18
Fig. 3: Lay-out of Tata Steel BET plant B. ................................................................................................................18
Fig. 4: Schematic of the Carrousel 2000 system waste water treatment plant at Tata Steel BET plant C. ............... 19
Fig. 5: Photograph of the Aquacell automatic composite sampler used in the project ECOWATER for sampling
coke oven waste water effluents...............................................................................................................................21
Fig. 6: Photograph of a DGT and schematic of a section through the DGT assembly. ............................................22
Fig. 7: Schematic of a Semi Permeable Membrane Device for passive sampling of organic contaminants in ambient
freshwaters. ..............................................................................................................................................................23
Fig. 8: Analysis of dissolved and particle-bound PAHs in cokemaking and steelmaking effluents. ..........................24
Fig. 9: Validation data for the analytical procedure for PAHs in cokemaking /steelmaking effluents. The graph
compares the mean concentrations of the 16 US EPA PAHs (N = 12 replicate analysis) with the certified values. . 24
Fig. 10: Schematic of the Agilent 7500cx ICP/MS used by Tata Steel for the analysis of trace metals in
steelmaking effluents. ...............................................................................................................................................26
Fig. 11: Principles of whole cell biosensors. .............................................................................................................28
Fig. 12: Specific / non specific biosensors for the detection of contaminants and toxicity. (A) toluene biosensor
quantitatively detected toluene in water. (B) Toxicity biosensor detected DNA damage caused by a mutagen
(Mytomycin C : MMC) or UV light. ............................................................................................................................29
Fig. 13: Use of human cytochrome P450 activation of PAHs into carconogens (epoxides) for construction of a PAH
biosensor using Acinetobacter baylyi ADP1_recA_lux system. ................................................................................30
Fig. 14: Detection of pyrene by the PAH toxicity biosensor Acinetobacter ADP1_P450_lux. Mitomycin C (MMC)
was used as a positive control. .................................................................................................................................30
Fig. 15: ADWPH_recA_sal450 and ADPWH-recA response to genotoxic compounds (pyrene and MMC) at three
different temperatures as a function of the cell culture time......................................................................................31
Fig. 16: ADWPH_recA_sal450 and ADPWH-recA response to genotoxic compounds (pyrene, B[a]P, Naphthalene
and MMC) at 30 as a function of the cell culture time..............................................................................................32
Fig. 17: A. Schematic outline of construction of alkane bioreporter Acinetobacter baylyi ADPWH_alk (DNA lengths
are not scaled). .........................................................................................................................................................33
Fig. 18: Genetic structure of alkane regulation part of ADPWH_alk. There are three mutation points at promoter
region of alkRM. The mutation points are highlighted in bold. The inverted repeats with two mismatches are marked
by arrows under the sequence. ................................................................................................................................34
Fig. 19: Dynamic ADPWH_alk response to n-alkanes with 100 mM of each compound. .........................................34
Fig. 20: Dynamic ADPWH_alk response to 100 mg / L Brent crude oil in pure water (PW) and seawater (SW). .....35
Fig. 21: Response of ADPWH_alk and ADPWH-lux to 100 M of n-alkanes ranging from n-hexane (C6) to
tetracontane (C40). ...................................................................................................................................................35
Fig. 22: Response of ADPWH_alk (induction ratios) to 100 M of n-alkanes /n-alkenes ranging from n-hexane to
tetracontane (C40). ...................................................................................................................................................36
Fig. 23: Acinetobacter baylyi ADPWH_alk natural affinity to oil droplets. Unlike E. coli which was unable to emulsify
and attach to oil droplets (A), ADPWH_alk, a subclone from ADP1, was able to emulsify and attached to the
surface of mineral oil (B, C) and crude oil (D) droplets. The scale bar is 5 mm. ....................................................... 37
Fig. 24: Bioluminescens bacteria (Vibrio fischeri) .....................................................................................................38
Fig. 25: Interpretation of the possible interaction between two factors using two levels. ..........................................39
Fig. 26: Comparison of the effects - single and combined parameters .....................................................................40
(Case 1: phenols and heavy metals at high concentrations, pH) ..............................................................................40
Fig. 27: Comparison of the effects - single and combined parameters .....................................................................40
(Case 2: phenols at high concentration and heavy metals at low concentration) .....................................................40
Fig. 28: Comparison of the effects - single and combined parameters .....................................................................40
(Case 3: pH, phenols and heavy metals at low concentration) .................................................................................40
Fig. 29: Comparison of the effects - single and combined parameters .....................................................................41
(Case 4: iron and heavy metals at low concentration) ..............................................................................................41
Fig. 30: Comparison of the effects - single and combined parameters .....................................................................41
(5case: phenols and salicilic acid at low concentration) ..........................................................................................41
Fig. 31: Comparison between real (x-axes) and estimated (y-axes) data for the variable EC50(30 min) ..................43
Fig. 32: Percentage of various trace metals found in the dissolved phase in the untreated coke oven liquor at Tata
Steel BET Plant A. ....................................................................................................................................................44
Fig. 33: Percentage of individual PAHs found in the dissolved and suspended particulate matter phase in the
untreated coke oven liquor at Tata Steel BET plant A. .............................................................................................44
Fig. 34: PAH concentrations in the untreated coke oven liquor and the treated effluents .........................................45
(Clarifiers 1 and 3, tidal reservoir) at Tata Steel BET plant A. ..................................................................................45
Fig. 35: Trace concentrations in the untreated coke oven liquor and the treated effluents .......................................46
(clarifiers 1 and 3, tidal reservoir) at Tata Steel BET plant A. ...................................................................................46
Fig. 36: Percentage abatement of trace metals at Tata Steel BET plant A. ..............................................................47
Fig. 37: Percentage abatement of PAHs at Tata Steel BET plant B. ........................................................................48
Fig. 38: Trace metal concentrations (ie. Al, Fe, Zn, Cr, Co, Ni, Cu, As, Cd and Pb) in untreated coke oven liquor
and the treated coke oven effluent at Tata Steel BET plant B. .................................................................................48

174

Fig. 39: Sum of the individual PAH emission concentrations at Tata Steel BET plant A for the 6 PAHs identified in
the BREF document with a BAT-AEL value of 50 g / l ( 6 PAHs). ........................................................................ 51
Fig. 40: Total and dissolved concentrations of Cd, Pb, Ni (c), Fe and As in effluents from Tata Steel Plant A in
2012..........................................................................................................................................................................52
Fig. 41: Percent of trace metals found in the dissolved phase in the combined final ................................................54
effluent (long sea outfall) of Tata Steel Plant B.........................................................................................................54
Fig. 42: Picture of organisms used in Direct Toxicity Assessment tests of industrial effluents for freshwater or
marine waters in the UK. (A) Daphnia Magna : 0.5 to 0.5 mm in length ; (B) Pseudokirchneriella subcapitata
freshwater algae ; (C) Tisbe battagliai and (D) Skeletonema costatum marine algae (size:43 70 m)..................55
Fig 43: Percent contribution of each effluent discharge point to the total trace metals emissions in 2011 at Tata
Steel Plant A. Metals considered are Cd, Pb, Ni, Cr, Fe, Cu, Zn and As..................................................................63
Fig. 44: Map of passive sampling locations at Tata Steel Plant A showing the sampling locations on (A) the
steelworks, and (B) the river. ....................................................................................................................................67
Fig. 46: Average concentrations of PS, PHS and specific pollutants in water bodies around Tata Steelworks A with
th
nd
respect to the Environmental Quality Standard (EQS) between 10 August - 2 November 2011. The error bars
represent the standard deviation between the four consecutive sampling periods. ..................................................69
Fig. 47: Mean concentrations of PAHs in water bodies around Tata Steelworks A, with respect to the Environmental
th
nd
Quality Standard (EQS), between the 10 August and the 2 November 2011. The error bars represent the
standard deviation between the four consecutive sampling periods. ........................................................................71
Fig. 48: Wastewater discharge points (green) and sampling points (yellow) near a coke oven plant in Italy. ........... 72
Fig. 49: PAHs in seawater ........................................................................................................................................73
Fig. 50: PAHs in sediments ......................................................................................................................................73
Fig. 51: Picture of the sediment samples collected in August 2012 (left) and April 2013 (right). ..............................73
Fig. 52: Group profile of PAHs in the sediments. 1, 2 and 3 represent respectively the data obtained in August
2012, April 2013 and July 2013. ...............................................................................................................................74
Fig. 53: Cross plot of the Phenanthrene/Anthracene versus Fluranthene/Pyrene ratios for source characterisation
of PAHs in marine sediments. ..................................................................................................................................74
Fig. 54: Results from genotoxicity tests carried out using the biosensor ADPWH_recA_SAL450 on discharge
effluent samples from cokemaking steelmaking operations and river samples near the final discharge point of Tata
Steel Plant A. ............................................................................................................................................................75
Fig. 55: Acinetobacter baylyi ADPWH_alk response to different concentrations of mineral oil and Brent, Chestnut
and Sirri crude oils in pure water and seawater. .......................................................................................................76
Fig. 56: Calibration curve for ADPWH_alkresponse to crude oil in soils. ..................................................................76
Fig. 57: Protein synthesis in ribosomes and role of 16S rRNA. ................................................................................82
Fig. 58: Typical molecular biology workflow to carry out a phylogenetic analysis of an environmental sample using
the 16s rRNA. ...........................................................................................................................................................83
Fig. 59: Stable Isotope Probing (SIP) for the identification of uncultivable microorganisms involved in the
degradation of organic contaminants in activated sludge systems. ..........................................................................84
Fig. 60: TEM pictures showing the functionalisation of a living bacterium (ie. Acinetobacter Baylyi ADP1) using 18
nm Fe2O3 MNPs (A and B, and picture showing how the bacterial cells could be spatially controlled by an external
magnetic field as a result of the magnetic funtionalisation (C). .................................................................................85
Fig. 61: Culture of 100L of the biosludge from Tata Steel BET plant C on minimal media agar supplemented with
phenol as a carbon source. Control plate contained no phenol. ...............................................................................85
Fig. 62: Phenol-degrading bacterial colonies in pure culture after re-streaking a single colony from the plates in Fig.
61. The control plate contained no phenol. ...............................................................................................................86
Fig. 63: Naphthalene-degrading bacterial colonies from Tata Steel Plant A in pure culture on minimal media agar
plates. The control plate contained no naphthalene. ................................................................................................86
Fig. 64: Growth of anthracene-degrading bacteria on MMA plates supplemented with anthracene from a culture
realised using an activated biosludge from Tata Steel Plant A. ................................................................................86
Fig. 65: Results from a DNA extraction of a biosludge from Tata Steel BET plant A. Lane 1: 5 l hypperladder used
to determine the size of DNA fragments; Lanes 5-8: Replicate DNA extractions of the activated sludge.................88
Fig. 66: Results from 16S-rDNA PCR of total DNA extracted from coke oven activated sludges. Lane 1: 5 l
hypperladder used to determine the size of DNA fragments; Lane 2: negative control; Lanes 3-5: Tata Steel BET
plant B; Lanes 6-8: Tata Steel BET plant C. .............................................................................................................88
Fig. 67: DGGE analysis of 16s rDNA products from biosludge samples of Tata Steel BET Plant A (lanes 1, 2 and
3), Tata Steel BET Plant C (Lanes 5, 6 and 7), CAS plant (lanes 8, 9 and 10) and Tata Steel BET Plant B (lanes
12, 13 and 14). .........................................................................................................................................................89
Fig. 68: DGGE analysis of biosludges from Tata Steel BET plants A and D. Lanes 1-3: Former Tata Steel BET
plant D (West stream); Lane 4: Blank; Lanes 5-7: Former Tata Steel BET plant D (East stream); Lane 9: Blank;
Lanes 10-13: Tata Steel BET plant A. ......................................................................................................................90
Fig. 69: DGGE analysis of biosludges from the former Tata Steel BET plant D. Lane 3: West stream during loss of
thiocyanate treatment; Lane 4: West stream before loss of treatment; Lane 5: East stream during loss of
thiocyanate treatment; Lane 6: East stream after loss of treatment. Lanes 1 and 2: BET plant E (External
cokemaking plant operating in South Yorkshire). .....................................................................................................91
Fig.70: Principle of the MNP-Mediated Isolation (MMI) technique developed in ECOWATER to isolate selectively
phenol- and naphthalene degrading micrororganisms for active biosludges. ...........................................................93
13
12
Fig. 71: Phenol degradation curves ( C and C) of the raw biosludges (G-BS and P-BS) and the MNP
functionalised biosludges..........................................................................................................................................94

175

Fig. 72: Phenol degradation curves obtained with the MNP-free cells isolated from the G-GS biosludge and the raw
G-BS biosluge indicating that the MNP-free cells exhibited the same phenol degradation characteristics as the raw
biosludge. .................................................................................................................................................................94
13
Fig. 73: Raman microspectroscopic analysis confirming that 78.7% of cells were C-labelled in MFCs from
13
biosludge with C-phenol as carbon source. (From Zhang et al 2014) ....................................................................95
Fig. 74: 16s-rRNA polygenetic pyrosequencing analysis on the 12-C and 13C fractions recovered after SIP carried
out on the biosludge (G-BS) sample. ........................................................................................................................96
Fig. 75: Use of a salicylate biosensor to demonstrate naphthalene-degrading activities in a series of experiments
carried out with MNP to isolate naphthalene degraders and without MNP (control). ................................................98
Fig. 76: PCR products observed for Comamonas-type naphthalene 1,2-dioxygenase in the MNP-free cells from
cultures S1 and S2 taken at time point t2. ................................................................................................................99
Fig. 77: Phenotypic microarray (Biolog PM1) of carbon utilisation of MNP-isolated cells (from Zhang et al 2014)
Highlighting: grey compounds inhibiting bacterial growth; red compounds promoting bacterial growth; blue
compounds not supporting bacterial growth. ..........................................................................................................100
TM
Fig. 78: Biolog PM1 MicroPlate Carbon Sources.................................................................................................100
TM
Fig. 79: Biolog PM3B MicroPlate Nitrogen Sources ............................................................................................101
Fig. 80: Phenotypic microarray (Biolog PM3B) showing the effects of various nitrogen sources on Ralstonia sp.
isolated using MMI. .................................................................................................................................................101
Fig. 81: Growth curve of MNP-free cells in presence of hydroxylamine, ammonia, nitrite and nitrate. ...................102
Fig. 82: Phenol concentrations after 20-h incubation of Ralstonia sp. with hydroxylamine, nitrite, ammonia and
nitrate showing severe phenol inhibition with hydroxylamine concentrations > 5 mg / L. .......................................103
Fig. 83: Schematic of the reactions involved in the photocatatytic activity of anatase (TiO2)..................................105
Fig. 84: Photograph of zeolites. ..............................................................................................................................106
Fig. 85: Schematic of the coke-oven water fluxes and sampling points. .................................................................107
Fig. 86: Picture of the three cokery wastewater samples........................................................................................107
Fig. 87: Trials with EQ2 sample in presence of TiO2 and sunlight (at start reaction time). ..................................... 109
Fig. 88: COD and BOD5 reduction observed during photo-oxidation trials (EQ2 sample). .....................................109
Fig. 89: Phenols trend during photo-oxidation trials (EQ2 sample).........................................................................110
Fig. 90: Nitrates and sulphates trend during photo-oxidation trials (EQ2 sample). .................................................110
Fig. 91: Evolution of the colour of the test solutions during photo-oxidation trials...................................................110
(Left after 1 h of reaction; Right after 5 h of reaction) .............................................................................................110
Fig. 92: Phenols and COD reduction during photo-oxidation trials with ICD3 sample. ...........................................111
Fig. 93: pH trend during the photo-oxidation tests. .................................................................................................112
Fig. 94: COD and phenols reduction.......................................................................................................................112
Fig. 95: Comparison between TiO2 aeroxide and TiO2 powder (Carlo Erba) abatement efficiency. .......................112
Fig. 96: Tests with Aeroxide powder and hydrogen peroxide. ................................................................................113
Fig. 97: Stainless steel support coated by Fotometal paint.....................................................................................113
Fig. 98: Test with TiO2 in a fixed support. ...............................................................................................................113
Fig. 99: COD and phenol reduction in function of water thickness over the TiO2 fixed bed. ...................................114
Fig. 100: Zeolite column. ........................................................................................................................................115
Fig. 101: Picture of coke oven wastewater samples at the exit of the zeolite column.............................................115
(on the left first samples, on the right final samples)...............................................................................................115
Fig. 102: COD concentrations at the exit of the zeolite column. .............................................................................115
Fig. 103: Phenols, ammonia and iron concentrations at the exit of the zeolite column...........................................115
Fig. 104: THC emissions during zeolite regeneration. ............................................................................................116
Fig. 105: COD, phenols and ammonia inlet / outlet concentrations during a laboratory trial with regenerated
zeolites. ..................................................................................................................................................................117
Fig. 106: Schematic of the zeolite / photooxidation pilot plant. ...............................................................................118
Fig. 107: Computed velocity map in the reactor (m/s). Reference case. ................................................................119
Fig. 108: Computed velocity map in the reactor (m/s).Check 1 ..............................................................................119
Fig. 109: Computed velocity map in the reactor (m/s).Check 2 ..............................................................................119
Fig. 110: Computed velocity map in the reactor (m/s).Check 3 ..............................................................................120
Fig. 111: Comparison between different scenarios dead zoned. ............................................................................120
Fig. 112: Pilot plant composed of a zeolite column connected in series to a photocatalytic reactor, with diagonal
configuration of the agitation system. .....................................................................................................................121
Fig. 113: Comparison between the colour of coke oven effluents with and without zeolite / photocatalytic treatment.
...............................................................................................................................................................................122
Fig. 114: Trend of COD and phenols during trials with zeolite column. ..................................................................122
Fig. 115: COD and phenols yield during trials with zeolite column. ........................................................................122
Fig. 116: Inlet / outlet trace metal concentrations obtained during the treatment of coke oven effluents using two
zeolite columns in series.........................................................................................................................................124
Fig. 117: Schematic (A) and pictures (B, C, D) of the pilot scale BET plant commissioned at Tata Steel Plant A in
July 2012. ...............................................................................................................................................................127
Fig. 117: (Continued). Schematic (A) and pictures (B, C, D) of the pilot scale BET plant commissioned at Tata Steel
Plant A in July 2012. ...............................................................................................................................................128
Fig. 117: (Continued). Schematic (A) and pictures (B, C, D) of the pilot scale BET plant commissioned at Tata Steel
Plant A in July 2012. ...............................................................................................................................................129
Fig. 118: Sludge age reduction program during the optimisation tests of the BET pilot plant commissioned by Tata
Steel. ......................................................................................................................................................................130

176

Fig. 119: Monitoring results for suspended solids, thiocyanate, COD, total phenols, free cyanide nitrite and
ammonia in inlet / outlet and return sludge / aeration cell samples collected at the pilot scale BET plant during the
12 weeks optimisation campaign. ...........................................................................................................................131
Fig. 119: (Continued) Monitoring results for suspended solids, thiocyanate, COD, total phenols, free cyanide nitrite
and ammonia in inlet / outlet and return sludge / aeration cell samples collected at the pilot scale BET plant during
the 12 weeks optimisation campaign. .....................................................................................................................132

Fig. 120: Pictures of the two HOK activated lignite adsorbents selected for testing in the ECOWATER project. . 133
Fig. 121: Graph showing the percent colour removal from treated coke oven effluent during laboratory experiments

using activated lignite products (HOK Pulverised and Super)...............................................................................135

Fig.122: Graphs showing the concentrations of Ni, As, Cd and Pb in the dissolved phase with increasing amounts

of HOK Super or Pulverised added. ......................................................................................................................135

Fig. 123: Schematic describing the activated lignite tests carried out at the pilot ...................................................136
scale BET plant of Tata Steel Plant A. ....................................................................................................................136
Fig. 124: Schematic of a typical fuzzy filter system and picture showing fuzzy filter balls. .....................................138
Fig. 125: Fuzzy filter operation. ..............................................................................................................................139
Fig. 126: Particle size removal for fuzzy filters in comparison with other wastewater treatment filtration technologies.
...............................................................................................................................................................................139
Fig. 127: Schematic showing that the compression rate of the fuzzy filter can be adjusted to collect smaller particles
(high compression rate) or larger particles (low compression rate). .......................................................................140
Fig. 128: Particle size distribution (in volume) of treated coke oven effluents from ................................................141
Tata Steel BET plants A (Left) and B (Right). .........................................................................................................141
Fig. 129: Pilot scale fuzzy filter unit installed at Tata Steel BET plant A for testing (November 2012)....................142
Fig. 129: (Continued) Pilot scale fuzzy filter unit installed at Tata Steel BET plant A for testing (November 2012) 142
Fig. 130: Graph indicating the time necessary for the pressure in the filter to reach the maximum set point of 350
psi as a function of compression and flow rates. ....................................................................................................143
3
Fig. 131: Picture of the fuzzy filter tower after 2-h operation (40% / 10 m / h) and immediately after a washing
cycle. ......................................................................................................................................................................144
Fig. 132: Pictures showing the appearance of liquid and suspended solid samples taken at the inlet, outlet and
inside the filtering tower (ie. wash water) during the fuzzy filter tests. ....................................................................144
Fig. 133: Graph showing the turbidity (NTU units) of the influent and effluent samples and the percent abatement
during the pilot scale fuzzy filtration tests. ..............................................................................................................145
Fig. 134: Graph showing the PAH concentrations (Sum of the 16 US EPA PAHs) of the influent and effluent
samples and percent abatement during the pilot scale fuzzy filtration tests at Tata Steel plant A. ......................... 146
Fig. 45: Deployment of passive samplers (ie. DGTs and SPMDs) at Tata Steel Works to monitor for the
concentrations of PAHs and trace metals in local rivers and internal water streams. .............................................168

177

List of Tables
Table 1. Work packages for ECOWATER. ...............................................................................................................13
Table 2. Environmental Quality Standards (EQS) for the PHS and PS identified in the WFD and list of the specific
pollutants (SP) which are of relevance for the iron and steelmaking industry. ..........................................................16
Table 3: Effluent discharge limits for Tata Steel Plant A. .......................................................................................... 19
Table 4: Effluent discharge limits for Tata Steel Plant B. .......................................................................................... 20
Table 5: Chemical composition of the four effluent streams treated at Tata Steel BET plant C................................20
Table 6: Wastewater discharge limits for Tata Steel BET plant C. ...........................................................................20
Table 7: Limit of detection (LOD) for the procedure developed by Tata Steel to analyse for 16 US EPA PAHs in
cokemaking and steemaking effluents. .....................................................................................................................25
Table 8: Validation data for the analytical procedure used to analyse trace metals in steelmaking effluents. The
graph compares the mean concentrations of trace metals with the assigned values. ..............................................26
Table 9: Limits of detection (LODs) for the procedure developed by Tata Steel to analyse for selected trace metals
in cokemaking and steemaking effluents. .................................................................................................................27
Table 10: Experimental conditions used to study the effects of interactions between organic compounds (phenols,
naphthalene, salicylic acid) and heavy metals upon toxicity responses. ..................................................................39
Table 11: Scheme of factorial experimentation.........................................................................................................42
Table 12: Variance analysis data ..............................................................................................................................42
Table 13: Parameters Estimation. ............................................................................................................................42
Table 14: PAH concentrations in the untreated coke oven liquor and the treated effluents at Tata Steel BET plant B
and abatement efficiency. .........................................................................................................................................47
Table 15: Summary of abatement efficiencies for trace metals at Tata Steel BET plant B. ...................................... 49
Table 16: Description of the 10 major effluent discharges at Tata Steel Plant A. .....................................................50
Table 17: Emission concentrations (expressed in g / l) of the 6 PAHs identified in the BREF reference document
for which a BAT-AEL value of 50 g / l has been proposed (sum of the 6 PAHs). ...................................................51
Table 18: PAH annual mean concentrations (expressed in g / l) in effluents from the main effluent discharge point
(long sea outfall) at Tata Steel Plant B. ....................................................................................................................53
Table 19: Concentrations of trace metals (total and dissolved concentrations (expressed in g/l) in the combined
final effluent (long sea outfall) at Tata Steel Plant B. ................................................................................................54
Table 20: Direct Toxicity Assessment for industrial effluents discharged to freshwater. ...........................................55
Table 21: Direct Toxicity Assessment for industrial effluents discharged to marine waters. .....................................56
Table 22: Details of the effluent samples submitted for Direct Toxicity Assessment. ...............................................56
Table 23: Results from freshwater ecotoxicity tests on steelmaking effluents. .........................................................57
Table 24: Results of marine ecotoxicity tests on cokemaking and steelmaking effluents. ........................................57
Table 25: Annual mass releases (kg/annum) of PAHs at Tata Steel Plant A (Period 2010-2013). ........................... 59
Table 26: Mean emission factors of PAHs (all expressed in mg / tonne liquid steel, except for DP10: expressed in
mg / tonne coke) for all the wastewater effluent discharges at Tata Steel Plant A. Releases from the cokemaking
process are highlighted in grey. ................................................................................................................................60
Table 27: Annual mass releases (expressed in kg / annum) of trace metals at Tata Steel Plant A (Period 20102013). .......................................................................................................................................................................62
Table 28: Mean emission factors (all expressed in mg / tonne liquid steel, except for DP10: in mg/tonne coke) of
trace metals at Tata Steel Plant A. ...........................................................................................................................62
Table 29: Annual mass releases of PAHs (expressed in kg / annum) and emission factors (expressed in mg / tonne
liquid steel) for the main effluent discharge point (long sea outfall) at Tata Steel Plant B in 2010 and 2013. ........... 64
Table 30: Annual mass releases (expressed in kg / annum) and emission factors (expressed in mg / tonne liquid
steel) of trace metals from the main effluent discharge point (long sea outfall) at Tata Steel Plant B during 2010 2013..........................................................................................................................................................................65
Table 31: Seawater and sediment characterization at sampling position .................................................................72
Table 32: Details of the effluent discharges assessed. .............................................................................................78
Table 33: List of Priority Hazardous Substances included in the Tiered approach. ..................................................79
Table 34: List of Priority Substances included in the Tiered approach. ....................................................................79
Table 35: Concentrations of trace metals used in the Tiered approach....................................................................79
Table 36: Concentrations of organic contaminants (PAHs and benzene) used in the Tiered approach. ..................80
Table 37: Results from Tier 0 and 1 assessment Priority Hazardous Substances. ...............................................80
Table 38: Results from Tier 0 and 1 assessment Priority Substances. .................................................................. 81
Table 39: Input parameters for the discharge test carried out in Tier 2 assessment. ...............................................81
Table 40: Identification of the key cultivable degraders isolated from Tata Steel coke oven BET plant A. ...............87
Table 41: Matching Pyrosequencing DNA with Ralstonia spp. .................................................................................95
13
Table 42: The various types of phenol hydroxylase genes found in (i) the MNPs-free and C fraction cells isolated
using MMI, (iii) the original raw biosludge and (iii) the bacteria isolated though cultivation techniques. ...................97
Table 43: Process involved in the photocatatytic activity of anatase (TiO2) ............................................................ 104
Table 44: Characteristic of three samples of coke oven wastewater. .....................................................................107
Table 45: PAH concentrations in EQ2 coke oven wastewater sample used for both photo-oxidation and zeolites
tests ........................................................................................................................................................................108
Table 46: Main characteristics of Carlo Erba and Aeroxide anatase selected for the ECOWATER project............108
Table 47: Ammonia and sulphide concentrations at start and end reaction time. ...................................................111
Table 48: Adsorption capacity of zeolites ...............................................................................................................116
Table 49: PAHs abatement efficiency in laboratory tests carried out using zeolites, anatase photocatalytic
treatment and combined zeolite / anatase treatment. .............................................................................................117

178

Table 50: Percent reduction of COD, phenols and BOD5 obtained during the pilot scale tests realised at CSM. ...122
Table 51: Percent reduction of PAHs obtained during pilot scale tests realised using two zeolite columns in series
and combined zeolite / photocatalytic treatment. ....................................................................................................123
Table 52: Trace metal concentrations and abatement efficiency in inlet / outlet coke oven effluent samples during
pilot scale tests using two zeolite columns in series. ..............................................................................................124
Table 53: Data on columns geometrical features and zeolite costs. .......................................................................125
Table 54: Cost-benefit analysis results for the treatment of coke oven effluents using zeolites. ............................126

Table 55: Physical characteristics of the two activated lignite products (HOK Super and Pulverised) tested for
PACT in the ECOWATER project. ..........................................................................................................................134

Table 56: Results from laboratory tests realised to study the effects of HOK Pulverised and Super for coke oven
effluent treatment with regard to colour removal efficiency. ....................................................................................134
Table 57: Abatement efficiencies of PAHs and trace metals with and without........................................................137
Table 58: Cost-benefit analysis results for the treatment of coke oven effluents using activated lignite. ................137
Table 59: Mean diameters particle size distribution (in volume) of treated coke oven effluents from Tata Steel BET
plants A and B. .......................................................................................................................................................140
Table 60: Suspended solids concentrations in inlet, outlet and wash water coke oven effluent samples during pilot
scale fuzzy filter tests at Tata Steel Plant A. ...........................................................................................................145
Table 61: Concentrations of trace metals in coke oven effluents after biological treatment and after fuzzy filtration
and abatement efficiency obtained using fuzzy filters. ............................................................................................146
Table 62: Cost-benefit analysis results for the treatment of coke oven effluents using fuzzy filtration at Tata Steel
BET plant A. ...........................................................................................................................................................147
Table 63: Details of the experiments carried out by CSM to investigate possible synergetic / antagonist effects
between trace metals and organic compounds upon toxicity (EC50) measured using Microtox test. .....................162
Table 64: Mean and range of PAH concentrations (expressed in g / l) in effluents from Tata Steel Plant A between
2010 and 2013........................................................................................................................................................164
Table 65: Mean concentrations of trace metals (total and dissolved concentrations; g / l) in effluents from Tata
Steel Plant A in 2012. .............................................................................................................................................166
Table 66: Sampling details for the passive samplers monitoring trial at Tata Steel Site A......................................168
Table 67: Primers used for amplification of 16s rRNA and phenol hydroxylase genes...........................................172

179

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The first objective of the project ECOWATER was to address the challenges
faced by the steel industry to fulfil the requirements of the Water Framework
Directive (WFD). Accordingly, research was carried out in this area to
characterise, quantify and measure the environmental impacts of key pollutants
including a series of trace metals and polycyclic aromatic hydrocarbons in
wastewater effluents from cokemaking and steelmaking operations. The
second aspect of ECOWATER was concerned with the investigation of the
potential of state-of-the-art molecular biology techniques for understanding
further the biodegradation processes taking place in coke oven biological
effluent treatment (BET) plants. In recent years, there has been an explosion
in the molecular biology techniques in environmental microbiology. Accordingly,
for the first time, a range of novel molecular biology techniques were used
and developed to study microbial communities in biosludges and identify
unculturable microorganisms that plays a major role in the degradation of
organic pollutants such as phenols and PAHs. The third important aspect
of the ECOWATER project was concerned with the development of innovative
technological solutions for the removal of key WFD substances in wastewater
effluents from the steel industry. Several innovative abatement solutions were
tested at the pilot scale including the application of a photocatalytic oxidation
process using anatase (TiO2), the use of sorbents such as powdered activated
lignite and zeolites, and a new high efficiency filtration technology (Fuzzy
filtration).

Studies and reports

ISBN 978-92-79-45179-9
doi:10.2777/77100