Environment International 31 (2005) 149 154

www.elsevier.com/locate/envint

Degradation of polycyclic aromatic hydrocarbons (PAHS) by a bacterial

consortium enriched from mangrove sediments
S.H. Yu, L. Ke, Y.S. Wong, N.F.Y. Tam*
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong, China
Available online 2 November 2004

Abstract
The biodegradability of a polycyclic aromatic hydrocarbons (PAHs) mixture consisted of fluorene (Fl), phenanthrene (Phe) and pyrene
(Pyr) by a bacterial consortium enriched from mangrove sediments under sediment-free and sediment slurry conditions was investigated. The
enriched consortium made up of three bacterial strains, namely Rhodococcus sp., Acinetobacter sp. and Pseudomonas sp., had a good PAH
degradation capability with 100% degradation of Fl and Phe in sediment-free liquid medium after 4 weeks of growth. The Fl and Phe
degradation percentages in sediment slurry were higher than that in liquid medium. Autochthonous microorganisms in sediments also
possessed satisfactory PAH degradation capability and all three PAHs were almost completely degraded after 4 weeks of growth.
Bioaugumentation (inoculation of the enriched consortium to sediments) showed a positive effect on PAH biodegradation after 1 week of
growth. Complete biodegradation of pyrene took longer time than that for Fl and Phe, indicating the enriched bacterial consortium had
preference to utilize low-molecular weight PAHs.
D 2004 Elsevier Ltd. All rights reserved.
Keywords: Polycyclic aromatic hydrocarbons; Bacterial consortium; Mangrove sediments

1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous
environmental pollutants generated from both natural and
anthropogenic processes, and pose a serious concern on the
health of aquatic life and human through bioaccumulation
(Hughes et al., 1997). They are hydrophobic and readily
adsorbed onto particulate matter, thus, coastal and marine
sediments become the ultimate sinks for PAHs (Hughes et
al., 1997). Mangrove ecosystems, important inter-tidal
estuarine wetlands along the coastlines of tropical and
subtropical regions, are closely tied to human activities and
are subject to contamination. Tam et al. (2001) reported that
the mean concentration of total PAHs (based on 16 USEPA
priority PAHs) in mangrove sediments in Hong Kong was
around 2000 ng g 1 dry weight, ranging from 356 to 11,098

* Corresponding author. Tel.: +852 27887793; fax: +852 27887406.

E-mail address: bhntam@cityu.edu.hk (N.F.Y. Tam).
0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2004.09.008

ng g 1, significantly higher than the marine coastal sediments in Hong Kong which had mean and range concentrations of 553 and 74420 ng g 1 (Zheng and Richardson,
1999). According to the sediment quality criteria for the
classification of sediments developed by the Hong Kong
SAR Government, the Lower Chemical Exceedance Level
(LCEL) and Upper Chemical Exceedance Level (UCEL) for
total PAHs are 2250 and 12,770 ng g 1 dry weight,
respectively (website: http://www.etwb.gov.hk), indicating
that some mangroves in Hong Kong have been contaminated by PAHs. Moreover, although the mean PAHs
concentration in Hong Kong mangrove sediments was
comparable to that recorded in mangrove sediments in
Puerto Rico receiving petroleum pollution (mean of 1820 ng
g 1), the range recorded in Hong Kong mangrove sediments
was significantly wider than that in Puerto Rico (5006000
ng g 1) (Klekowski et al., 1994).
Microbial degradation is believed to be one of the major
processes to clean up PAH-contaminated sediments (Hughes
et al., 1997). Previous studies have reported that indigenous

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S.H. Yu et al. / Environment International 31 (2005) 149154

microbial communities could have a considerable potential

to remedy the oil-contaminated sediments (Ramsay et al.,
2000) and remove phenanthrene from aqueous solution
(Tam et al., 2002, 2003). The efficiency of PAH biodegradation in sediment was different from that in liquid
medium. Some studies showed that PAH biodegradation
was reduced by sorption to sediments (Ramirez et al., 2001)
as highly lipophilic PAH tended to sorb tightly on sediments
and limited its availability to microorganisms. However,
other studies reported that sediments did not inhibit and
even enhanced the biodegradation of PAHs (Guerin and
Boyd, 1992, 1997; Laor et al., 1999). Several possible
explanations for enhancing degradation by sediments were
proposed by Poeton et al. (1999), including (i) bacteria
might expose to higher concentrations near the particle
surface due to PAH desorption and a diffusion concentration
gradient could exist between the surface and bulk liquid,
and (ii) bacteria might be able to consume PAHs at the
particle surface without the need to desorb PAH and
transport into the bulk liquid due to attachment and release
of extracellular enzymes. Another possible reason is that the
sediment provides nutrients, carbon sources and growth
factors for microorganisms. Bioaugmentation (supplementation of microorganisms to contaminated sediments) is
often used to enhance the rate of biodegradation of organic
substances (Vogel, 1996). However, other studies showed
that bioaugmentation did not improve and even slow down
the degradation processes (Launen et al., 2002; Saponaro et
al., 2002). The present study therefore aims to (1) determine
the degree of PAH contamination in mangrove sediments;
(2) examine the capability of a bacterial consortium
enriched from the sediments to degrade a mixture of three
PAHs (fluorene, phenanthrene and pyrene); (3) investigate
the degradation capability of the autochthonous bacterial
communities in the sediments on the degradation of a
mixture of three PAHs; and (4) study the effect of
augmentation of the enriched bacterial consortium on the
degradation of a mixture of three PAHs.

2. Materials and methods

2.1. Collection of sediments and analysis of PAHs in
sediments
Three random replicate surface sediments (03 cm) were
collected at the landward region (close to the discharge point
of a public sewer) of the Ho Chung mangrove swamp in
HKSAR, China during low tides. This swamp covers a
mangrove area of 2.37 ha and has been affected by vehicle
exhausting deposition, and discharge of industrial, livestock
and household waste and wastewater. Each sediment
sample, around 23 kg fresh weight, was actually a
composite sample made up by thoroughly mixing sediments
collected from several sampling points. Sub-sample of each
composite fresh sediments (20 g fresh weight) was

immediately used for microbial enrichment, around 100 g

was used for sediment slurry degradation studies, 200 g was
freeze-dried, ground and passed through a 180-Am sieve for
PAH analyses, and the remaining was air-dried for
determination of other sediment properties. The mean and
standard deviation values (based on three replicates) of
silt+clay percentage, organic matter content and pH in this
mangrove sediment were 34F10%, 5.49F1.25% dry weight
and 7.16F0.35, respectively. The concentrations of each of
the 16 USEPA priority PAH compounds in sediments were
analyzed by a standard protocol (Tam et al., 2001) using
dichloromethane and methanol for extraction, m-terphenyl
as an internal standard, and detected by GC-FID (HewlettPackard 5890 gas chromatography with a flame ionization
detector).
2.2. Enrichment, isolation and identification of the
PAH-degrading microbial consortium
Fluorene (Fl), phenanthrene (Phe) and pyrene (Pyr)
purchased from Sigma (USA, purity N96%) were used as
the model PAH mixture. Mineral salt medium (MSM) at a
salinity of 10x (parts per thousands) and pH 7 was used as
the culture medium (Tam et al., 2002). An aliquot of 10 ml
sediment supernatant obtained from shaking 1:5 sediment/
10x seawater (w/v) for 15 min was added to a 250-ml
conical flask containing 90 ml MSM with the addition of Fl,
Phe and Pyr, each at 10 mg l 1, gave a total contamination
of 30 mg l 1 of PAH mixture (equivalent to 1350 ng g 1
fresh sediment). Although this concentration was higher
than the real contamination level in Ho Chung sediments, it
was common to use an elevated concentration for enrichment to ensure that the PAH degraders were selected.
Actually a wide range of PAH concentrations for enrichment
and degradation (from 5 to 100 mg l 1) had been adopted
by previous workers (e.g. Yuan et al., 2000), 10 mg l 1 for
each PAH was employed in our previous studies (Tam et al.,
2002, 2003) and also by another researcher (Zaidi and
Imam, 1999). The flask was shaken in an orbital shaker (150
rpm) at 20F2 8C for 2 weeks for microbial enrichment.
After 2 weeks, an aliquot of 10 ml enriched culture was
inoculated into another 250-ml conical flask containing 90
ml MSM with the same amount of mixed PAH for the
second enrichment. Five consecutive enrichments were
carried out to reach a PAH-degrading microbial consortium.
The bacterial colonies were isolated by streaking the
enriched consortium on nutrient agar plates containing a
mixture of three PAHs. Each individual colony was first
identified by its color and morphology. The purified
bacterium was then identified by 16S rDNA gene sequence
after amplification of the gene by PCR using the set of
primers 27F (Escherichia coli position 827, 5V-AGA GTT
TGA TCC TGG CTC AG-3V) and 1492R (E. coli position
15101492, 5V-GGC TAC CTT GTT ACG ACT T-3V)
(Lane, 1991; Ikenaga et al., 2002). Almost a full length of
the bacterial 16S rDNA sequence was amplified by this set

S.H. Yu et al. / Environment International 31 (2005) 149154

of primers. The PCR reaction mixture (final volume= 50 Al)

was prepared in a single tube as follows: 1PCR buffer
containing 1.5 mM MgCl2 (SuperTherm), 200 AM dNTPs
(Invitrogen), 200 nM of each primer, 50 ng genomic DNA
from isolates, and 1.5 U Taq DNA polymerase (SuperTherm). Thermal cycling was performed in a PTC-200
DNA Engine (MJ Research) with a profile consisting of 35
cycles of 94 8C for 30 s, 55 8C for 30 s and 72 8C for 1.5
min. PCR products (about 1.5 kb) were gel-purified and
subsequently cloned into the pGEM-T Easy vector (Promega) for automated DNA sequencing. Database searches
with the determined 16S rDNA gene sequences were
conducted using the BLASTN program (Altschul et al.,
1997) maintained by National Center of Biotechnology
Information (NCBI).
2.3. Biodegradation studies
The experimental set up for biodegradation in liquid
medium was the same as that used for enrichment, with
initial Fl, Phe and Pyr concentrations, each at 10 mg l 1.
The residual PAH concentrations in the cultures were
determined after 0, 1, 2, 4 and 6 weeks of growth by GCFID after extracting with distilled dichloromethane (Tam et
al., 2002). For sediment slurry biodegradation, sediments
were divided into sterile and non-sterile portions. For the
sterile sediment slurry, 4-g fresh sediments were added into
a 100-ml conical flask containing 40 ml MSM and
autoclaved at 121 8C for 30 min. The autoclaving step
was omitted for the non-sterile sediment slurry. Mixed PAHs
(400 Ag) from the stock PAH mixture standard diluted in
distilled acetone (1000 mg l 1) were added into the
sediment slurry and shaken overnight to let the PAH
adsorbed onto the sediments. To examine the effect of
augmentation, 1 ml of the enriched bacterial consortium was
inoculated into each flask containing either sterile or nonsterile sediment slurry to give an initial inoculum size of
2.3105 cell ml 1 at the beginning of each degradation
experiment. Another sets of flasks were prepared in the
same way for both sterile and non-sterile sediment slurry but
without bacterial inoculum, the former sterile flask acted as
the control for determination of any abiotic loss and the non-

151

sterile one indicated the PAH degradation capability of

microorganisms naturally present in sediments (i.e. the
autochthonous microbes). The flasks, in triplicate for each
treatment and control, were retrieved at the same time
interval as that for liquid biodegradation studies and
measured for residual PAHs followed the same method
described in Section 2.1. Biodegradation percentage was
calculated as: (Residual PAH concentration in the control Residual PAH concentration in the degradation flask)/
(Residual PAH concentration in the control)*100%.
2.4. Enumeration of PAH-degrading microorganisms
The population size was determined by the most
probable number (MPN) method, modified from a technique
reported by Kiyohara et al. (1982). Every MSM agar plate
was divided into half and each half representing a 10-fold
serial dilution factor was further divided into five divisions
(five replicates). Then, a drop of 20 Al mixed PAH standard
prepared in acetone (1000 mg l 1) was added in the middle
position of each division. The acetone was evaporated
immediately and a white spot was left on the plate. An
aliquot of 10 Al diluted culture was dropped onto the spot.
The plates were incubated at 28 8C for 3 weeks. The spot
was marked as positive if the colony was surrounded by a
clear zone. The MPN result was then calculated using a GW
Basic Version 3.20computer program developed by Tam
(1982).

3. Results
3.1. PAH concentrations in Ho Chung surface mangrove
sediments
The total concentrations of 16 USEPA priority PAHs in
mangrove sediments varied from 1162 to 3322 ng g 1 dry
weight, whereas Pyr had the highest concentration among
the three PAHs examined (Table 1). These values were
unlikely to cause any adverse biological effects as they were
below the Effects Range-Low values (Long et al., 1995),
and they were lower than the exceedance levels set by the

Table 1
Concentrations of total PAHs and mixed PAHs, Fluorene (Fl), Phenanthrene (Phe) and Pyrene (Pyr), total concentrations of low-molecular weight (LMW) and
high molecular weight (HMW) PAHs, and ratios of Phenanthrene/Anthracene (Phe/Ant) and Flu/Pyr in surface Ho Chung mangrove sediments (mean and
standard deviation of three replicates are shown; NA: not applicable)
1

Sediment

PAH concentrations (ng g

Total

Fl

Phe

Pyr

Ho Chung sediments
ERL (Long et al., 1995)
HKEPD guidelinea: LCEL
HKEPD guidelinea: UCEL

2202.3F959.8
4022
2250
12,760

52.1F6.6
19
NA
NA

92.0F17.0
240
NA
NA

180.7F51.7
665
NA
NA

dw)

LMW-PAHs

HMW-PAHs

232.3F42.97
NA
550
1700

1969.8F827.41
NA
3160
9600

Specific PAH ratios

Phe/Ant

Flu/Pyr

4.7F1.8
NA
NA
NA

0.3F0.17
NA
NA
NA

a
Sediment quality criteria for the classification of sediments adopted by the Environmental Protection Department of the Hong Kong SAR Government,
LMW-PAHs include naphthalene, acenaphthene, acenaphthylene, anthracene, fluorene and phenanthrene, while HMW-PAHs include fluoranthene, pyrene,
benzo(a)anthracene, benzo(a)pyrene, chrysene, dibenzo(a,h)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, indeno(1,2,3-c,d)pyrene and
benzo( g,h,i)perylene. LCEL: lower chemical exceedance level, UCEL: upper chemical exceedance level (website: http://www.etwb.gov.hk).

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S.H. Yu et al. / Environment International 31 (2005) 149154

Hong Kong SAR Government. The PAH profile was

dominated by high molecular weight (HMW) PAHs (46
rings PAHs). Table 1 also shows that the Phe/Ant in Ho
Chung sediments was 4.7, less than the ratio of 10 for
pyrolytic source of contamination proposed by Soclo et al.
(2000), on the contrary, their Flu/Pyr were less than 1
suggesting petrogenic source of contamination. These
results indicate PAHs in Ho Chung mangrove sediments
might come from a mixture of pyrolytic and petrogenic
sources.
3.2. PAH biodegradation in liquid medium by the enriched
bacterial consortium
The enriched bacterial consortium contained three
bacterial isolates, namely Rhodococcus sp., Acinetobacter
sp. and Pseudomonas sp. During 6 weeks of the degradation
experiment in sediment-free liquid cultures, total abiotic
losses of Fl and Phe were 12% and 4%, respectively, and no
Pyr was lost. The biodegradation percentages of Fl and Phe
after 2 weeks of growth were greater than 99% but only
around 10% of Pyr was degraded (Fig. 1). After 4 weeks of
growth, all three PAHs were completely degraded, indicating that the consortium had a good PAH degradation
capability and preferred to utilize low-molecular weight
PAHs.
3.3. Effect of sediment and bioaugmentation on PAH
biodegradation
In sterile sediment slurry with inoculation of the
enriched bacterial consortium, the biodegradation percentages of Fl, Phe and Pyr after 1 week of growth were
54.29F2.59%, 35.07F2.56% and 15.29F1.18%, respec-

tively (Fig. 1), significantly higher than that in sedimentfree liquid culture (two-way ANOVA F 3,47 values showing
significant differences among treatments were 117.9, 25.9
and 0.67 for Fl, Phe and Pyr, respectively), indicating that
sediments enhanced PAH biodegradation in the first week.
The highest Fl and Phe degradation percentages after 1
week of growth were found in the non-sterile sediment
slurry with an inoculum (bioaugmentation), followed by
the inoculated sterile slurry (just inoculum without autochthonous microbes), whereas the non-sterile sediment slurry
without any inoculation (only the autochthonous microorganisms) had the smallest degradation (b10% for all
three PAHs). These results suggested that the addition of an
enriched consortium could enhance the efficiency of Fl and
Phe biodegradation but not Pyr. After 2 weeks of growth,
all treatments achieved similar biodegradation percentages
which were around 99% for Fl and 97% for Phe. Very little
Pyr biodegradation took place in the first 2 weeks of
growth. Nevertheless, all three PAHs were completely
degraded after 4 weeks of degradation. The abiotic losses
of Phe and Pyr in sediment slurries were negligible, similar
to that in liquid culture. In contrast, around 30% Fl was
lost abiotically.
3.4. Growth of PAH-degraders
In sediment-free liquid culture with inoculation of the
enriched consortium, log10MPN of the PAH-degraders
increased from an initial value of 5.36 to 6.36 after 6
weeks of growth, indicating that PAH was used as the
carbon and energy source for bacterial growth (Table 2).
The log10MPN value of the PAH-degraders in sterile
sediment slurry inoculated with the enriched bacterial
consortium reached the highest number, around 7.61 after

Fig. 1. Percentages of degradation of Fl, Phe and Pyr by enriched bacterial consortium and/or autochthonous microbes in liquid culture and different sediment
slurry treatments during 6 weeks of growth. Mean and standard deviation values of three replicates are shown. Percentage biodegradation=(Residual PAH
concentration in the sterile control Residual PAH concentration in degradation flask)/(Residual PAH concentration in the sterile control)*100%. According to
parametric two-way ANOVA test, significant differences in Fl and Phe biodegradation percentages were found between treatments as well as degradation time,
and results were (i) Fl: between treatments ( F 3,47 =117.9, Pb0.001) and between times ( F 3,47 =2038.6, Pb0.001); and (ii) Phe: between treatments
( F 3,47 =25.9, Pb0.001) and between times ( F 3,47 =557.9, Pb0.001); but for (iii) Pyr, there was no significant difference between treatments ( F 3,47 =0.673,
P =0.575) and significantly different between times ( F 3,47 =1351.5, Pb0.001).

S.H. Yu et al. / Environment International 31 (2005) 149154

153

Table 2
Changes in population sizes of PAH-degraders (log10MPN) in different treatments during 6 weeks of growth (mean and standard deviation of three replicates
are shown)
Treatments

Weeks
0

Sediment-free liquid culture with enriched consortium

Sterile sediment slurry with enriched consortium
Non-sterile sediment slurry without enriched consortium
Non-sterile sediment slurry with enriched consortium

5.36F0.00
5.36F0.00
3.36F0.00
5.37F0.00

7.29F0.15
6.36F0.00
4.46F0.09
6.42F0.09

6.58F0.10
7.61F0.11
6.59F0.28
7.61F0.11

5.96F0.12
7.61F0.11
6.33F0.21
6.52F0.16

6.36F0.00
7.61F0.11
6.37F0.46
6.72F0.38

Two-way ANOVA results

Between treatments
Between time
Interaction (treatmentstime)

F 3,59=109.8 ( Pb0.0001)
F 4,59=161.5 ( Pb0.0001)
F 12,59=23.5 ( Pb0.0001)

2 weeks of growth and maintained at a similar level

thereafter. In the inoculated non-sterile slurry (consisted
both autochthonous microbes and enriched consortium), the
log10MPN declined from the peak value of 7.61 to around
6.5, suggesting that other microorganisms existed in the
sediment slurry might compete with the enriched consortium leading to smaller number of PAH degraders towards
the end of the experiment. The slurry without any
inoculum, just the autochthonous microbes had lower
number of PAH-degraders, explaining why the initial
removal percentages of Fl and Phe were lower in this
treatment. According to two-way ANOVA, the population
sizes of PAH-degraders in the sterile sediment slurry with
inoculation of the enriched consortium were significantly
higher than that in the non-sterile sediment slurry (Table 1),
indicating that the autochthonous microbes might compete
with the incoulum.

4. Discussion
Similar to previous studies (Ramsay et al., 2000; Tam et
al., 2002), surface sediments in a typical mangrove swamp
in HKSAR supported a group of bacteria (the enriched
consortium) which had a good PAH degradation capability
and could be used to remedy PAH contaminated sediments.
However, PAH biodegradation in sediments containing
mixed PAH was different from that in aqueous solution.
The interactions between sediment matrix and PAHs such as
their sorption to sediment particles would inhibit biodegradation (Hughes et al., 1997; Ramirez et al., 2001). Ramirez
et al. (2001) showed that pyrene biodegradation rates of a
consortium and Mycobacterium sp. in sediment slurry were
much lower than that in aqueous solutions. On the contrary,
Poeton et al. (1999) reported that the degradation rates of
phenanthrene and fluoranthene in the presence of sediment
were 2.1 to 3.5 and 2.1 to 5.3 times faster than that in
sediment-free aqueous medium, respectively. Guerin and
Boyd (1992) discovered sediments enhanced the rate and
extent of naphthalene mineralization by Pseudomonas
putida ATCC 17874 but mineralization was reduced when
another species, NP-Alk, was used, suggesting that sediment

effects varied from species to species. Guerin and Boyd

(1997) further demonstrated that naphthalene associated
with forest soils was less available than naphthalene sorbed
to agricultural soils or river sediments, indicating the
binding characteristics of the sediments might control the
bioavailability. Humic substances were found to stimulate
microbial metabolisms (Laor et al., 1999). Phenanthrene
mineralization was substantially enhanced upon sorption to
mineral humic acid complexes as the sorbed contaminants
were still bioavailable and the presence of surfaces might
even stimulate mineralization (Laor et al., 1999). The
present study also demonstrated that mangrove sediment
with around 5% organic matter had higher initial Fl and Phe
biodegradation than that in the liquid culture. These findings
revealed that sediment effects were complicated.
Bioaugmentation with an enriched consortium is a
common practice to enhance the rate and extent of
bioremediation, especially in combination with other sitespecific amendments (Vogel, 1996). Trzesicka-Mlynarz and
Ward (1996) showed that bioaugmentation of mixed
cultures enhanced fluoranthene degradation over 9 weeks.
However, many studies showed that bioaugmentation was
not necessary. Launen et al. (2002) demonstrated that
bioaugmentation did not affect the efficient remediation of
PAH-contaminated dredged sediment in aerated slurry phase
bioreactors. Similarly, Saponaro et al. (2002) reported that
bioaugmentation did not seem to speed up bioremediation of
the manufacturing gas plant aged soil in slurry phase. In the
present study, inoculation of the enriched consortium to
sediment slurry showed some synergistic effect on degradation of Fl and Phe after 1 week of growth only. The
bioaugmentation process might be species- as well as sitespecific. More studies should be done with different
consortia and concentrations of the initial inoculum to
elucidate the significance of bioaugmentation.

Acknowledgement
The work described in this paper was fully supported by
a grant from the Research Grant Council of the Hong Kong
SAR (Project No. RGC Ref: CityU 1110/02M).

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S.H. Yu et al. / Environment International 31 (2005) 149154

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