Water Research 89 (2016) 321e329

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Water Research
journal homepage: www.elsevier.com/locate/watres

Monitoring sulde-oxidizing biolm activity on cement surfaces using

non-invasive self-referencing microsensors
Liqiu Cheng a, *, Mitch W. House b, W.Jason Weiss b, c, M.Katherine Banks a
a

Zachry Department of Civil Engineering, Texas A&M University, 3136 TAMU, College Station, TX 77843-3136, USA
Lyles School of Civil Engineering, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907-2051, USA
c
Bindley Bioscience Center, Physiological Sensing Facility, Discovery Park, Purdue University, 1203 W. State Street, West Lafayette, IN 47907-2057, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 April 2015
Received in revised form
15 August 2015
Accepted 28 November 2015
Available online 14 December 2015

Microbially inuenced corrosion (MIC) in concrete results in signicant cost for infrastructure maintenance. Prior studies have employed molecular techniques to identify microbial community species in
corroded concrete, but failed to explore bacterial activity and functionality during deterioration. In this
study, biolms of different sulfur-oxidizing bacteria compositions were developed on the surface of
cement paste samples to simulate the natural ecological succession of microbial communities during MIC
processes. Noninvasive, self-referencing (SR) microsensors were used to quantify real time changes of
oxygen, hydrogen ion and calcium ion ux for the biolm to provide more information about bacterial
behavior during deterioration. Results showed higher transport rates in oxygen consumption, and
hydrogen ion at 4 weeks than 2 weeks, indicating increased bacterial activity over time. Samples with
ve species biolm had the highest hydrogen ion and calcium ion transport rates, conrming attribution
of acidophilic sulfur-oxidizing microorganisms (ASOM). Differences in transport rates between three
species samples and two species samples conrmed the diversity between Thiomonas intermedia and
Starkeya novella. The limitations of SR sensors in corrosion application could be improved in future
studies when combined with molecular techniques to identify the roles of major bacterial species in the
deterioration process.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Self-referring microsensors
Microbially inuenced corrosion
Biolm
Sulde-oxidizing bacteria

1. Introduction
Concrete has been widely used as a construction material for
centuries due to its advantages in strength, durability, plasticity and
low cost. However, application of concrete is facing enhanced
deterioration caused by carbonation (Lin et al., 2013), chloride
corrosion (Abd El Haleem et al., 2013; Hartt, 2013) and biological
reactions which lead to high nancial costs for operation and
maintenance (Beider et al., 2002). Microbially inuenced corrosion
(MIC) usually occurs in harsh environments, such as water distribution systems, sewage structures and wastewater treatment
plants, and signicantly reduces the service time of the infrastructure and endangers water quality (Coleman and Gaudet, 1993;
Marleni et al., 2012; O'Connell et al., 2012; Zhang et al., 2008).
Previous studies (Herisson et al., 2013; Islander et al., 1991; Parker,
1951) established MIC mechanism in sewer systems involving a

* Corresponding author.
E-mail address: liqiucheng@tamu.edu (L. Cheng).
http://dx.doi.org/10.1016/j.watres.2015.11.066
0043-1354/ 2015 Elsevier Ltd. All rights reserved.

sulfur cycle beginning with anaerobic production of hydrogen

sulde (H2S) by sulfate-reducing microorganisms (SRM) in submerged part of sewer. Released H2S in the pipe atmosphere (Jiang
et al., 2014; Joseph et al., 2012; Lahav et al., 2006; Yongsiri et al.,
2003) is absorbed by the moist concrete wall (original pH 12e13)
and then converted to elemental sulfur or partially oxidized sulfur
species and eventually to sulfuric acid which corrodes the concrete,
with the assistance of sulde-oxidizing microorganisms (SOM).
To better understand the corrosion processes and the interaction between concrete structure and attached biolm, various
studies have been conducted to identify the microbial species
involved in concrete degradation. Acidihiobacillus spp. (Milde et al.,
1983; Parker, 1951) were identied using conventional culturedependent methods in corroded sewer pipes. With the development of molecular techniques like polymerase chain reaction (PCR)
and cloning of 16S rRNA gene fragments, microbial communities in
the corroded area were characterized and more SOM species were
identied such as Thiothrix, Thiomicrospira, and Beggiatoa, along
with other coexisting heterotrophic bacteria as a-, b- and g-Proteobacteria, and Acidobacteria (Bielefeldt et al., 2010; Little et al.,

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L. Cheng et al. / Water Research 89 (2016) 321e329

2000; Nica et al., 2000; Vincke et al., 2001). In recent years universal small-subunit (SSU) rRNA gene amplicon pyrosequencing
was used to character microbial community is concrete sewer
system (Cayford et al., 2012; Gomez-Alvarez et al., 2012; Ling et al.,
2014, 2015; Santo Domingo et al., 2011). While some eld studies
still conrmed Acidithiobacillus spp. dominating the microbial
community (>50%) (Ling et al., 2014), other studies showed
extremely low abundance (<3%) of Acidithiobacillus spp., suggesting
that they may not be as important as people used to believe
(Cayford et al., 2012). Succession of microbial community was also
explored as results showed high variation and abundances of heterotrophic types under neutral pH while low diversity biolms
dominated by acidophilic species were discovered in severe
corrosion sites (Islander et al., 1991; Okabe et al., 2007). Although
molecular techniques provided detailed information about microbial community composition, the link between the species and
their real-time function in the corrosion process is still not well
understood. Furthermore, sensitivity and detection range of these
techniques is challenged by the environment as high calcium and
heavy metals inhibit nucleic acid amplication (Vincke et al., 2001;
Wilson, 1997).
Non-invasive self-referencing (SR) microsensors have been
successfully used for biochemical and agricultural studies
(McLamore et al., 2010b; Portereld and Smith, 2000) and also in
the environmental eld, focusing on characterization and quantication of real time biolm behavior (McLamore et al., 2011;
2010c). SR sensors are capable of detecting change of various analytes, including inorganic ions (H, Ca2, K, NH4 , Cl) (McLamore
et al., 2009; 2011; 2010c; Sun et al., 2012), organic compounds
(indole-3-acetic acid and glucose) (McLamore et al., 2010a; Shi
et al., 2011) and metabolic oxygen (McLamore and Portereld,
2011; McLamore et al., 2010c) in the biolm boundary layer. The
background noise is eliminated by sensor oscillation, hence the
descriptor of self-referencing. With the advantages of being noninvasive and non-destructive, SR sensors are powerful tools in
detecting real time changes in electrochemical signals related to
biological activity, while maintaining biolm functionality. In this
study, biolms of ve well-documented and closely related Acidithiobacillus species were developed to explore the application of
SR sensors in MIC detection. With real time measurements of key
elements, including oxygen, hydrogen ion (H) and calcium ion
(Ca2) in the biolm, information about bacterial metabolism and
their impact on cement deterioration was collected to better understand the corrosion process.

and were obtained from American Type Culture Collection (ATCC,

Manassas, VA). Each of these ve species was grown individually in
specic medium following ATCC instructions. Nutrient solution was
developed from ATCC Medium 290 S6 for Thiobacilli and ATCC
Medium 152 for Thiobacillus containing (in g/L): 1.2 Na2HPO4, 1.8
KH2PO4, 0.02 FeCl3, 0.02 MnSO4, 0.5 MgCl2, 1.0 NH4Cl, 1.0 Yeast
Extract, and 10.0 Na2S2O3$7H2O. Each species was harvested,
concentrated and transferred into one or more up-ow aerated
bioreactors (volume of 3 L) following species composition listed in
Table 1. Cell numbers were counted to insure that in every bioreactor, each species added had a similar number of cells around
1  108. The bioreactors were placed in a biological controlled room
at 30  C. Cement paste samples afxed to glass slides were suspended in growth media in bioreactors when cell numbers in the
bioreactors reached steady state. Biolms after 2 weeks and
4 weeks of growth on the cement surface were considered as
immature and mature biolm respectively (Huang et al., 2013;
McLamore et al., 2009) and were used in the following analysis.
Cement samples placed in nutrient solution without bacterial
inoculation were used as controls, and they were stored in a
biosafety cabinet with constant air ow and UV disinfection 3e5
times a day to make sure the solution was not contaminated.
2.3. Sensor structure

2. Material and methods

Fiber optic oxygen microsensors (optrodes), with 5e7 mm tip

diameter, were constructed following published procedures
(Chatni et al., 2009; Chatni and Portereld, 2009; McLamore and
Portereld, 2011) with platinum tetrakispentauorophenyl
porphyrin (PtPFPP) as uorophore. For each chosen uorophore,
there is a constant relationship between oxygen concentration and
the measuring phase angle. Calibration of the optrodes were conducted at both air purged O2 saturation (21%) and 0% O2 saturation
(nitrogen purged) solutions. Measured phase angle was transduced
to a current using a lock-in amplier and no reference electrodes
were needed. Micro ion-selective electrodes (mISE) were used to
measure proton and calcium transport of the biolm based on the
recognition-transduction mechanism. Both types of electrodes
used the borosilicate glass capillaries with 2e5 mm tip diameter and
were constructed following standard methods (McLamore et al.,
2009; 2010c; Portereld et al., 2009). The specic liquid ion exchange membranes (LIX) (hydrogen ionophore I and calcium
Ionophore IV, SigmaeAldrich, St. Louis, MO) lled in the capillary's
tips were used to recognize the target ions (H and Ca2). The realtime changes of the voltage signals between the mISE and the Ag/
AgCl reference electrode were recorded.

2.1. Cement paste samples

2.4. Experimental section

The samples were prepared at water to cementitious materials

ratios (w/c) of 0.42 with ordinary Type I Portland Cement (OPC,
BuzziUnicem USA), containing (weight %) 64.4% of CaO, 20.9% of
SiO2, 5.2% Al2O3, and 0.3% Fe2O3. The size of the sample was 1.91 cm
(3/4 inch) in diameter and 0.32 cm (1/8 inch) in thickness. After
setting for 24 h, samples were sealed in plastic bags and placed at
25  C in 100% humidity chamber for 60 days. Samples were then
glued with epoxy onto microscope glass slides and used for surface
biolm development.

The diagram of the self-referencing sensor system for biolm

ux measurement is presented in Fig. 1. Three linear stepper motors
placed at the X, Y, and Z-axis control sensor movement. Each ux
reading was collected after measurements were completed at both
initial position (DX from sample surface) and sample position (at
sample surface). The values of data acquisition frequency and DX
were set to maintain the concentration gradient in the boundary
layer during sensor movement, and to eliminate signicant sensor
drift and background noise (Kuhtreiber and Jaffe, 1990; McLamore
et al., 2009). Before measurements, samples were taken out of the
bioreactors and placed in specically designed sample holders lled with sterile nutrient solution to keep samples in the vertical
plane which is perpendicular to the movement direction of the
microsensor tip for 60 min to allow for stabilization. Final values of
ux were calculated as average of readings recorded at 3e5
different locations along sample surface.

2.2. Biolm development

Acidithiobacillus thiooxidans (ATCC 19377), Halothiobacillus
neapolitanus (ATCC 23638), Thiobacillus thioparus (ATCC 23646),
Thiomonas intermedia (ATCC 15466) and Starkeya novella (ATCC
8093, formerly Thiobacillus novellus) were used in the experiment

L. Cheng et al. / Water Research 89 (2016) 321e329

Table 1
Microbial species composition.
Bioreactor

Species

1
2
3
4
5

T.
T.
T.
T.
T.

sp.
sp.
sp.
sp.
sp.

thioparus
thioparus,
thioparus,
thioparus,
thioparus,

S.
S.
S.
S.

novella
novella, T. intermedia
novella, T. intermedia, T. neapolitanus
novella, T. intermedia, T. neapolitanus, A. thiooxidans

323

where ra analyte transport rate (mol/m2$sec), Qa integrated

analyte ux (mol/m2), and t calculation time (sec). When Q was
plotted against time, the transport rate should be the slope of the
curve.
The difference between the calcium transportation rate of test
specimens and the control samples may be due to surface biolm
activity. Therefore calcium transportation rate by biolm was
adjusted to better present the bacteriaecement interaction:

2.5. Flux calculation and integration

rbio rTot  rCtrl

Fick's rst law of diffusion is employed as fundamental theory

for sensor measurement and ux calculation. The real-time ux
was calculated as

where rbio calcium transportation rate by biolm, rTot total

calcium transportation rate, and rCtrl calcium transportation rate
of control sample.

J D

DC
DX

(1)

where J analyte ux (mol/m2$sec), D molecular diffusion

constant (m2/sec), C analyte concentration (mol/m3), and
X diffusion distance (m). The sign of J indicates the transport
direction as a positive sign indicates the analyte is moving away
from the biolm (efux); while a negative sign means the analyte is
moving towards biolm (inux). Each ux point was average during the sampling period (Dt). The total amount of analyte released
or absorbed by biolm (Q) could be determined through ux
integration:

t
X

JDt

(2)

(4)

2.6. Cell counting

Bacterial cell numbers on sample surface was quantied using
Beckman Multisizer 4 Coulter Counter (Beckman, Pasadena, CA)
(Zhang et al., 2013). 20 mm aperture tube was selected to detect
particles ranging from 0.4 mm to 12 mm diameter according to
bacterial sizes (Adachi and Suzuki, 1977; London, 1963; Milde et al.,
1983; Sievert et al., 2000; Waksman and Joffe, 1922). After oxygen
ux measurement, biolm on the according sample was detached
by vortexing the sample in 30 mL of PBS solution for 5 min (Vortex
Genie 2, Scientic Industries, Bohemia, NY). The solution with detached particles was diluted to the desired concentration with
Isoton II solution (Beckman, Pasadena, CA) and measured
immediately.

Transport rate (r) was used to represent analyte transport during steady-state:

ra

dQa
dt

(3)

2.7. SEM imaging

Biolm samples were xed for SEM imaging to determine the
growth of biolm over time. Primary xation procedures used a
xative contained 2.5% glutaraldehyde (w/v), 2.0% paraformaldehyde (w/v) and 0.1 mM sodium cocadylate buffer (pH 6.8).

Fig. 1. Schematic diagram of self-referencing sensor system for ux measurement of microbial biolms developed on cement paste surface.

324

L. Cheng et al. / Water Research 89 (2016) 321e329

Chemical drying using hexamethyldisilazane (HMDS) was applied

after xation. Samples were transferred from 100% ethanol to 100%
HMDS through a graded series of ethanol-HMDS mixtures, and
dried overnight at room temperature. Biolms were then sputter
coated with gold-palladium and viewed on FEI NOVA nanoSEM
eld emission scanning electron microscopes. Identical samples
were washed and placed in 70% ethanol for sonication of 10 min to
remove biolm. Structural changes in the cement surface was
observed on SEM as well.

better characterize the biological corrosion process following previous equations (Eqs. (1)e(4)) and the calculation procedure and
results are presented in Fig. 2. Plot (a)e(c) shows typical real-time
ux results for each analyte, and the integrated ux is in plot
(d)e(f). Average transport rate was the slope of the integrated ux
curve and are shown in plot (g)e(i) with the error bar representing
the actual values. Controls were samples in nutrient solution
without microbial inoculation.

3.2. Oxygen consumption rate

3. Results

0.4

10

(b)

0.2
0.1

50

20

2 Weeks
4 Weeks

0.3

40

10

15

(c)

2 Weeks
4 Weeks

30
20
10
0

10

15

Time (min)

20

TIme (min)

1000

10
8

20

10

15

20

Time (min)

(e)

6
4
2
0

5000

10

Time (min)

(f)

4000

15

20

2 Weeks
4 Weeks

3000
2000
1000
0

10

15

Time (min)

20

(g)

60

2 Weeks
4 Weeks

40
20
0

5 sp

0.4

2 Weeks
4 Weeks

0.0

Ca Efflux (pmol/cm sec)

15

TIme (min)

H Efflux (pmol/cm sec)

0.5

2000

80

0.3

(h)

3000

H Transport Rate (pmol/cm sec)

-50

2 Weeks
4 Weeks

4000

(d)

0.2

Ctrl
2 Weeks
4 Weeks

0.1
0.0
-0.1

5 sp

Ctrl

-0.2

Ca Transport Rate (pmol/cm sec)

50

5000

100

H Integrated Flux (pmol/cm )

2 Weeks
4 Weeks

150

Ca Integrated Flux (pmol/cm )

(a)

O2 Influx (pmol/cm sec)

200

O2 Integrated Flux (pmol/cm )

Flux of oxygen, H and Ca2 were chosen to represent biolm

metabolism level, net acid transport and specimen degradation
respectively. Prior to measurement, sensor performance within
3 mm from biolm surface was tested and the distance of biolmliquid mass boundary layers was calculated for each analyte to
verify the sampling point.
Original ux readings were further integrated and adjusted to

Oxygen ux is the primary assessment used to monitor biolm

metabolism. Since the species selected are chemoautotrophic, the
direction of oxygen ux (negative, inux) conrmed that biolm
was consuming dissolved oxygen from the bulk solution.
Comparing the consumption rate of 2-week and 4-week biolm,
the latter showed little increase from 24.65 pmol/(cm2$sec) to
29.63 pmol/(cm2$sec) (Fig. 2 Plot(g)).
Fig. 3(a) represents calculated average oxygen consumption rate
(rO2 ) at 3e5 positions along the biolm (three replicate biolms
samples each). The mature biolms of 4-week samples showed

O2 Consumption Rate (pmol/cm sec)

3.1. Flux integration and transport rate calculation

50

(i)

40

2 Weeks
4 Weeks

30
20
10
0

5 sp

Ctrl

Fig. 2. Original ux values for (a) oxygen, (b) proton and (c) calcium of the biolm. Integrated ux values for (d) oxygen, (e) hydrogen and (f) calcium of the biolm. The calculated
transport rate for (g) oxygen, (h) hydrogen and (i) calcium of the biolm. Error bars represent actual value range.

L. Cheng et al. / Water Research 89 (2016) 321e329

3.3. Calcium ion transport rate

50

(a)

2 Weeks
4 Weeks

40
30
20
10

4sp

3sp

2sp

1sp

(b)

18
12
6
0

5sp

4sp

3sp

2sp

1sp

Fig. 3. Plots of (a) oxygen consumption rate and (b) cell numbers for samples with
different species composition. The values were calculated average ux at three to ve
positions along the biolm surface with three replicate samples, and error bars
represent standard error of the arithmetic mean.

(a)

2 Weeks
4 Weeks

25
20
15
10
5
0

Ctrl
2 Weeks
4 Weeks

30

20

5sp

24

Hydrogen ion ux was analyzed as complementary to the

Biofilm Corrosion Rate (pmol/cm sec)

0
30

Cell No. (x10 /mL)

3.4. Hydrogen ion transport rate

O2 Consumption Rate (pmol/cm sec)

Oxygen consumption only indicates biolm metabolism, which

may not provide enough information about the bacteriaecement
corrosion interaction. Calcium ux is correlated to representing
corrosion given that Ca2 is dominant in cement and leaches out of
the structure during deterioration. The overall calcium transport
rate (calculated from original calcium ux) from 2 weeks to 4
weeks showed mixed trends for all test samples (Fig. 4(a)). For the

ve species biolm, the rate increased while the transport rates

decreased or remained the same for the other samples. Since
growth media (pH 6.8) also underwent neutralization reactions
with cement (surface pH 12), control samples showed a decrease in
total corrosion rate at 4 weeks from 2 weeks.
Transport rate by biolm are shown in Fig. 4(b) which was an
attempt to separate the chemical leaching from the biolm
response. The increase in biolm calcium transport rate (rbio) over
time indicated that the activity of the microbial communities was
connected to structural deterioration. It should be noted that while
low rbio suggests low bacterial impact, it is the total calcium ux
that quanties the overall damage. The rbio for ve species sample
was 4.56 pmol/(cm2$sec) and 11.10 pmol/(cm2$sec) for 2 weeks and
4 weeks; while the rbio for four species sample was 0.50 pmol/
(cm2$sec) and 4.32 pmol/(cm2$sec) respectively. Two species
samples and single species samples showed negative biolm
corrosion rates at 2 weeks, indicating lower calcium efux
compared to the controls.

Ca Transport Rate (pmol/cm sec)

higher oxygen consumption than the 2-week immature samples,

indicating an increase in microbial activity. The test results for
control samples with no bacterial incubation were low at both 2
weeks and 4 weeks, representing only the background caused by
sensor movement near sample surface. To determine whether the
changes in oxygen ux resulted from biolm activity or the initial
bacterial concentration differences in bioreactors, number of cells
attached on each sample was counted and the results are shown in
Fig. 3(b). All samples used the same mixture design (0.42 w/c) and
materials, resulting in similar surface chemistry, and hence similar
capability for bacterial attachment, which explains the close cell
numbers on test samples obtained from different bioreactors.

325

5sp

4sp

3sp

2sp

1sp

(b)

Ctrl
2 Weeks
4 Weeks

15

2 Weeks
4 Weeks

10
5
0
-5
-10

5sp

4sp

3sp

2sp

1sp

Fig. 4. Plots of (a) total calcium transport rate and (b) biolm calcium transport rate
with different species composition. The values were calculated average ux at three to
ve positions along the biolm surface with three replicate samples, and error bars
represent standard error of the arithmetic mean.

326

L. Cheng et al. / Water Research 89 (2016) 321e329

H Transport Rate (pmol/cm sec)

0.4

2 Weeks
4 Weeks

0.3

2 Weeks
4 Weeks

0.2
0.1
0.0

for ve species samples. The two species sample and single species
samples also had similar rH, which were lower than the rates of four
and three species samples. Decrease of these two groups is due to
the difference of Thiomonas intermedia and Starkeya novella which
is further explained in discussion. The same trend was observed in
rH for the 2 weeks samples. Measurements from both 2 weeks and 4
weeks samples presented differences between ve species biolm
and four/three species biolm, indicating successful inoculation of
different species. The rH of ve species biolm at 2 weeks and 4
weeks were 0.12 pmol/(cm2$sec) and 0.20 pmol/(cm2$sec)
respectively, which were two orders of magnitude of lower than
oxygen consumption rates.

-0.1
-0.2

5sp

4sp

3sp

2sp

1sp

Ctrl

Fig. 5. Hydrogen transport rate for samples with different species composition. The
values were calculated average ux at three to ve positions along the biolm surface
with three replicate samples, and error bars represent standard error of the arithmetic
mean.

oxygen and calcium ux tests. The results are presented in Fig. 5.

The direction of ux showed H diffused from the biolm surface
into solution. Flux results for control samples, however, were
negative due to the neutralization reactions between samples and
nutrient solution, which was consistent with the calcium release of
control samples. For ve species mixed biolms, the H transport
rate (rH) nearly doubled. The rH of samples containing four species
and three species at 4 weeks were similar and lower than the rate

3.5. SEM imaging

In order to observe biolm structure and the microstructure of
cement paste samples, SEM was used and the results were shown
in Fig. 6. After 4 weeks, the entire surface of the cement sample was
covered by a dense layer of biolm (Fig. 6(b)). Plot (a) and (c)
showed the surface of the control samples in air, and the surface of
the test sample after the biolm was removed, respectively. For the
control sample, there are large areas of dense unreacted cement
paste with sharp edges, and crystal-like hydration productions lled within the gaps. After bacterial corrosion, the sample surface
was transformed from compact to a loose structure with many
micropores. The area of cement paste decreased and edges became
smooth. The crystal-shaped products became thin and loose both at
the sample surface and inside the pores, indicating bacterial
penetration during the corrosion period.

Fig. 6. SEM images of (a) control sample, (b) test sample with 4 weeks of biolm growth and (c) test sample after biolm was removed.

L. Cheng et al. / Water Research 89 (2016) 321e329

4. Discussion
4.1. Analyte transport rate
For control samples without bacteria inoculation, the transport
rates of calcium and hydrogen ion both decreased from 2 week to 4
week measurement, which may result from the initial nebulization
reactions between nutrient solution and the cement, consuming
surface calcium ions and producing corresponding salts that prevents further penetration of bulk liquid into the sample core as
barriers (Monteny et al., 2000).
As for the highly articial experiment set-up, the characterizations of each species were well documented, hence could be of
great help to explain the data from SR sensor measurements.
All ve species have the ability to decrease surface pH of the
cement samples. Based on their tolerance pH ranges, SOM were
further categorized into acidophilic sulfur-oxidizing microorganisms (ASOM), such as A. thiooxidans, and neutrophilic sulfuroxidizing microorganisms (NSOM), such as Thiomonas intermedia
and the other species. Oxygen consumption rates decreased as the
biolm composition went from ve species to single species, while
the cell numbers attached on different samples were similar
(Fig. 3), which could be explained by the difference in sulfuroxidation abilities among selected species (Table 2) (Bielefeldt
et al., 2010; Islander et al., 1991). Since A. thiooxidans could fully
oxidize sulfur (S0) and other sulfur compounds (S2 O3 2 , S4 O6 2
and SO3 2 ) to produce sulfuric acid (H and SO4 2 ) while the rest
of the species could only oxidize S2 or S0 to partial oxidized intermediates S4 O6 2 =S2 O3 2 , more oxygen was needed for
A. thiooxidans comparing to other NSOM, which also explains the
differences of H and Ca2 transport rate between biolm samples
containing ve species and samples containing four in Figs. 4 and 5.
Previous studies also conrmed that concrete exposed to mixed
cultures of both ASOM and NSOM corroded signicantly faster than
the concrete exposed only to NSOM (Bielefeldt et al., 2010).
The absence of T. intermedia was the reason for the differences of
corrosion rates and acid production rates between 3 species biolm
and 2 species biolm samples. At the early stages, T. intermedia has
similar sulfur metabolism as S. novella (Chandra and Shethna, 1977;
English et al., 1995; London, 1963). However, later studies found
that T. thioparus, Thiobacillus neapolitanus and A. thiooxidans could
not grow on glucose, and T. intermedius uses an inducible EntnerDoudoroff pathway similar to Thiobacillus ferrooxidans, while
S. novella follows a non-cyclic pentose phosphate pathway along
with the Krebs cycle (Matin and Rittenbe, 1971). Another study
(Barridge and Shively, 1968) evaluated the phospholipids and found
that T. intermedia, T. neapolitanus, and A. thiooxidans contained the
same phospholipids as phosphatidyl glycerol (PG), diphosphatidyl
glycerol (DPG), and phosphatidyl-N-monomethylethanolamine
(PME); S. novella, however, had more types and different composition of phospholipids, indicating potential function diversity between S. novella and other acidithiobacillus species.
Two major different pathways for thiosulfate oxidation were
conrmed, leading to identication and reclassication of

Table 2
Optimal growth pH range and oxidization reaction for selected species.
Species

Optimal pH

Oxidization reactions

T. thioparus
S. novella
T. intermedia
T. neapolitanus
A. thiooxidans

4.5e7.8
5e9.2
1.9e7
6
<4

Organic S; H2 S; S2 /S0

S2 /S4 O2
6
S2 /S4 O2
6
0
2
2
S ; S2 O3 ; S4 O2
6 /SO4
2
S0 ; S2 O2
/SO
3
4

327

Acidithiobacillus species (Kelly, 2008; Kelly et al., 1997). S. novella

uses the paracoccus sulfur oxidation pathway to oxidize thiosulfate into sulfur and sulde and then to sulfate; while T. neapolitanus
and A. thiooxidans can produce tetrathionate or trithionate as intermediates. The role of T. intermedia in thiosulfate oxidation has yet
to be identied, but based on the similarity in enzyme and phospholipids types as well as polyamine distribution patterns (Hamana
and Matsuzaki, 1990), it was reasonable to suggest that T. intermedia
is closely aligned with the typical Thiobacillus sp. The 5S rRNA homology test (Lane et al., 1985) also conrmed that T. thioparus,
A. thiooxidans and T. intermedia were in the same group and related
to Rhodopseudomonas gelatinosa and Pseudomonas cepacia; while S.
novella was similar to T. versutus and T. acidophilus and related to
Rhodospirillumrubrum and Pseudomonas diminuta. Therefore,
T. intermedia and T. thioparus could be closely connected and the
intermediates produced from the latter would be linked to
T. intermedia than to S. novella. The diversity difference of these two
species may be the main reasons for gaps of H and Ca2 transport
rate rates between the three species biolm and two species biolm.
However, concerns have been raised against the total calcium
transport rate measured at the biolm surface (Fig. 4(a)). While 5
species biolm had increased calcium ux from 2 weeks to 4
weeks, the rest of the biolm samples experienced decrease. Since
the numbers of cells attached on sample surfaces were close with
each other, the difference could be explained by the variation of
bacteria species composition and the actual abundance of each
species, which was not covered in current study. Furthermore,
calcium ux of the control samples at 2 weeks were larger than the
calcium ux measured from two species and single species samples. Since no calcium chemicals were added in the nutrient solution, the only calcium source for bacteria growth is the cement. One
explanation might be for biolm of ve, four, and three species with
stronger ability to oxidize sulfur compounds and produce acid,
more calcium was degraded from the cement structure and was
more than enough to meet their growth needs; while for two and
single species biolm, they acted more like a barrier (or partial
barrier depending on coverage) to reduce the direct contact between cement and nutrient solution, than a major destroyer for
the cement structure. Again the results would be much better
explained with the help of molecular techniques after identifying
each species abundance.
4.2. Sensor application in corrosion assessment
Existing test methods employed to evaluate corrosion, such as
mass loss, thickness change, surface pH, and compressive strength
(Ehrich et al., 1999; Monteny et al., 2003; Sand, 1987; Torii and
Kawamura, 1994), focuses mainly on the properties and performance of concrete and may not be appropriate to address the
bacterialeconcrete interaction. Other testing methods, measuring
the concentration change of sulfate (SO4 2 ) and calcium (Ca2) in
solution (Fisher et al., 2008; Vincke et al., 1999) during the testing
period, are indirect representation of the biological sulfate cycle
during corrosion and still failed to represent the actual bacterial
activity. Molecular techniques provided detailed information
about microbial community composition, and could also be used
to study long term microbial community species succession
through careful design. The MIC biolm development depends on
various factors, including environmental conditions and
geographic locations, and the molecular techniques stop the
corrosion process and capture the corrosion situation as it is.
Pieces are still missing on connecting microbial activities with real
time corrosion monitoring.
The ideal methodology is to measure the direct interaction between the biolm and concrete structures at the interface, but the

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L. Cheng et al. / Water Research 89 (2016) 321e329

effort was limited by the non-conducting nature of concrete materials. The SR microsensor method was an attempt and an alternative approach to character the microbial activity at the biolm
surface. With its advantages in measuring real-time oxygen demand and calcium transport rate from biolm surface to the bulk
solution, there are clear limitations within this application that
need to be fully addressed.
MIC is usually a slow process. Previous studies (Mori et al., 1991;
Vincke et al., 1999) detected an average thickness change of
1e5 mm per year for corroded sewer pipes. The oxygen consumption rate of 2-week and 4-week samples from ve species
reactors was 24.65 pmol/(cm2$sec) and 29.63 pmol/(cm2$sec),
respectively. Biological oxidation rates of pyrite (Biglari et al., 2009;
Elberling et al., 1994) from previous studies ranged from 0.25 to
0.46 pmol/(cm2$sec) and were nearly two orders of magnitude
lower than the oxygen consumption rates. The high oxidation rate
indicated a high acceleration factor. However, when considering
the total calcium transport rate, corrosion rate for the testing
samples was around 1.7 mm/year (assuming calcium ions were
released from the concrete structure at the same rate as measured
at 4 weeks), which still fell into the range of natural deterioration.
The reasons for this comparison highlighted the limitations for SR
sensor techniques in MIC study. The rst reason is the bacteria
species selected for the test. The original goal was to explore the
application of SR sensors in MIC detection, so ve well-documented
and closely related Acidithiobacillus species were used, which was
not the actual representative of the sewer environment. More
bacteria species and different compositions are highly desired in
the future experiment design. The second is the parameter selection of the test. The objective was to determining the acid production ability of the biolm and to associating with cement
corrosion results, but the H ux used in this experiment was
indeed the net accumulation rate, not direct measure of acid production. Furthermore, the calcium transport rate at biolm surface
represent concrete degradation at certain degree, but still didn't
take into account the amount of calcium used by biolm. With the
detailed study at the cell metabolism level, more specic parameters including different ion (K, Na, SO4 2 etc.), amino acids and
proteins are needed to monitor activity and function of target
species. The third is the duration of the set-up. Based on the previous mono-species biolm development experience, the test was
set only for four weeks, monitoring short-term changes, and the
calcium transport rate is believed to increase along with time,
which was not suitable for charactering the longevity of MIC. Future
development should consider long-term continuous monitoring of
biolm activity with more selective parameters. Molecular techniques should also be added into the experiment design to associate the real-time activity with the actual species composition
which was not included in the current study.
5. Conclusion
Linking the bacterial function and interspecies action to the
actual concrete deterioration is critical to determine the mechanism of MIC. SR sensors are proven to be powerful to characterize
detailed microbial activity while maintaining biolm functionality.
Application and combination of SR sensors and molecular techniques could help to identify the roles of major bacterial species in
the concrete deterioration process.
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