Article

pubs.acs.org/est

Wastewater-Enhanced Microbial Corrosion of Concrete Sewers

Guangming Jiang,*,† Mi Zhou,† Tsz Ho Chiu,† Xiaoyan Sun,† Jurg Keller,† and Philip L. Bond†
†
Advanced Water Management Centre, The University of Queensland, St. Lucia, Queensland 4072, Australia
*
S Supporting Information

ABSTRACT: Microbial corrosion of concrete in sewers is known

to be caused by hydrogen sulfide, although the role of wastewater
in regulating the corrosion processes is poorly understood.
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Flooding and splashing of wastewater in sewers periodically

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inoculates the concrete surface in sewer pipes. No study has

systematically investigated the impacts of wastewater inoculation
on the corrosion of concrete in sewers. This study investigated the
development of the microbial community, sulfide uptake activity,
and the change of the concrete properties for coupons subjected
to periodic wastewater inoculation. The concrete coupons were
exposed to different levels of hydrogen sulfide under well-
controlled conditions in laboratory-scale corrosion chambers
simulating real sewers. It was evident that the periodic inoculation
induced higher corrosion losses of the concrete in comparison to
noninoculated coupons. Instantaneous measurements such as surface pH did not reflect the cumulative corrosion losses caused
by long-term microbial activity. Analysis of the long-term profiles of the sulfide uptake rate using a Gompertz model supported
the enhanced corrosion activity and greater corrosion loss. The enhanced corrosion rate was due to the higher sulfide uptake
rates induced by wastewater inoculation, although the increasing trend of sulfide uptake rates was slower with wastewater.
Increased diversity in the corrosion-layer microbial communities was detected when the corrosion rates were higher. This
coincided with the environmental conditions of increased levels of gaseous H2S and the concrete type.

1. INTRODUCTION activity of sulfide-oxidizing bacteria (SOB) on the exposed

Sewer networks are one of the most critical components of the
urban infrastructure of modern societies. Hydrogen sulfide
(H2S) is the primary cause for the shortened service life of
these essential, costly installations due to microbial-induced
concrete corrosion. The U.S. Environmental Protection Agency
(EPA) states that severe hydrogen sulfide induced corrosion
can reduce the 50 to 100 year expected life of sewer
infrastructure to less than 10 years.1 It is reported that the
corrosion rate of sewer pipes is between 1 and 10 mm per
year.2
Corrosion causes loss of concrete mass, cracking of the sewer
pipes, and, ultimately, structural collapse. Sewer systems
suffering from corrosion often require premature replacement
or rehabilitation of damaged pipes, manholes, and pump
stations, which involves very high costs. Sewer replacement and
rehabilitation is estimated to cost several billion dollars every
year globally.3,4 This cost is expected to increase as the aging
infrastructure inevitably continues to fail.5,6
Corrosion of concrete sewer is a complex process, involving
microbial sulfide oxidation to produce sulfuric acid, chemical
reactions between acids and cementitious materials, and
physical changes of the concrete structure. In the anaerobic
sections of the sewer, sulfate-reducing bacteria (SRB) will thrive
in the sewer biofilms and sediments, and their activity generates
H2S. The H2S will repartition from the liquid into the gas phase
of the sewer pipes. In the presence of oxygen and through the
surface of the concrete, sulfuric acid produced by the oxidation
of H2S gas reacts with cement in the concrete to form gypsum
and ettringite. The reactions weaken the structural integrity of
the concrete and reduce the load-bearing capacity, eventually
resulting in the collapse of the sewer.
Our recent study, using advanced mineral analytical
techniques, has established a conceptual model of the
physiochemical processes for concrete sewer corrosion.7 The
environmental factors impacting the initiation and corrosion
rate have been investigated through long-term exposure tests in
simulated and real sewers.8−10 These studies enhance the
prediction of sewer service life using mathematical models
based on key environmental parameters that include gaseous
H2S concentrations, relative humidity, and temperature.
However, microorganisms in corroding sewers are proposed
drivers of concrete corrosion by catalyzing sulfuric acid
production from the oxidation of hydrogen sulfide, which is
ubiquitous in sewers due to its production in the anaerobic
regions. Recent studies identified various microorganisms,
including both bacteria and fungi, which are involved in
sulfuric acid production from H2S in the sewer gas.11−14 These
Received: April 27, 2016
Revised: July 1, 2016
Accepted: July 8, 2016
Published: July 8, 2016

© 2016 American Chemical Society 8084 DOI: 10.1021/acs.est.6b02093

Environ. Sci. Technol. 2016, 50, 8084−8092
Environmental Science & Technology
microorganisms, in particular the sulfide-oxidizing bacteria, are
portrayed to be key to the corrosion process of sewer concrete.
The sulfide uptake rate (SUR) by the corrosion-causing
microorganisms is shown to be affected by many factors,
including H2S level and fluctuations, temperature, humidity,
and sewer-headspace air flow.15−17 SUR is proportional to the
corrosion rate by assuming that a constant part of sulfide
uptake will lead to actual corrosion due to the formation of
sulfuric acid.18−21 One important factor that has lacked
investigation is the impact of sewage inoculation on the
corrosion microbial community and on the corrosion processes.
Sewage inoculation onto the corroding sewer surfaces occurs
naturally due to transient high-flow (flooding) events or splash
caused by abrupt flow changes (e.g., slope and flow-direction
changes or water-drop structures). Traditionally, it is believed
that the concrete surface near the sewage level in gravity sewers
has much higher corrosion than other exposed surfaces. This
has been confirmed by long-term concrete corrosion experi-
ments conducted in laboratory corrosion chambers8 and
indicated the importance of sewage to the corrosion in sewers.
In general, the sewer surface is wetted by condensation that
is related to the high humidity of the headspace and the low
temperature of sewer walls. At certain places on the sewer wall,
wetting by wastewater will occur occasionally. Surface wetting
by wastewater not only provides water, nutrients, and trace
elements for the development of corrosion biofilm but will also
inoculate the concrete with various microorganisms present in
wastewater. Depending on the inoculation duration and the
hydraulic strength, the sewage might neutralize the acid
produced by microorganisms in the corrosion layer or even
scour the biofilm together with the corrosion products. This
research aims to investigate the microbial corrosion commun-
ity, corrosion development, and corrosion rates of concrete
coupons that are exposed to a simulated sewer environment
and are being inoculated repetitively with real wastewater at
different frequencies. The expected outcomes can be utilized to
mitigate corrosion through the optimization of the sewer
operations.
2. MATERIALS AND METHODS
2.1. Concrete Coupons. To represent both newly built
and existing sewer pipes, coupons were prepared from new and
old sewer concrete pipes. The so-called fresh concrete coupons
were cut from a new sewer pipe (1.2 m in diameter and 0.07 m
in thickness) obtained from a sewer-pipe manufacturer
(HUMES; Sydney, Australia). The precorroded coupons were
prepared from corroded concrete sewer slabs that have been in
service for over 70 years (Sydney Water Corporation; Sydney,
Australia). After cutting, the coupons were washed in fresh
water and then dried at 60 °C for 3 days to achieve similar and
stable initial water contents. The coupons were embedded as
pairs (one fresh and one precorroded) in stainless steel frames
using epoxy (R180 epoxy and H180 hardener; Fiber Glass
International; Queensland, Australia), with the steel frame
providing a reference point for determining the change in
thickness due to corrosion.8 The coupons were exposed to the
gas phase of laboratory corrosion chambers, as described below
in section 2.2.
Using advanced mineral analysis, it was previously reported
that quartz (aggregates and sand particles) and calcium silicate
account for around 57.3% and 13.1% and 47.8% and 22.2% of
the fresh and precorroded concrete, respectively.22 The
remaining major compounds in the concrete were other
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calcium−iron−potassium aluminum silicates (e.g., amphibole,
orthoclase, and plagioclase). The results indicate that the
abundance of the major mineral compounds of the precorroded
and fresh coupon were similar. The corrosion products, such as
gypsum and ettringite, were present on the exposed surface area
of the precorroded coupons.
2.2. Corrosion Chamber and Exposure Conditions. A
total of two corrosion chambers were operated with gas-phase
levels of 10 and 25 ppm of H2S to simulate the corrosive
environment of real sewers. Details of the chamber operation
and control of conditions has been described previously.9 To
achieve the specified H2S gaseous concentrations in the
corrosion chamber, we employed a program logic controller
to trigger the corrosion-resistant solenoid pump (Bio-Chem
Fluidics model 120SP2440-4TV; Boonton, NJ) for the addition
of Na2S solution to acid (13% HCl), according to the
monitored H2S concentrations using a OdaLog type 2 gas
detector with a range between 0 and 200 ppm (App-Tek
International Pty Ltd.; Brendale, Australia). The temperature
and relative humidity were controlled at around 25 °C and
100%. Temperature and H2S variations were within 1 °C and 2
ppm, respectively. The environmental factors were checked
regularly to ensure the correct operation of the corrosion
chambers. The coupon pairs were arranged in the corrosion
chambers with the original internal surface of the pipe,
designated as the surface exposed to the chamber gas phase.
For each chamber, five coupon pairs (one fresh and one
precorroded concrete coupon) were used for different
treatment purposes in this study. One coupon pair was used
as the control with no wastewater inoculation. The other
coupon pairs were inoculated regularly, as described in section
2.3; two were inoculated with wastewater by flooding, and the
other two were inoculated by spraying. After 12 months of
exposure in the corrosion chambers, the coupons were
destructively sampled, with half of each coupon being used
for the measurement of corrosion loss and sulfur compounds
(350 day) and the other half used for analysis of the corrosion
microbial communities (480 day).
2.3. Wastewater Inoculation of the Coupons. Before
the inoculation and exposure tests, the coupons were washed
with high-pressure water to remove the existing corrosion layer
(including associated biofilm) and ensure that all coupons were
starting at similar initial states. After washing, the surface pH
and H2S uptake rates were measured (as described in section
2.4) to confirm the comparable status of the coupons. A total of
two types of inoculation, namely flooding with diluted
wastewater (50% strength) or spraying with wastewater, were
used in this study. Domestic wastewater collected from a local
wet well at the Robertson Park pump station (Indooroopilly,
Brisbane, Australia) was used for the inoculation. The sewage
typically contained sulfide (<3 mg-S/L), sulfate (10−25 mg-S/
L), volatile fatty acids (50−100 mg-COD/L), and total COD
and soluble COD (450−600 mg/L and 260−450 mg/L,
respectively). The sewage was stored in a cold room at 4 °C
and heated to 20 °C before being used. The inoculation
approaches mimicked sewer situations when rain-diluted
wastewater floods the sewer pipe surface or when wastewater
splash occurs due to flow turbulence. The experimental
concrete coupons were removed from the corrosion chambers
and inoculated by flooding monthly or by spraying weekly, as
described below. After inoculation, the coupons were replaced
in the chambers for continuous exposure to sewer conditions.
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To perform the flooding inoculation of coupons, we used a
wastewater-recirculation system (Figure SI-1). This was an
enclosed pipe of Φ14 cm × 100 cm long that housed four sets
of coupons in steel frames. A wastewater flow mimicking a
sewer flood event was used to flood the coupons placed within
the pipe. This was achieved by passing diluted wastewater (1:1
with dechlorinated tap water) through the pipe, using a Husky
1040 pump, at a velocity of 0.8 m/s. Each flooding inoculation
lasted for 4 h to simulate a transient high flow in sewers due to
heavy rainfalls.
For the spray inoculation, a hand-pump sprayer was used to
deliver a calibrated amount of 2−4 mL of wastewater to the
coupon surface. This provided a water film of 30−60 μm in
depth to the coupon surface. The wastewater was filtered to
remove solids that might block the sprayer nozzle (Whatman
coarse filter paper, 10 μm nominal pore) prior to being
uniformly applied to the coupons with a hand sprayer.
2.4. Monitoring the Corrosion of the Coupons. To
monitor the corrosion development on the coupons, we
measured the surface pH using a flat surface-pH electrode
(Extech PH150-C concrete pH kit, Extech Instruments). A
total of four measurements were made on randomly selected
spots on the coupon surface to determine an average value. The
sulfide uptake rate of the corroding concrete coupon pairs was
measured using a specifically designed SUR reactor to obtain
H2S profiles.23
From the fresh coupons, scraping samples were taken as
described by Jiang, Sun, Keller, and Bond9 to determine the
concentrations of sulfur compounds. For the precorroded
concrete, the exposed surface of coupons was washed using a
high-pressure washer (Karcher K 5.20 M). Samples were taken
from the wash-off water and preserved with a sulfide
antioxidant buffer solution.24 A Dionex ICS-2000 IC with an
AD25 absorbance (230 nm) and a DS6 heated conductivity
detector (35 °C) was used to measure the soluble sulfur
species.
The actual loss of concrete from the coupons due to
corrosion was determined using a photogrammetry approach.25
Photos (five for each coupon) at different orientations were
used to construct a 3D image of the exposed surface of the
coupon. The relative distance of the surface to the stainless
steel frame (as a reference plane) was obtained before and after
the exposure test in the corrosion chambers. The decrease in
thickness after exposure was then calculated by subtracting the
average thickness after washing from the average thickness
before exposure. It was shown that the photogrammetric
process can reliably detect corrosion loss as small as 0.1 mm in
a calibration process.26
2.5. Microbial Community Analysis. The corrosion
layers on the surface of each coupon were scraped with a
sterile surgical scalpel directly into sterile 50 mL polypropylene
containers. Only loose corrosion layers were collected; concrete
and large aggregate were avoided. The wet samples were stored
at 4 °C for less than 24 h until used for DNA extraction.
Prior to DNA extraction, the microbial cells were separated
from the corrosion layer matrix, essentially as previously
described for separation of cells from mine tailings.27 To loosen
the microbial cells from the corrosion layer matrix, the samples
were first sonicated after the addition of 25 mL of a 0.2%
sodium pyro-phosphate solution (pH ∼ 4.0). This pH was used
to match the sample pH (ranging from pH 2 to 4) and avoid
cell lysis. The sonication was performed on ice for 30 s with
output level 7 followed by 30 s resting on ice, and then the
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sonication and resting process was repeated twice. Sonicated
samples were then loaded onto a sucrose density gradient and
treated as described by Li et al.27 After the cells were
concentrated from the sucrose gradient, they were washed in
pyro-phosphate buffer (pH 4) and centrifuged to pellet the cells
(18000g for 10 min at 4 °C). The cell pellets were finally
resuspended in pyrophosphate buffer (pH 7.5), and then cells
were lysed and DNA extracted using the Fast DNA SPIN Kit
for Soil.
To perform 16S rRNA gene amplicon pyrosequencing,
extracted DNA samples were provided to the Research and
Testing Laboratory (Lubbock, Texas). PCR amplifications were
conducted in 25 μL reactions with Qiagen HotStar Taq master
mix (Qiagen Inc.; Valencia, California). Modified universal
primers 28F and 519R were used for the amplification of
approximately 500 bp of the variable regions V1−V3 of the 16S
rRNA gene.28 Amplification products were confirmed with
eGels (Life Technologies; Grand Island, New York), and the
confirmed products were sequenced on the Genome Sequencer
FLX instrument using Titanium protocols and reagents
(Roche; Indianapolis, IN).29,30
Data generated from high-throughput sequencing were
analyzed via a pipeline that consisted of denoising and chimera
detection followed by microbial diversity analysis.31,32 Denois-
ing was performed to remove short sequences, singleton
sequences, and noisy reads. Chimera detection was then
performed using the UCHIME chimera detection software
executed in de novo mode on the clustered data.33 The
denoised sequence data, excluding chimeras, were then
screened for quality so that sequences must be at least half
the expected length given the primer sets used and contain a
valid error-free barcode. Sequences that passed the quality
control screening were assigned to samples on the basis of their
nucleotide barcode. Following that, dereplication of sequences
was performed using the USEARCH prefix dereplication
method.34 Next, all singleton clusters were removed, and the
remaining sequences were sorted by cluster size from largest to
smallest and trimmed down to the same size. The UPARSE
algorithm was then employed to determine the operational
taxonomic units (OTUs) using the trimmed sequences on the
basis of their sequence similarity at 97%.35 Taxonomic
assignments were determined using USEARCH with the
centroid sequence from each cluster against the NCBI
database.31,36
α diversity within each sample was computed from the
normalized data sets by phylogenetic diversity whole tree index
in QIIME.32 Next, rarefaction curves were generated. β
diversity was also computed from the trimmed taxonomic
data sets using QIIME, and a principal component analysis
(PCA) plot was generated using the weighted UniFrac metric,
which includes phylogenetic β diversity measures. Differences
in the structure of microbial communities were visualized by a
heat map via R (version 3.1.0), as described previously.14
2.6. Gompertz Model for the Development of Sewer
Corrosion. For the modeling, the corrosion activity is
represented by SUR data because they reflect the potential
maximum corrosion rate as reported previously.23 Due to the
constant level of relative humidity in the corrosion chambers, a
fixed percentage of sulfuric acid (a part of SUR) had been lost
with the dripping of condensation water. Also, the fluctuation
of H2S concentrations during the experimental period was
minimal, which implies that the primary product of sulfide
oxidation would be sulfuric acid.15,37 These assumptions
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Figure 1. Surface pH of concrete coupons subjected to periodic flooding or spray inoculation with wastewater in corrosion chambers. The dot-and-
dashed line indicates the time of coupon retrieval (350 days) for the analysis of corrosion loss and sulfur compounds.

Figure 2. Sulfide uptake rates of concrete coupons exposed to sewer conditions and subjected to periodic flooding or spray inoculation with
wastewater in corrosion chambers. The curves are best-fit lines using the Gompertz model. The dot-and-dashed line indicates the sample retrieval for
the analysis of corrosion losses and sulfur compounds.

support the proportionality between SUR and the corrosion
rate. An empirical model based on Gompertz equation was
employed to model the development of corrosion activity, i.e.,
sulfide uptake rate, on concrete coupons exposed to sewer
conditions. A similar model was used to describe the recovery
of sulfate-reduction activity of sewer biofilms after exposure to
elevated pH and nitrite.38,39
−α(t − t0)
rSUR = rSUR,0 + rSUR,maxe(−e )
(1)
where rSUR is the SUR of concrete in sewers (mgS/m2·h); t is
the time since the initiation (day); rSUR,0 is the SUR due to
physicochemical processes (mgS/m2·h); α is the relative
increasing rate of SUR at time t0 (d−1); rSUR is the maximum
sulfide uptake rate achieved at steady state (mgS/m2·h); and t0
is the time (day) when rSUR is at its maximum. The model
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adopted here represents the growth of corrosion biofilm and
the associated sulfide-oxidation activity on concrete sewer when
exposed to a H2S-containing environment. For each set of
parameters estimated for the Gompertz equation, the increasing
rate of SUR (K) could be determined using equations below:40
α(rSUR, max − rSUR, 0)
K=
2.7183 (2)
where 2.7183 is the Euler’s constant (e). To identify the effects
of wastewater inoculation on the corrosion activity on the basis
of SUR, the Gompertz equation was fitted to the experimental
data, and model parameters were determined using SigmaPlot
version 12.0. rSUR,max is used to compare the potential corrosion
activity when microbial biofilm is fully developed. K is used to
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Figure 3. Corrosion rate (A) and the expansion level of the corrosion layer (B) for concrete coupons exposed to sewer conditions and subjected to
periodic flooding or spray inoculation with wastewater in corrosion chambers.

Figure 4. Heatmap representing the relative abundance of the predominant microbial types (>0.05%) detected in the corrosion layers of the sewer
concrete coupons subjected to sewer conditions for 480 days and periodic wastewater inoculation. The labels of the columns at the bottom indicate
the sample parameters, such that P or F indicates a precorroded or fresh coupon; either 10 or 25 indicates the ppm of H2S exposure level; and F, C,
or S represent inoculation by flooding, no inoculum, or inoculation by spraying, respectively (e.g., the label of P25F indicates a precorroded coupon,
exposed to 25 ppm of H2S, and inoculated by flooding).

indicate the growth rate of the corrosion biofilm, which might Table 1. Change of Microbial Abundance Induced by the
be heavily affected by the wastewater inoculation. Periodic Flooding and Spray-Inoculation of Concrete
Coupons with Wastewater in Comparison to the Control
3. RESULTS AND DISCUSSION Coupon
3.1. Surface pH. During microbially induced corrosion, the inoculation species change of abundance
decrease of surface pH is mainly caused by the continuous acid flooding Acidiphilium sp 10% increase (fresh concrete; 10 and 25
ppm of H2S)
production by sulfide-oxidizing bacteria and the subsequent
At. caldus 30% increase (precorroded concrete; 25
neutralization of alkaline cement materials. Surface pH is thus a ppm of H2S)
good indicator of the corrosion development. Although the At. thiooxidans 30% decrease (precorroded concrete; 25
starting levels of surface pH varied, the surface pH decreased ppm of H2S)
clearly with time when the coupons were exposed to 10 and 25 spray At. caldus 20% increase (fresh concrete; 25 ppm of
H2S)
ppm of H2S (Figure 1). It reached a level of around 2−3 after Leptospirillum sp 10% decrease (precorroded concrete; 25
350 days when coupons were sampled for the measurement of ppm of H2S)
corrosion losses. Such a low surface pH was favorable for the Mycobacterium sp 15% increase (precorroded concrete; 25
colonization of acidophilic sulfide-oxidizing bacteria (SOB) and ppm of H2S)
indicated an active corrosion activity.
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Figure 5. Principle component analysis of the microbial community
detected on the concrete samples exposed to different levels of H2S
(A), the two types of concrete exposed to 10 ppm of H2S (B), and the
two types of concrete exposed to 25 ppm of H2S (C). The arrows
indicate the predominant microbial species (>0.05%) detected in the
corrosion layers of the concrete coupons, subjected to sewer
conditions for 480 days and periodic wastewater inoculation.
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For concrete coupons subjected to wastewater inoculation,
the surface pH was affected by the wastewater, which had a pH
around 7.5. Therefore, wastewater sprayed on the corroding
concrete surface would have some effect of neutralizing the
sulfuric acid produced in the corrosion biofilm. However,
flooding inoculation might remove some of the SOB biofilm all
together with other corrosion products. The observed surface
pH would reflect the combined effects of both biological acid
production and the multiple effects of wastewater inoculation.
Although the decreasing trend of surface pH is clear, a
difference between the control and inoculated coupons was not
observed. Consequently, the surface pH was not indicating that
inoculation was contributing to more rapid levels of concrete
corrosion. It should be noted that surface pH is an
instantaneous measurement of the corrosion status, which
reflects the conditions at that time point. Thus, it serves well as
an indicator of the corrosion progression but should be
assessed together with other parameters to quantitatively assess
the level of corrosion.
3.2. Sulfide Uptake Rate. Sulfide uptake rate has been
used as an indicator of concrete corrosion development
through the measurement of the activity of the microbial
biofilms in situ of the concrete corrosion layers mediating the
oxidation of hydrogen sulfide.23 With the corrosion develop-
ment, the SUR of all concrete coupons increased gradually with
exposure time (Figure 2). The SUR of the fresh coupons
showed similar SUR values for the two different H2S
concentrations (Figure 2A,C), indicating that H2S is not a
limiting factor for these conditions. For precorroded coupons,
the SUR is slightly higher for the 25 ppm of H2S than the 10
ppm of H2S conditions (Figure 2B,D). Also, the sulfide-
oxidizing activity on precorroded coupons were more active
than fresh coupons; this is likely due to the lower starting levels
of surface pH on the precorroded coupons, which would
facilitate a faster establishment of SOB biofilms.
To further identify the effects of wastewater inoculation on
the SUR, the Gompertz model parameters were determined
and shown in Table SI-1. It is clear that the maximum SUR
(rSUR,max) was increased due to wastewater inoculation for both
types of concrete at the two different level of H 2 S
concentrations. This implies that the actual corrosion losses
and the ultimate corrosion rates would be higher on concrete
coupons subject to wastewater inoculation. Also, the control
coupons reached the maximum SUR at an earlier exposure time
than did the inoculated concrete coupons (especially the
precorroded concrete), which were still increasing at the end of
500 days (Figure 2). For another important parameter, K,
which is an indicator of the growth rate of corrosion-inducing
biofilm, wastewater inoculation increased the growth of biofilm
on fresh concrete and exerted the opposite effects to the
precorroded concrete. This is likely due to the overall balance
of positive and negative effects discussed below.
Wastewater inoculations might promote the biological
activity (positive effects) by microbial inoculation and provide
necessary nutrients and microelements. However, it may reduce
the SUR (negative effects) if biofilm scouring of the loose and
soft corrosion layer on concrete surface occurred with
wastewater flooding. Also, the sprayed water residing in the
corrosion layer could possibly temporarily reduce the SUR by
blocking the mass transfer of H2S from the gas to the corrosion
layer. It seems for the fresh concrete that the positive effects of
wastewater inoculation exceeded the negative effects. This
suggests that when the surface pH is higher (near neutrality in
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this case), repeated inoculum aids the development of the
neutrophilic and acidophilic SOB.
For precorroded concrete, the corrosion biofilm growth rate
K was actually reduced for inoculated coupons in comparison
to the control. This might be caused by the overall higher
negative effects than positive effects. It is observed that
precorroded coupons with obvious corrosion layers were
more susceptible to the biofilm-scouring effect (flooding) and
also had higher water-retention capacity (spray) compared to
the fresh coupons. Additionally, the acidophilic biofilm is likely
more readily established on these coupons with a lower starting
surface pH, and thus, the advantage of repeated inoculum was
not evident.
3.3. Corrosion Losses. The mass loss of concrete during a
certain time period is the ultimate measure of the corrosion
occurring. It was found that inoculated coupons constantly
showed higher corrosion losses than control coupons for both
fresh and precorroded concrete (Figure 3A). The observed
corrosion losses were comparable to ranges reported in other
laboratory and field studies,10,41 although those studies did not
report the effects of wastewater inoculation. Unlike surface pH,
the SUR data clearly support this observation. This is likely due
to the instantaneous nature of surface pH measurements, and
thus, they are inadequate for quantifying the long-term
corrosion.
It was seen that the precorroded coupons had consistently
higher corrosion rates than the fresh coupons. This is likely due
to the different levels of microbial development on the two
types of coupons. The relatively low microbial colonization on
fresh coupons means its SUR and corrosion activity was limited
by the abundance of microbes in the corrosion layer. It is also
interesting to note that the corrosion losses of fresh coupons
were similar for both 10 and 25 ppm of H2S. In contrast, the
corrosion rates were about 2 mm per year greater for the
inoculated precorroded coupons at the higher levels of H2S.
Consequently, the SUR and corrosion activity of the well-
established SOB biofilms on the precorroded coupons were
limited by the availability of the substrate, i.e., H2S. This is
supported by the different SUR detected of the precorroded
concrete coupons with different levels of H2S (Figure 2) and
the higher levels of sulfate detected on the corrosion layers with
increased H2S (Figure SI-2).
Expansion of the concrete coupons was detected, and the
sprayed coupons showed a higher expansion percentage than
did flooded coupons (Figure 3B). The expansion of concrete is
caused by the formation of various corrosion products, mainly
gypsum and ettringite, increasing the volume of the original
concrete by 124% to 700%.7,42,43 The difference in expansion
detected here may be due to the flooding inoculum washing off
some of the loose corrosion products.
3.4. Microbial Community. With higher corrosion rates
measured on concrete coupons subjected to periodic waste-
water inoculation, it is highly relevant to examine the microbial
communities that developed between the inoculated and
control coupons. Examination of the within community
diversity (richness and evenness of microbial species) showed
that inoculation increased slightly the phylogenic diversity of
the microbial community on the concrete coupons (Figure SI-
3). Other factors leading to slight increases in diversity were
spraying in comparison to flooding inoculum, precorroded
concrete in comparison to fresh concrete, and higher H2S
concentration (Figures SI-4 and SI-5). It is apparent that
increased community diversity occurred in conditions when
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increased SUR and corrosion rates were detected. However, the
increase in community diversity is limited and not statistically
significant, likely due to the complicated effects of different
factors including wastewater inoculation approaches, the level
of H2S, and types of concrete.
The relative abundance of the predominant microbial types
(>0.05%) was detected on the coupon corrosion layers (Figure
4). The corrosion community detected on the concrete
coupons is mainly composed of acidophilic SOB (in particular,
those of the Acidithiobacillus genera). A recent study on
microbial communities associated with corrosion fronts also
identified acidophilic Acidithiobacillus spp. as the dominant
sulfur oxidizer after the microbial succession from a community
including heterotrophic, nitrogen-fixing, and sulfur-oxidizing
genera in conjunction with decreasing pH.44 These are
chemolithoautotrophic γ-proteobacteria deriving their energy
from the oxidation of inorganic sulfur compounds and ferrous
iron.11 At. thiooxidans was the most dominant species observed
on the corroding coupons, accounting for 80−95% of the total
microbial community for concrete coupons exposed to 10 ppm
of H2S. Another species, At. caldus, was detected at 35−50% of
abundance, comparable to that of At. thiooxidans for some
coupons exposed to 25 ppm of H2S. Other Acidithiobacillus
species, At. ferrooxidans and Acidithiobacillus sp., were detected
at relatively high levels, i.e., 1.5−9%, on coupons exposed to 25
ppm of H2S. The dominance of Acidithiobacillus spp. in a sewer
corrosion environment is consistently reported by many
previous studies using both culture-dependent and molecular
techniques.12,45−48
Acidiphilium species were detected on the fresh concrete
coupons exposed to both H2S levels. Acidiphilium are obligate
aerobic, sulfur-oxidizing heterotrophs.49 Many acidophilic SOB,
including the dominant species At. thiooxidans, are inhibited by
the presence of organic molecules.12,45,50 Heterotrophic
microorganisms such as Acidiphilium play an important role
in scavenging the organic compounds inhibitory to sulfur-
oxidizing autotrophs. The coexistence of many SOB and
acidophilic heterotrophic bacteria such as Acidiphilium was
likely important for the colonization of the fresh concrete
coupons. Acidiphilium was not detected at meaningful
abundancies on the precorroded coupons. In that instance,
other heterotrophs might take over such an organic scavenger
role in those highly developed corrosion communities.
Mycobacterium spp. were also detected at notable abundan-
cies, especially on the precorroded coupons; these were at 17%
for the P25S sample (Figure 4). They were reported previously
in concrete corrosion samples from real sewer environments
and are known to be able to perform sulfur oxidation.14,45
Leptospirillum ferriphilum and Leptospirillum sp. were detected
at 2−12% on concrete coupons exposed to 25 ppm of H2S.
These iron-oxidizing bacteria normally existing in acid mine
drainage have been detected at notable levels in concrete-
corrosion samples previously.12
Differences in the community structure of the corrosion
layers were detected between the inoculated and control
coupons (Table 1). The differences include the abundance of
key sulfide-oxidizing bacteria (At. thiooxidans and At. caldus),
the heterotrophic Acidiphilium sp., Mycobacterium sp., and
Leptospirillum sp. The greatest change detected between the
control and the inoculated coupons was the increased
abundance of the Acidithiobacillus species (Table 1), implicating
that these were important for the increased corrosion rates
measured on the inoculated coupons. The change of abundance
DOI: 10.1021/acs.est.6b02093
Environ. Sci. Technol. 2016, 50, 8084−8092
Environmental Science & Technology
also depended on the type of concrete and the level of H2S,
thus implying that these other factors played a role on top of
microbial inoculation.
Using principle component analysis, we compared variation
in the microbial community compositions from the coupon
corrosion layers to detect clustering and relatedness among
those with respect to the different H2S levels and the different
types of concrete (Figure 5). Samples with similar microbial
community structures tend to cluster together.
It was clear that the coupon microbial communities were
separated according to the level of H2S exposure (Figure 5A). It
was also evident that the coupons exposed to 10 ppm of H2S
had very similar microbial community structures, and those
exposed to 25 ppm of H2S had more diverse populations
(Figure 5A). The fast establishment of SUR activity on coupons
exposed to 25 ppm of H2S means the SOB biofilm was at a
more mature state than those exposed to 10 ppm of H2S. Due
to the mass transfer of two important gases, i.e., H2S and
oxygen, from sewer gas to the corrosion layer, high H2S levels
(25 ppm) would lead to a deeper penetration depth than the
low H2S level (10 ppm). It was also found that the pH increases
with the depth.7 Thus, a higher H2S level lead to a wider range
of pH and sulfide levels suitable to SOB proliferation, implying
a higher microbial diversity in those samples exposed to 25
ppm of H2S.
Figure 5B,C also show that the microbial community
detected on the concrete coupons clustered separately for the
two concrete types when exposed to both 10 and 25 ppm of
H2S. The two types of concrete used were reported to have
different ratios of silicate to calcium, acid-neutralization
capacity, and pore-size distribution.7,10 These properties will
directly impact the chemical composition of corrosion layer,
pH, and moisture content; all of these can contribute as
selection force of different microbial species.
3.5. Implications for Sewer Corrosion Management.
The findings from this study support the idea that periodic
wastewater inoculation promotes sewer corrosion. This may be
partially due to the inoculum inducing a more-diverse microbial
community in addition to other physiochemical processes
enhancing corrosion. Due to climate change, extreme weather
events such as floods and droughts are occurring more often.
These create new challenges for the management of sewer
assets, as these events would cause increased exposed-concrete
surfaces (drought and reduced water use) and chances of
wastewater inoculation (more floods in sewers due to high
rainfall). The service life of sewers might be severely shortened
due to the climate change and weather extremes, which vary
from region to region.
To minimize the corrosion of concrete in sewers, it would be
desirable to minimize the inoculation of exposed surfaces. This
study indicated that monthly flooding actually promoted
corrosion. However, the maintenance of a constant water
level in sewers is difficult to achieve and might lead to high
corrosion activity right above the water line.8 It is also difficult
to avoid the negative effects caused by wastewater splash.
However, improved sewer design can be made to minimize the
sharp change of slope or flow directions and sewer drops.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.6b02093.
8091
Article
Figure S1: wastewater-recirculation system for flooding
inoculation. Figure S2: sulfate detected on concrete
surface. Figure S3−S5: rarefaction-community diversity
detected on coupons exposed to sewer conditions. Table
S1: parameters determined for Gompertz model based
on the experimental data of sulfide uptake rates measured
over 500 days. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +61 7 3346 3217; fax: +61 7 3365 4726; e-mail: g.jiang@
awmc.uq.edu.au and guangming.jiang@gmail.com.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors acknowledge the financial support provided by the
Australian Research Council and many members of the
Australian water industry through LP0882016, the Sewer
Corrosion and Odor Research (SCORe) Project (www.score.
org.au). Dr. Guangming Jiang is the recipient of a Queensland
State Government Early Career Accelerate Fellowship.
■
REFERENCES
(1) USEPA. Detection, Control, and Correction of Hydrogen Sulfide
Corrosion in Existing Wastewater System; Office of Wastewater
Enforcement and Compliance: Washington, DC, 1992.
(2) Zhang, L.; De Schryver, P.; De Gusseme, B.; De Muynck, W.;
Boon, N.; Verstraete, W. Chemical and biological technologies for
hydrogen sulfide emission control in sewer systems: A review. Water
Res. 2008, 42 (1−2), 1−12.
(3) Pikaar, I.; Sharma, K. R.; Hu, S.; Gernjak, W.; Keller, J.; Yuan, Z.
Reducing sewer corrosion through integrated urban water manage-
ment. Science 2014, 345 (6198), 812−814.
(4) Jiang, G.; Sun, J.; Sharma, K. R.; Yuan, Z. Corrosion and odor
management in sewer systems. Curr. Opin. Biotechnol. 2015, 33, 192−
197.
(5) Sydney, R.; Esfandi, E.; Surapaneni, S. Control concrete sewer
corrosion via the crown spray process. Water Environ. Res. 1996, 68
(3), 338−347.
(6) USEPA. Hydrogen Sulphide Corrosion in Wastewater Collection and
Treatment System; USEPA: Washington, DC, 1991.
(7) Jiang, G.; Wightman, E.; Donose, B. C.; Yuan, Z.; Bond, P. L.;
Keller, J. The role of iron in sulfide induced corrosion of sewer
concrete. Water Res. 2014, 49, 166−174.
(8) Jiang, G.; Keller, J.; Bond, P. L. Determining the long-term effects
of H2S concentration, relative humidity and air temperature on
concrete sewer corrosion. Water Res. 2014, 65, 157−169.
(9) Jiang, G.; Sun, X.; Keller, J.; Bond, P. L. Identification of
controlling factors for the initiation of corrosion of fresh concrete
sewers. Water Res. 2015, 80, 30−40.
(10) Wells, T.; Melchers, R. E. Modelling concrete deterioration in
sewers using theory and field observations. Cem. Concr. Res. 2015, 77,
82−96.
(11) Kelly, D. P.; Wood, A. P. Reclassification of some species of
Thiobacillus to the newly designated genera Acidithiobacillus gen.
nov., Halothiobacillus gen. nov and Thermithiobacillus gen. nov. Int. J.
Syst. Evol. Microbiol. 2000, 50, 511−516.
(12) Okabe, S.; Odagiri, M.; Ito, T.; Satoh, H. Succession of sulfur-
oxidizing bacteria in the microbial community on corroding concrete
in sewer systems. Appl. Environ. Microbiol. 2007, 73 (3), 971−980.
(13) Ling, A. L.; Robertson, C. E.; Harris, J. K.; Frank, D. N.; Kotter,
C. V.; Stevens, M. J.; Pace, N. R.; Hernandez, M. T. Carbon dioxide
and hydrogen sulfide associations with regional bacterial diversity
patterns in microbially induced concrete corrosion. Environ. Sci.
Technol. 2014, 48 (13), 7357−64.
DOI: 10.1021/acs.est.6b02093
Environ. Sci. Technol. 2016, 50, 8084−8092
Environmental Science & Technology
(14) Cayford, B. I.; Dennis, P. G.; Keller, J.; Tyson, G. W.; Bond, P.
L. High-throughput amplicon sequencing reveals distinct communities
within a corroding concrete sewer system. Appl. Environ. Microbiol.
2012, 78 (19), 7160−7162.
(15) Sun, X.; Jiang, G.; Bond, P. L.; Keller, J. Impact of fluctuations in
gaseous H2S concentrations on sulfide uptake by sewer concrete: The
effect of high H2S loads. Water Res. 2015, 81 (0), 84−91.
(16) Nielsen, A. H.; Hvitved-Jacobsen, T.; Vollertsen, J. Effect of
Sewer Headspace Air-Flow on Hydrogen Sulfide Removal by
Corroding Concrete Surfaces. Water Environ. Res. 2012, 84 (3),
265−273.
(17) Joseph, A. P.; Keller, J.; Bustamante, H.; Bond, P. L. Surface
neutralization and H2S oxidation at early stages of sewer corrosion:
Influence of temperature, relative humidity and H2S concentration.
Water Res. 2012, 46 (13), 4235−4245.
(18) Apgar, D.; Witherspoon, J.; Easter, C. Minimization of Odor and
Corrosion in Collection Systems Phase 1; Water Environment Research
Foundation: Alexandria, VA, 2007
(19) USEPA. Process Design Manual for Sulfide Control in Sanitary
Sewer Systems; USEPA: Washington, DC, 1974.
(20) Pomeroy, R. D. The Problem of Hydrogen Sulphide in Sewers, 2nd
ed.; Clay Pipe Development Association Limited: London, 1990; p 31.
(21) De Belie, N.; Monteny, J.; Beeldens, A.; Vincke, E.; Van Gemert,
D.; Verstraete, W. Experimental research and prediction of the effect
of chemical and biogenic sulfuric acid on different types of
commercially produced concrete sewer pipes. Cem. Concr. Res. 2004,
34 (12), 2223−2236.
(22) Sun, X.; Jiang, G.; Chiu, T. H.; Zhou, M.; Keller, J.; Bond, P. L.
Effects of surface washing on the mitigation of concrete corrosion
under sewer conditions. Cem. Concr. Compos. 2016, 68, 88−95.
(23) Sun, X.; Jiang, G.; Bond, P. L.; Wells, T.; Keller, J. A rapid, non-
destructive methodology to monitor activity of sulfide-induced
corrosion of concrete based on H2S uptake rate. Water Res. 2014,
59, 229−238.
(24) Keller-Lehmann, B.; Corrie, S.; Ravn, R.; Yuan, Z.; Keller, J.
Preservation and simultaneous analysis of relevant soluble sulfur
species in sewage samples. In Proceedings of the Second International
IWA Conference on Sewer Operation and Maintenance; Vienna, Austria,
26−28 October, 2006.
(25) Wells, T.; Melchers, R. E.; Bond, P. Factors involved in the long
term corrosion of concrete sewers. In Corrosion & Prevention,
Australasian Corrosion Association Proceedings of Corrosion and
Prevention, Coffs Harbour, Australia, Nov 15−19; Australasian
Corrosion Association: Victoria, Australia, 2009.
(26) Wells, T.; Melchers, R. E. An observation-based model for
corrosion of concrete sewers under aggressive conditions. Cem. Concr.
Res. 2014, 61−62 (0), 1−10.
(27) Li, X.; Huang, L.; Bond, P. L.; Lu, Y.; Vink, S. Bacterial diversity
in response to direct revegetation in the Pb−Zn−Cu tailings under
subtropical and semi-arid conditions. Ecol. Eng. 2014, 68, 233−240.
(28) Kautz, S.; Rubin, B. E. R.; Russell, J. A.; Moreau, C. S. Surveying
the Microbiome of Ants: Comparing 454 Pyrosequencing with
Traditional Methods To Uncover Bacterial Diversity. Appl. Environ.
Microbiol. 2013, 79 (2), 525−534.
(29) Dowd, S. E.; Callaway, T. R.; Wolcott, R. D.; Sun, Y.;
McKeehan, T.; Hagevoort, R. G.; Edrington, T. S. Evaluation of the
bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-
encoded FLX amplicon pyrosequencing(bTEFAP). BMC Microbiol.
2008, 8, 125.
(30) Ishak, H. D.; Plowes, R.; Sen, R.; Kellner, K.; Meyer, E.; Estrada,
D. A.; Dowd, S. E.; Mueller, U. G. Bacterial diversity in Solenopsis
invicta and Solenopsis geminata ant colonies characterized by 16S
amplicon 454 pyrosequencing. Microb. Ecol. 2011, 61 (4), 821−31.
(31) RTL Genomics Data Analysis Methodology (Last Updated: 04/
29/2016). http://www.researchandtesting.com/docs/Data_Analysis_
Methodology.pdf (accessed June 21, 2016).
(32) Kuczynski, J.; Stombaugh, J.; Walters, W. A.; Gonzalez, A.;
Caporaso, J. G.; Knight, R. Using QIIME to analyze 16S rRNA gene
sequences from microbial communities. Current Protocols in Bio-
8092
Article
informatics Editorial Board 2011, DOI: 10.1002/
0471250953.bi1007s36.
(33) Edgar, R. C.; Haas, B. J.; Clemente, J. C.; Quince, C.; Knight, R.
UCHIME improves sensitivity and speed of chimera detection.
Bioinformatics 2011, 27 (16), 2194−200.
(34) Edgar, R. C. Search and clustering orders of magnitude faster
than BLAST. Bioinformatics 2010, 26 (19), 2460−1.
(35) Edgar, R. C. UPARSE: highly accurate OTU sequences from
microbial amplicon reads. Nat. Methods 2013, 10 (10), 996−8.
(36) Bokulich, N. A.; Rideout, R. J.; Kopylova, E.; Bolyen, E.;
Patnode, J.; Ellett, Z.; McDonald, D.; Wolfe, B.; Maurice, C. F.;
Dutton, R. J.; Turnbaugh, P. J.; Knight, R.; Caporaso, J. G. A
standardized, extensible framework for optimizing classification
improves marker-gene taxonomic assignments. PeerJ PrePrints 2015,
3(e1502), https://doi.org/10.7287/peerj.preprints.934v2.
(37) Nielsen, A. H.; Hvitved-Jacobsen, T.; Jensen, H. S.; Vollertsen, J.
Experimental Evaluation of the Stoichiometry of Sulfide-Related
Concrete Sewer Corrosion. J. Environ. Eng. 2014, 140 (2), 04013009.
(38) Gutierrez, O.; Park, D.; Sharma, K. R.; Yuan, Z. Effects of long-
term pH elevation on the sulfate-reducing and methanogenic activities
of anaerobic sewer biofilms. Water Res. 2009, 43 (9), 2549−2557.
(39) Jiang, G.; Gutierrez, O.; Sharma, K. R.; Yuan, Z. Effects of nitrite
concentration and exposure time on sulfide and methane production
in sewer systems. Water Res. 2010, 44 (14), 4241−4251.
(40) Ukuku, D. O.; Zhang, H.; Huang, L. Growth Parameters of
Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and
Aerobic Mesophilic Bacteria of Apple Cider Amended with Nisin−
EDTA. Foodborne Pathog. Dis. 2009, 6 (4), 487−494.
(41) Vollertsen, J.; Nielsen, A. H.; Jensen, H. S.; Wium-Andersen, T.;
Hvitved-Jacobsen, T. Corrosion of concrete sewers-The kinetics of
hydrogen sulfide oxidation. Sci. Total Environ. 2008, 394 (1), 162−
170.
(42) Parande, A. K.; Ramsamy, P. L.; Ethirajan, S.; Rao, C. R. K.;
Palanisamy, N. Deterioration of reinforced concrete in sewer
environments. Proceedings of the Institution of Civil Engineers-Municipal
Engineer 2006, 159 (1), 11−20.
(43) Monteny, J.; Vincke, E.; Beeldens, A.; De Belie, N.; Taerwe, L.;
Van Gemert, D.; Verstraete, W. Chemical, microbiological, and in situ
test methods for biogenic sulfuric acid corrosion of concrete. Cem.
Concr. Res. 2000, 30 (4), 623−634.
(44) Ling, A. L.; Robertson, C. E.; Harris, J. K.; Frank, D. N.; Kotter,
C. V.; Stevens, M. J.; Pace, N. R.; Hernandez, M. T. High-Resolution
Microbial Community Succession of Microbially Induced Concrete
Corrosion in Working Sanitary Manholes. PLoS One 2015, 10 (3),
e0116400.
(45) Vincke, E.; Boon, N.; Verstraete, W. Analysis of the microbial
communities on corroded concrete sewer pipes - a case study. Appl.
Microbiol. Biotechnol. 2001, 57 (5−6), 776−785.
(46) Islander, R. L.; Devinny, J. S.; Mansfeld, F.; Postyn, A.; Shih, H.
Microbial Ecology of Crown Corrosion in Sewers. J. Environ. Eng.
1991, 117 (6), 751−770.
(47) Sand, W.; Bock, E. Concrete corrosion in the Hamburg Sewer
system. Environ. Technol. Lett. 1984, 5 (12), 517−528.
(48) Santo Domingo, J. W.; Revetta, R. P.; Iker, B.; Gomez-Alvarez,
V.; Garcia, J.; Sullivan, J.; Weast, J. Molecular survey of concrete sewer
biofilm microbial communities. Biofouling 2011, 27 (9), 993−1001.
(49) Dopson, M.; Johnson, D. B. Biodiversity, metabolism and
applications of acidophilic sulfur-metabolizing microorganisms. Envi-
ron. Microbiol. 2012, 14 (10), 2620−2631.
(50) Clark, D. A.; Norris, P. R. Acidimicrobium ferrooxidans gen nov,
sp nov: Mixed-culture ferrous iron oxidation with Sulfobacillus species.
Microbiology 1996, 142, 785−790.
DOI: 10.1021/acs.est.6b02093
Environ. Sci. Technol. 2016, 50, 8084−8092