Construction and Building Materials 248 (2020) 118609

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Construction and Building Materials

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Microbial enhanced corrosion of hydraulic concrete structures under

hydrodynamic conditions: Microbial community composition and
functional prediction
Yi Li a, Mingyue Wan a, Jiming Du a, Li Lin b, Wei Cai a, Longfei Wang a,⇑
a
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Xikang Road #1,
Nanjing 210098, PR China
b
Basin Water Environmental Research Department, Changjiang River Scientific Research Institute, Wuhan, Hubei 430010, PR China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

The adverse hydraulic effects and

microbial action of HCS corrosion
were analyzed.

Microbial corrosion of HCS were
quantified by mass losses and visual
inspection.

Flowing water accelerated the
succession of bacterial community on
HCS.

Nitrogen and sulfur metabolism were
actively involved in microbial actions
on HCS.

a r t i c l e i n f o a b s t r a c t

Article history: Microbial corrosion has long been a threat to engineered structures, but the effects of microbial action on
Received 6 August 2019 hydraulic concrete structures (HCSs), particularly those in freshwater environments, have not been sys-
Received in revised form 25 February 2020 tematically investigated. In this study, the composition and succession of bacterial communities in bio-
Accepted 27 February 2020
films attached to HCSs and the mass loss of concrete were analyzed in a simulated hydrodynamic reactor.
Available online 19 March 2020
After a 335-day experiment, the mass losses of aseptic concrete in flowing and static water were 3.55%
and 2.96%, respectively. Biofilms clearly formed on concrete cultured in representative freshwater, and
Keywords:
the mass losses of biofilm-attached concrete placed in flowing and static water were 4.65% and 4.04%,
Hydraulic concrete structures
Biofilm
respectively, at the end of the experiment. These results indicate that microbial action was actively
Corrosion involved in the corrosion process, contributing 32.3–36.6% of the total mass loss. Obvious differences
Functional prediction were found in the microbial community distribution under flowing and static water conditions, in which
Microbial action temperature and surface pH were the main factors. The formation of a relatively stable community struc-
ture of attached biofilms was observed in flowing water since day 165, whereas the community structure
plateaued and was prolonged to 225 days for concrete cultivated in static water. A functional prediction
analysis revealed that functional bacteria related to nitrogen and sulfur metabolism accounted for 70% of
all functionally metabolizing bacteria cultivated under both conditions. The abundances of sulfate reduc-
ers and nitrite reducers decreased remarkably with the succession of microbial communities in flowing
and static water, while the percentages of sulfur oxidizers and sulfide oxidizers increased gradually. Our

⇑ Corresponding author at: College of Environment, Hohai University, Nanjing 210098, PR China.
E-mail address: lfwang@hhu.edu.cn (L. Wang).

https://doi.org/10.1016/j.conbuildmat.2020.118609
0950-0618/Ó 2020 Elsevier Ltd. All rights reserved.
2 Y. Li et al. / Construction and Building Materials 248 (2020) 118609
results provide future hypotheses for more quantitative studies focusing on special groups and functional
genes in HCSs and contribute to optimizing microbial control in water conservancy projects for
freshwater environments.
1. Introduction
Hydraulic concrete structures (HCSs) are one of the most critical
components in the infrastructure of water conservancy projects
[1]. The surfaces of HCSs generally suffer from serious corrosion
during long-term service [2,3]. The annual rehabilitation cost for
existing HCS infrastructures has been reported to be hundreds of
millions of dollars [4]. Corrosion of concrete is a complex process,
during which chemical reactions between multiple environmental
factors and cementitious materials, and the effects of microbial
activity are involved [5]. Previous studies have reported that
approximately 40% of concrete failure in sewer systems can be
directly attributed to microbial-induced corrosion [6], but knowl-
edge of this process involved in HCSs from freshwater systems is
lacking.
HCSs in freshwater systems experience distinct hydrodynamic
conditions, e.g., flowing/static, flux/reflux and alternation of dry
and wet conditions, providing suitable habitats for colonization
of microbes [7]. Specific microbes easily corrode the pores of con-
crete by secreting biogenic acids and other metabolites, resulting
in increased permeability and seepage [8,9]. Previous studies con-
cerning microbial-induced concrete corrosion were mainly con-
ducted in wastewater environments, such as sewer systems
[10,11]. However, some studies have implied that severe microbial
corrosion also occurs in HCSs from freshwater environments. Zap-
ata et al. [12] and Shang et al. [13] reported that biofilms that
attach to the underwater surfaces of concrete play a significant role
in material degradation. None of the tested materials entirely
resisted this type of biogenic acid attack, and all materials failed
to reach their projected operating lifetime [14]. Nevertheless,
although corrosion by microbes is very common, only a few studies
have investigated the effects of microbes on HCSs in freshwater
systems.
Experimental studies have suggested that microbes pose long-
term negative effects on HCSs through microbial corrosion, and
bacteria with specific functions constitute the main body of the
potentially corrosive groups. Some studies have classified the
potentially corrosive bacterial groups as sulfate reducing bacteria
(SRB), sulfur oxidizing bacteria (SOB), acid producing bacteria,
and nitrifying bacteria according to their functions [12,15]. The
mainstream hypothesis in sewer systems has been accepted and
proved, in which acidophilic microorganisms act on submerged
areas of sewer pipelines [16,17]. SOB convert H2S gas into sulfuric
acid through oxidation on the exposed surface of the concrete [10].
Then, the acid reacts with the concrete to form gypsum and ettrin-
gite. These reactions deteriorate the structural integrity of the con-
crete and reduce load-bearing capacity, eventually resulting in a
weakened structure [18,19].
The structure and diversity of the microbial communities in the
aquatic environment are susceptible to various factors [20]. HCSs
are continuously subjected to alternating flowing and static condi-
tions, which remarkably affect the colonization and growth of
microbes. Simulated hydrodynamic tests indicate that hydraulic
conditions play an important role in the succession of microbial
communities [21,22]. Flowing water generally enhances the mass
transfer of inorganic nutrients and carbon sources from the liquid
phase to the surface of the solid substrate, altering the aggregation
Ó 2020 Elsevier Ltd. All rights reserved.
and adhesion degree of microorganisms, and, subsequently, chang-
ing the structural distribution of the biofilms attached to concrete
surfaces [23–25]. The formation, structure, and function of micro-
bial communities and the mass transfer process of nutrients in the
communities can easily alter concrete in response to different
hydrodynamic conditions [26,27].
In one of our previous field studies, distinct microbial commu-
nity compositions were observed among various areas of HCSs,
e.g., gate piers, land walls, and bank slopes, which were domi-
nated by a variety of hydrodynamic conditions [28]. That study
provided evidence that flowing and static hydraulic conditions
are the most prominent hydrodynamic conditions of HCSs. In
the present study, we determined the effects of microorganisms
on the erosion and mass loss of concrete in a lab-scale hydrody-
namic reactor. The composition and distribution of the bacterial
communities attached to the HCS surfaces under both conditions
were revealed. A functional prediction analysis was performed to
explore the functional bacteria and potential functions related to
nitrogen and sulfur metabolism. The results of this study will
deepen our understanding of microbial-induced concrete corro-
sion on HCSs. They will hopefully provide insight to prevent
and control microbial corrosion of hydraulic structures and offer
a theoretical basis for the scientific operation and maintenance
in freshwater conservation projects, finally reducing the occur-
rence of accidents and maintaining stability of the local
ecosystem.
2. Materials and methods
2.1. Study sample description
The concrete specimens were prepared by mixing water,
cement, sand, and stone at a weight ratio of 0.38:1:1.11:2.72,
according to the C30 standard for HCSs commonly used in the Chi-
nese Specification for the mix proportion design of ordinary con-
crete (JGJ55-2000) (J64-2000). The cement (Portland cement
grade 42.5) used was made by grinding the mixture with 0–5%
limestone and the proper amount of gypsum and Portland cement
clinker. Its compressive strength was 42.5 MPa. The resulting paste
was placed in a homemade frame (5  5  2 cm) for about 24 h and
allowed to solidify, then cured in a standard curing room at
20 ± 2 °C and 95% humidity for 28 days. After curing, the concrete
specimens were drowned in deionized water for 30 days to reduce
the experimental errors caused by high surface pH and mineral
leaching of early concrete.
Then, the concrete specimens were immersed half-way in a
simulated hydrodynamic reactor. The reactor was divided into
flowing and static water areas by a glass clapboard, and the bottom
water was connected (Fig. S1). The concrete specimens were
divided into two groups and placed in the reactor. The hydrody-
namic conditions in the flowing water area were controlled by an
immersible pump, and the current velocity of the water on the sur-
face of the concrete specimens was about 3 m/s [29]. Water in the
reactor of the experimental group was taken from Qinhuai River,
Nanjing, China, and the reactor for the aseptic blank group was
operated with sterile deionized water. The water used in both reac-
tors was changed every 24 h.
 Y. Li et al. / Construction and Building Materials 248 (2020) 118609 3

2.2. Sample collection
Biofilm samples were scraped from a surface area of about
2 cm2 from four replicate biofilms at the air-water interface of
the concrete specimens using sterilized metal spatulas carefully
washed with sterile deionized water. The wet samples were placed
into 2 mL aseptic centrifuge tubes and stored at 20 °C for no more
than 3 days until DNA extraction. Two parallel samples were col-
lected from the flowing (FW) and static (SW) water groups at the
same time and labeled FW1-1, FW1-2, SW1-1, and SW1-2 respec-
tively. The samples were collected at about 8-week intervals. A
total of 24 samples was obtained after six samplings (Fig. 1a).
The flowing water-blank group and the static water-blank group
used 70% alcohol and a 12 h daily UV treatment to remove
microorganisms attached to the concrete surfaces.
2.3. Determining the physicochemical parameters
The physicochemical indices of the water in the reactor were
measured using the Multi-Parameter Water Quality Detector
(HQ40d; HACH, Loveland, CO, USA), including temperature, dis-
solved oxygen concentration, and conductivity. Total nitrogen,

Fig. 1. (a) Schematic illustration of main processes involved in the experiment; (b) Visual evolution of the specimens exposed in both groups and mass loss.
4 Y. Li et al. / Construction and Building Materials 248 (2020) 118609

total phosphorus, ammonium nitrogen, chemical oxygen demand,
and biochemical dissolved oxygen were measured according to
the National Environmental Quality Standards for Surface Water
(GB 3838-2002). To monitor the pH changes on the specimen
surface, we measured surface pH using a flat surface pH electrode
(E-201-P, Shanghai INESA Scientific Instruments, Shanghai, China).
These indices were measured three times (Table 1).
The concrete specimens were dried at 75 °C for 48 h prior to the
experiment, and the initial mass was measured after cooling to
room temperature. After the experiment, the concrete specimens
were sterilized with 70% alcohol, rinsed with deionized water for
30 min to remove microbial residues, and drowned in deionized
water for 48 h to eliminate soluble substances on the surfaces
[30]. Finally, the mass loss of the concrete specimens was deter-
mined after drying to constant weight. Eight concrete specimens
were measured from each experimental group, and the average
mass loss of concrete was obtained. Considering the relatively
small volume of specimens, the mass of the concrete was deter-
mined by a balance of accuracy of 1/1000. The results are shown
in Table S1.
2.4. Bacterial community analysis
The biofilm samples were sent to Shanghai Biozeron
Science and Technology Ltd. (Shanghai, China) for DNA
extraction, 16S rRNA gene amplification, and Illumina MiSeq
sequencing. Among them, the 341F (50 -CCT AYG GGR BGC ASC
AG-30 ) and 806R (50 -GGA CTA CNN GGG TAT CTA AT-30 ) primers
targeting the V3–V4 variable region of the 16S rRNA gene were
used for amplification. The open-source bioinformatics pipeline
QIIME (Quantitative Insights into Microbial Ecology, version
1.9.0) was used to analyze the raw FASTQ data files [31]. The paired
reads were joined using the default settings in QIIME, and low-
quality sequences of the counter condition were removed. The
remaining chimeric sequences were removed using USEARCH
and the Genomes OnLine Database [32]. Then, the sequences were
clustered into operational taxonomic units (OTUs) using the pick-
ing script of pick_open_reference_otus.py with 97% similarity. Taxo-
nomic assignments were determined based on the SILVA database
(SILVA 128).
Alpha diversity within each sample was calculated from the
normalized data using the default QIIME script. Beta diversity
was also calculated using QIIME, and a principal coordinates anal-
ysis (PCoA) plot was generated using the unweighted UniFrac met-
ric, which included phylogenetic b diversity measures. Differences
in the structure of the microbial communities were detected using
R v. 3.3.0 packages, including ‘vegan’, ‘ggplot2’, and ‘pheatmap’ (R
Foundation for Statistical Computing, Vienna, Austria).
2.5. Statistical analysis
A canonical correspondence analysis (CCA) with 999 permuta-
tions was performed with Canoco 5.0 software to understand the
associations between microbial community composition and the
environmental variables. A p-value < 0.05 was considered signifi-
cant. The linear discriminant analysis (LDA) effect size (LEfSe)
was used to identify which specific bacterial taxa were signifi-
cantly more abundant in the two sample types, including signifi-
cant LEfSe results using the Kruskal-Wallis test (P < 0.05) and an
LDA score > 2.0. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA)
was used for the calculations and processing. The abundance-
weighted relatives with the weighted bNTI in combination with
RCbray were used to quantify the ecological processes [33] that
influence bacterial composition using packages in R v. 3.3.0
[34,35].
2.6. Functional prediction
The QIIME results were imported into METAGENassist [36]
(www.metagenassist.ca). Functional studies using the
METAGENassist database provided a preliminary prediction of
the metabolic potential based on automatic taxonomy-to-
phenotype mapping of the biofilm samples. The Phylogenetic
Investigation of Communities by Reconstruction of Unobserved
States (PICRUSt) was utilized to infer functional predictions based
on 16S rRNA gene content and explain the metabolic changes in
nitrogen and sulfur in the bacterial community. Prior to further
analysis, the .biom file of the OTUs was regenerated according to
the GreenGens databases (v13.5). Next, the results were predicted
using the PICRUSt pipeline in QIIME [37]. The nearest sequenced
taxon index (NSTI) scores were also calculated at the same time
and reflected the availability of reference genomes that were clo-
sely related to the most abundant microbes in the samples. In addi-
tion, the type of functional predictions was assessed using the
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways data-
base. The data characterization was based on differences in the
predicted functional gene family composition.
3. Results and discussion
3.1. Biofilm formation and mass loss in the concrete
Fig. 1a and b show the experimental processes and visual evo-
lution of the concrete during the experiment, respectively. A
mature biofilm was clearly observed in both groups during the
335-day experiment. In comparison, the biofilm formation process
was more rapid on concrete cultured in flowing water. The biofilm

Table 1
Physicochemical parameters of the water samples.

ID Surface T DO Cond NH3-N TN TP CODMn BOD5

pH (°C) (mg L1) (ls cm1) (mg L1) (mg L1) (mg L1) (mg L1) (mg L1)
FW1 SW1 8.42 17.4 5.59 642 2.21 2.75 0.69 4.58 2.26
(0.26) (0.2) (0.56) (18) (0.09 (0.08) (0.04) (0.18) (0.11)
FW2 SW2 8.13 21.5 4.43 484 1.18 2.91 0.48 5.52 3.39
(0.58) (0.3) (0.65) (24) (0.06) (0.13) (0.08) (0.22) (0.13)
FW3 SW3 7.8 23.2 3.96 553 1.84 1.72 0.32 3.02 1.95
(0.31) (0.4) (0.41) (31) (0.14) (0.18) (0.05) (0.31) (0.24)
FW4 SW4 7.83 21 4.03 411 1.67 2.29 0.53 5.36 2.15
(0.21) (0.3) (0.58) (13) (0.11) (0.09) (0.04) (0.24) (0.16)
FW5 SW5 7.86 19.6 6.46 789 2.82 2.43 0.58 5.66 3.08
(0.34) (0.2) (0.78) (21) (0.08) (0.12) (0.07) (0.28) (0.12)
FW6 SW6 7.91 15.2 7.12 704 2.39 2.52 0.78 5.14 3.19
(0.46) (0.4) (0.63) (17) (0.05) (0.05) (0.06) (0.33) (0.18)

Water quality based on Environmental Quality Standard for Surface Water of the People’s Republic of China (GB3838-2002). Values are mean ± standard error (n = 3).
 Y. Li et al. / Construction and Building Materials 248 (2020) 118609
that formed in the flowing water covered a larger area of the con-
crete surface along the vertical region. This phenomenon was
ascribed to the elevated dissolved oxygen concentration at the
air-water interface due to the flowing condition. Dissolved oxygen
is commonly proposed as an important driving force for microbial
community composition [38]. Flowing water also changes the oxy-
gen transfer rate, the material diffusion rate, humidity, and other
conditions at the concrete air-water interface, making it easier
for microbes to colonize on concrete surfaces [25].
The mass loss of concrete is one of the basic principles for deter-
mining the degree of corrosion [4]. Table S1 shows that the mass
losses of all concrete samples ranged from 2.96% to 4.65%. Consid-
ering the differences in raw materials, preparation methods, speci-
fic surface area, and testing period of the concrete specimens, the
total mass loss of concrete in a simulated freshwater environment
in this study was relatively higher than that under real conditions.
For aseptic concrete cultivated in sterile deionized water, the total
mass losses of concretes were 2.96% and 3.55%, under static and
flowing water conditions, respectively, at the end of the experi-
ment. The total mass loss for concrete with attached with biofilms
increased to 4.04% in static water and 4.65% in flowing water
(Fig. 1b).
These results suggest that the concrete cultivated in flowing
water suffered the highest mass loss (4.10–5.13%) and the average
value was about 15.1% higher than concrete cultured in the static
water condition. Hydrodynamic factors are closely related to the
increase in structural damage on the surface of HCSs [39]. The
aseptic concrete cultivated in static water experienced the lowest
mass loss (2.27–3.64%) among the samples, which was predomi-
nantly ascribed to mineral dissolution and precipitation of the con-
crete specimens. The mass losses of concretes in flowing and static
freshwater increased by 36.6% and 32.3%, respectively, confirming
that microbial action was responsible for the increased mass loss of
the HCSs. Flowing water generally affects the permeability of con-
crete, promoting the action of microbes on the concrete surface
[40]. Researchers have found that microbes that attach to HCSs,
such as underwater surfaces of dams or reservoirs, play a signifi-
cant role in concrete corrosion [41,42]. Therefore, the long-term
Fig. 2. Relative abundance of bacteria from biofilms classified at the phylum level as the top 10.
5
interaction between hydrodynamic factors (planned water storage
and drainage) and microbial action (long-term adhesion of biofilm)
poses a damage threat to the entire structure during service cycles.
Our results provide quantitative evidence that microbial action
contributes to the corrosion of HCSs in a simulated hydrodynamic
reactor.
3.2. Diversity and composition of the bacterial communities in the HCS
A total of 24 biofilm samples were collected in this study, and a
biological information analysis was performed. The effective
sequence reads of each biofilm sample ranged from 30,208 to
59,429, with a mean of 43,237.3. After the database annotation,
30 microbial phyla and 724 microbial genera were identified. The
biofilm bacterial communities were numerically dominated by
Proteobacteria and Actinobacteria with respective average relative
abundances of 65.2% and 15.1% (Fig. 2). The distribution of bacte-
rial phyla was approximately the same as reported previously
[2,43,44]. The abundance and diversity of samples changed dra-
matically over time and distinct abundances of some bacteria were
observed between the flowing and static water conditions. The
abundance of Actinobacteria increased from 1.5% at the initiation
of cultivation to 30.4% on day 335 in the static water group, while
no significant change in abundance was observed when concrete
was cultivated in flowing water. The relative abundance of Saccha-
ribacteria increased from 0% to 30.8% in flowing water and from
0.1% to 16.05% under static water conditions within 48 to 335 days.
Due to the pathogenic potential of Saccharibacteria [45], flowing
water might significantly increase the pathogenicity of the micro-
bial communities in biofilms attached to concrete in this study.
Major differences in microbial community composition were
detected between concrete cultured in flowing and static condi-
tions using the LEfSe analytic method (Fig. S3). The cladogram pro-
vided six levels (from kingdom to genus) and identified 53
differentially abundant bacterial taxa with LDA values > 2
(Fig. S3). The present results suggest that an unknown reason
might be responsible for the high abundance of Chloroflexi in the
flowing water group, although the majority of members in the
6 Y. Li et al. / Construction and Building Materials 248 (2020) 118609
phylum Chloroflexi are relatively unexplored [28]. The Nitrospirae
flora was highly abundant in the static water group, suggesting a
more robust nitrogen cycling capacity of biofilms formed in static
water. Clear differences in the distribution of the taxonomic groups
along with microbial community succession are found in Fig. S2. It
was apparent that genus diversity varied significantly along the
cultivation period (Fig. S2). Halomonas, known as a salt-tolerant
taxon, was the main genus detected in biofilms from the beginning
to 110 days of cultivation (samples FW2 and SW2) [46]. During this
early stage, the higher abundance of Halomonas was probably
owing to the higher salt and alkali levels due to concrete secretion.
In comparison, Thauera was the dominant genus in the attached
biofilms, accounting for 20–50% of the total microbial community
after 165 days of cultivation (samples FW3 and SW3). Thauera
has been commonly identified as an appropriate group in wet soil
and polluted freshwater, which may be closely related to surveying
a polluted urban river [47].
A PCoA with beta diversity and the unweighted UniFrac metric
was used to compare the variations in microbial community com-
position from the biofilm samples cultured under flowing and sta-
tic conditions in Fig. 3a. The results showed that 22.95% of the
variation in each sample was explained by PC1 and 15.81% of the
variation was explained by PC2. Fig. 3a provides evidence that a
stable community structure gradually formed during cultivation
in all biofilm samples. It was clear that differences between parallel
samples decreased gradually as a function of cultivation time. A
PCoA analysis was performed using the biofilm samples cultured
under both flowing and static conditions and collected on days
280 (samples FW5 and SW5) and 335 (samples FW6 and SW6)
(Fig. S4) to reveal the differences in the bacterial communities
between the flowing and static water after the microbial commu-
nity structure stabilized. PC1 and PC2 explained 30.68% and
22.89% of the variation, respectively. The cluster of microorganisms
was clearly separated among samples cultivated in flowing and
static water. The PCoA analysis indicated that flowing water not
only regulated the community succession process but also the
community composition and structure during the plateau stage.
Fig. 4 shows the Pearson’s correlation coefficient test results of
bacterial communities in the biofilms cultured in flowing and sta-
tic water. The similarity of the bacterial communities was marked
by the fullness of the red circle between the two samples [48]. As
shown in Fig. 4, an increase in the similarity of the bacterial com-
munities between the flowing and static conditions was observed
Fig. 3. Results of the principal coordinate analysis (PCoA) and canonical correspondence analysis (CCA) biplots. (a) The unweighted UniFrac distances for microbial
communities across biofilms at different periods; (b) the relationship between environmental factors and the overall bacterial community composition in all samples.
with community succession, consistent with the results in
Fig. 3a. Fig. 4a shows that the bacterial community structure culti-
vated in flowing water increasingly became stable at about
165 days of cultivation (sample FW3), while the bacterial commu-
nity cultured in static water began to stabilize after a longer period
of 225 days (sample SW4). These results indicate that the hydrody-
namic condition accelerates succession of the bacterial community
and shortens the time for the bacterial community to reach the
plateau state. Cai et al. [28] reported that the HCS structures, e.g.,
dam piers, land walls, and bank slopes, which have been affected
by flowing water for a long period during service, may be subject
to a more serious damage threat. Our study confirmed that flowing
water accelerated the succession of microbial communities, and
that the HCS would suffer from earlier microbial corrosion after
the community structure gradually became stabilized.
3.3. Effect of water parameters on the bacterial community
A CCA was used to examine the relationship between the water
parameters during sampling and overall bacterial community com-
position (Fig. 3b). The environmental factors and all microbes were
involved in the calculation, accounting for 35.7% of the variation in
the bacterial communities. Species-environment correlation coeffi-
cients indicated that species composition was closely related to the
environmental variables. The eigenvalues of the first two CCA axes
were 0.5556 and 0.4045 (pseudo-canonical correlation values:
0.912 and 0.785). The results of a CANOCO analysis showed that
surface pH and temperature had the most significant effect on
microbial community composition. Both parameters were nega-
tively correlated with the first species axis. Surface pH was the
most significantly correlated factor (r = 0.3279, p < 0.05), fol-
lowed by temperature (r = 0.1932, p < 0.05). The weighted bNTI in
combination with the RCbray analysis showed that the probabili-
ties of homogenous selection and variable selection were 60.87%
and 32.80% (Fig. 5b), and demonstrated the relative importance
of environmental selection during the formation and composition
of the microbial communities. The CCA and phylogenetic analyses
showed that temperature and surface pH were the main factors
affecting distribution of the microbial community (Fig. 3b).
During the 335-day experimental period, seasonal variations in
temperature largely affected microbial community succession [49].
Studies have confirmed that the succession of early bacterial com-
munities in attached biofilms is controlled by the pH value of the
 Y. Li et al. / Construction and Building Materials 248 (2020) 118609 7

Fig. 4. Pearson’s correlation coefficient analysis of the bacterial communities among different groups of samples. (a) Succession of bacterial communities cultured in flowing
water; (b) succession of bacterial communities cultured in static water.

Fig. 5. (a) The main nitrogen and sulfur metabolic functional groups in the bacterial communities among the biofilms at different periods according to the METAGENassist
analysis; (b) ecological processes governing bacterial community turnover in the hydraulic concrete structures (HCSs). The percentages are relative contributions of the
processes to community turnover as indicated by different colors.

surrounding environments [50]. Many researchers have suggested
that the high alkaline surface of concrete is the fundamental reason
explaining the difficulty for microbes to colonize concrete on the
surface and inward [11]. Ling et al. [6] investigated the microbial
communities associated with concrete corrosion in sewer systems.
Some sulfur oxidizers were identified as the dominant bacteria
after the pH decreased during microbial succession, suggesting
that pH is an important factor affecting the compositional distribu-
tion of bacterial communities.
Previous studies concerning microbial-induced corrosion have
explored the diversity and succession of bacterial communities in
the corrosion layer of concrete materials [2,16]. Grengg et al. [51]
found that microbial activity is not limited to the oxygen-rich cor-
rosion layers near the surfaces, but could expand throughout the
entire deterioration zone. Ongoing mineral dissolution and re-
precipitation and subordinated microbial distribution are con-
trolled by pH and diffusion in the microsystem of the concrete
deterioration zone [2,51]. We deduced that the microbes in the
concrete deterioration zone might form a community that was
suitable with the micro-system environmental factors, directly
affecting the concrete calcium silicate hydrate and adhesion pro-
cesses in the concrete [52,53]. This process might eventually accel-
erate the penetration of concrete and seriously deteriorate the
safety of the internal concrete structure under the dual influence
8 Y. Li et al. / Construction and Building Materials 248 (2020) 118609

of hydrodynamic factors and microbial action. Further studies on
concrete diffusion microsystems in freshwater under hydrody-
namic conditions are required to verify this speculation.
3.4. Functional prediction of bacterial communities in HCS
Functional prediction methods, e.g., PICRUSt, have been fre-
quently utilized to predict the functional composition of a meta-
genome through marker gene data and a reference genome
database [37]. The prediction results from METAGENassist merged
with parallel samples on average, and 25 major bacterial metabolic
activities were identified. The results revealed that a range of func-
tional bacteria related to nitrogen and sulfur metabolism
accounted for the majority, i.e., 70% of all functionally metabolizing
bacteria culturing in flowing and static water. As shown in Fig. 5a,
the percentages of sulfate reducers and nitrite reducers decreased
significantly with succession of the microbial communities in both
groups. Sulfate reducing bacteria (SRB) are confirmed dominant
species contributing to corrosion in sewer environments, and play
key roles in the succession and stabilization of microbial commu-
nities during the early stage [16]. In contrast, the increase in the
abundances of sulfur oxidizers and sulfide oxidizers indicate pro-
motion of the oxidation process. These bacterial groups have been
detected in samples from sewer pipelines [18] or concrete dams
[41] and their oxidation products are known to corrode concrete.
Notably, no significant differences were observed in the relative
abundances of bacterial communities cultured in flowing and sta-
tic water (Fig. 6).
NSTI scores were calculated to quantify the usability of nearby
genomic representatives for each microbial sample [37]. In this
study, the NSTI scores of each sample from PICRUSt ranged from
0.030 to 0.135, indicating the availability of closely related refer-
ence genomes and a high prediction accuracy for the dataset.
Fig. 6 shows that functional genes with a copy number higher than
1000 were examined according to the nitrogen and sulfur meta-
bolic pathways in the KEGG. The relatively stable functional gene
abundance for sulfur metabolism suggests that the metabolic
activities of microbes in the corrosion layer were not influenced
by environmental factors (Fig. 6a). SRB have been confirmed to suf-
fer under highly alkaline conditions [54]. In this study, the secre-
tion of alkalinity during the initial cultivation stage might favor
enrichment of these strains. The sulfur metabolic processes might
participate in microbial action, contributing to the corrosion and
deterioration processes [54,55].
It is noteworthy that clear nitrogen metabolism gene abun-
dance appeared on day 165 (samples FW3 and SW3), suggesting
more active nitrogen metabolic activity after a period of cultivation
(Fig. 6b). At the same time, the increased abundance of NRT genes
responsible for transporting nitrate and nitrite showed that stron-
ger nitrogen metabolic processes started to be involved in the

Fig. 6. Relative abundances of PICRUSt predicted genes relevant to sulfur metabolism (a) and nitrogen metabolism (b). PICRUSt predicted functional data are based on the
predicted functional gene family composition with only genes classified in ‘‘metabolism”. Genes with copy number less than 1000 in the whole dataset were discarded.
 Y. Li et al. / Construction and Building Materials 248 (2020) 118609
microbial actions. The functional prediction indicated that
microbial-induced corrosion of concrete was driven by a series of
functional bacteria and genes related to nitrogen and sulfur meta-
bolism. Pagaling et al. [56] reported that pH is significantly corre-
lated with bacterial communities. In this study, an extreme surface
pH could not be produced due to continuous water exchange at the
concrete interface; thus, limiting participation of more corrosive
bacteria [57]. By comparing the predicted values in static and flow-
ing water, we observed higher variations in most of the predicted
metabolic capacities of the bacterial community cultured under
the static water condition. These results suggest that corrosive bac-
teria play a more profound role on HCSs in static water. By using
functional prediction, a mechanistic understanding of the potential
metabolic capability of microbial action on HCS was obtained.
Understanding how hydrodynamic conditions affect the biofilms
on HCSs will be useful to control microbial corrosion of hydraulic
structures and prolong their service lives.
4. Conclusion
The findings of this study provide insight into the diversity and
predicted function of the bacterial communities in HCSs under
hydrodynamic conditions. The conclusions drawn from the present
study can be summarized as follows: (i) During the 335-day culti-
vation experiment, the mass losses of aseptic concrete in static and
flowing water were 2.96% and 3.55%, respectively. The mass loss of
concrete increased by 32.3% and 36.6% when the HCSs were cul-
tured in freshwater under static and flowing water conditions, sug-
gesting that microbial action of the attached biofilms was actively
involved in the concrete corrosion process; (ii) two phyla, i.e., Pro-
teobacteria and Actinobacteria, were predominant species in the
biofilms that formed on the concrete. Halomonas was the dominant
genera detected in biofilms during the initial 110 days of the
experiment, while Thauera was the dominant genera after 165 days
of cultivation. Surface pH and temperature were the predominant
factors driving bacterial community composition; (iii) functional
bacteria related to nitrogen and sulfur metabolism accounted for
the majority, i.e., 70% of all functionally metabolizing bacteria
and the process was actively involved in the microbial actions on
the HCSs. The percentages of sulfate reducers and nitrite reducers
decreased significantly with succession of the microbial communi-
ties in flowing and static water. The increased abundances of sulfur
oxidizers and sulfide oxidizers, as well as NRT genes responsible for
transport of nitrate and nitrite appeared on day 165. Our results
quantify the contribution of microbial activity to the corrosion of
HCSs in freshwater environments and suggest future hypotheses
for more quantitative and reliable studies, focusing on special
groups and functional genes on the HCSs. This study might help
us develop a corresponding strategy for safer operation and main-
tenance of water conservancy projects in freshwater systems. For
example, regular monitoring of HCSs might be focused on sites
with complex hydrodynamic conditions or biofilm formation for
signs of corrosion. Some of the current corrosion management
techniques would be useful for controlling or inhibiting bacterial
activities.
CRediT authorship contribution statement
Yi Li: Conceptualization, Funding acquisition. Mingyue Wan:
Data curation, Writing - review & editing. Jiming Du: Data cura-
tion, Writing - original draft. Li Lin: Visualization, Investigation.
Wei Cai: Software, Validation. Longfei Wang: Supervision, Writing
- review & editing.
9
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
This study was supported by National Natural Science Founda-
tion of China [grant number 51779076], Foundation for Innovative
Research Groups of the National Natural Science Foundation of
China [grant number 51421006], and Fundamental Research Funds
for the Central Universities [grant number 2016B10614], the Prior-
ity Academic Program Development of Jiangsu Higher Education
Institutions (PAPD) and the Top-Notch Academic Programs Project
of Jiangsu Higher Education Institutions (TAPP) [grant number
PPZY2015A051].
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.conbuildmat.2020.118609.
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