AEM Accepts, published online ahead of print on 27 July 2012 Appl
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Short communication: 1
High-throughput amplicon sequencing reveals distinct communities within a corroding 2
concrete sewer system. 3
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Barry I. Cayford1*, Paul G. Dennis1,2, Jurg Keller1, Gene W. Tyson1, 2, Philip L. Bond1* 5
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1Advanced Water Management Centre, The University of Queensland, QLD 4072, Australia. 7
2Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The 8
University of Queensland, QLD 4072, Australia. 9
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*Corresponding authors. 11
Barry Cayford: [email protected] Philip Bond: [email protected] 12
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Keywords: sewer, biocorrosion, concrete, Acidithiobacillus, SSU rRNA gene 14
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01582-12 AEM Accepts, published online ahead of print on 27 July 2012
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Abstract 15
Microbially-induced concrete corrosion (MICC) is an important problem in sewers. Here, 16
SSU rRNA gene amplicon pyrosequencing was used to characterize MICC communities. 17
Microbial community composition differed between wall- and ceiling-associated MICC 18
layers. Acidithiobacillus spp. were present at low abundance and the communities dominated 19
by other sulfur-oxidising associated lineages. 20
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Introduction 22
Microbially-induced concrete corrosion (MICC) is an important problem in sewers. Under 23
anaerobic conditions in wastewater, sulfur-reducing microorganisms convert sulfate and 24
other oxidised sulfur compounds to soluble sulfide and hydrogen sulfide (H2S) gas [2]. 25
Above the sewage level in the pipe, under aerobic conditions, microorganisms associated 26
with concrete surfaces oxidise H2S to sulfuric acid, which causes concrete corrosion [2, 14]. 27
This corrosion results in premature infrastructure degradation or failure, is expensive to repair 28
and mitigate, and presents a potential significant environmental health hazard [14]. 29
Consequently, it is critical to improve our understanding of the roles of microbial 30
communities in the corrosion process in order to predict and effectively manage MICC in 31
sewer systems. Recent developments in microbial community analysis techniques have 32
resulted in an opportunity to test the hypothesis that the breadth of microbes associated with 33
concrete sewer pipe corrosion layers is higher than has been previously reported. The finding 34
that organisms other than Acidithiobacillus thiooxidans are key community members may 35
also suggest that processes alternate to those currently known contribute to sewer pipe 36
corrosion. 37
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To date, the majority of MICC studies in sewers have used culture-dependent methods to 38
characterize microbial diversity and consistently report that At. thiooxidans is the key 39
protagonist [1, 4, 5, 10, 13-15, 17]. Three recent studies have used clone libraries of the 40
bacterial 16S rRNA gene to characterise the diversity and composition of microbial 41
communities associated with MICC layers [11, 16, 17]. These studies all indicate that while 42
Acidithiobacillus spp. can represent major components of some MICC layer communities 43
they are not ubiquitously dominant. These studies were limited, however, either being based 44
on low sequencing depth [11, 17], or focusing on samples taken from regions where 45
conditions are likely to be quite different to those in sewer pipe corrosion layers, such as 46
manholes [16] or cement coupons in manholes [11]. Given that culture-dependent methods 47
often poorly represent microbial diversity, and that culture-independent methods have 48
focussed on samples that may differ from sewer-pipes, knowledge of the microbial 49
communities associated with MICC layers in sewer pipes needs to be improved. 50
In this study the diversity of microbial communities associated with well-established MICC 51
layers in two adjacent but independent sewer pipes receiving the same input wastewater was 52
characterised using universal small subunit ribosomal ribonucleic acid (SSU rRNA) gene 53
amplicon pyrosequencing (Supplementary Information). We characterized multiple samples 54
with the goal of investigating the variability within these environments and to identify novel 55
microbes associated with MICC. 56
Ten samples were collected from random positions within the two sewer pipes 57
(Supplementary Table S1 & Supplementary Figure S1). Analysis of environmental 58
monitoring of gas phase temperatures and H2S levels (data not shown) indicated no statistical 59
difference between the two pipes, with average temperatures of 17.3-17.9 degrees Celsius 60
and H2S levels of 1-4 parts per million. A minimum of 1,462 amplicon sequences was 61
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obtained per sample resulting in all datasets being sub-sampled to a level of 1,400 sequences 62
each for all comparative analyses of diversity. The richness and evenness of microbial 63
communities did not differ between pipes (P > 0.05, Generalised Linear Modelling (GLM); 64
Supplementary Table S1 & Supplementary Figure S2). Likewise there was no difference in 65
the composition of microbial communities between pipes (P > 0.05, Redundancy Analysis 66
(RDA)), though Principal Component Analysis (PCA) revealed that the composition of three 67
samples was distinct from that of the others (Figure 1). Interestingly, these three samples 68
were taken from MICC layers on the pipe walls, whereas all other samples were collected 69
from sewer pipe ceilings. This difference was significant (P = 0.013, RDA) and was related 70
to a greater relative abundance of SSU rRNA gene sequences from an Acidiphilium sp. and a 71
Mycobacterium sp. in ceiling- relative to wall-associated microbial communities, and a 72
greater abundance of Burkholderiales spp., Sphingobacteriales spp. and Xanthomonadales 73
spp. in wall- relative to ceiling-associated microbial communities (Figs. 1 & 2). It is possible 74
that the lower variation in wall samples is the result of occasional inundation during flood 75
events that may act to homogenise the wall environment and limit the development of niche 76
communities. Bacterial populations, closely related to the abundant species detected here, 77
have been reported, albeit at lower abundances (<3%), in other culture-independent studies of 78
sewer-associated MICC layers [11, 16]. Importantly, however, sequences derived from 79
Acidithiobacillus spp. were present at <3% relative abundance in all samples except for one 80
(Figure 2). This finding is in stark contrast with the majority of previous studies, which 81
indicate that At. thiooxidans is the key protagonist of MICC in sewers [10-12, 17]. 82
The Acidiphilium sp. which dominated the ceiling communities was closely related to 83
Acidiphilium acidophilum, which is a known sulfur oxidiser [2] and has been previously 84
reported in sewer associated MICC layers [11]. The other dominant population in ceiling-85
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associated MICC layers, a Mycobacterium sp., shared 99% nucleotide identity across a 350 86
base pair region of the SSU rRNA gene with a Mycobacterium sp. capable of 87
chemolithoautotrophic growth at pH 3.6 by oxidising sulfur compounds to sulfuric acid [7]. 88
The high abundance of these bacteria, and their potential sulfur oxidation activities indicates 89
that both the Acidiphilium sp. and Mycobacterium sp. are likely protagonists of MICC in 90
sewers. Due to the obligate autotrophic and facultative autotrophic nature of At. thiooxidans 91
and A. acidophilum, respectively [2], the local level of organic carbon in the corrosion layers 92
likely influenced the relative abundances of at least these organisms. 93
The relationship between Burkholderiales spp., Sphingobacteriales spp. and 94
Xanthomonadales spp. and MICC in sewers has not been defined. One of the dominant wall-95
associated Burkholderiales sequences showed a high degree of homology to sequences from 96
the genus Thiomonas, which are known for their ability to oxidise reduced forms of sulfur to 97
sulfate [9]. The wall-associated Xanthomonadales spp. clustered with a deep-rooted clade of 98
sequences from organisms found in acidic environments, such as acid mine drainage sites 99
(results not shown). This group of Xanthomonadaceae spp. appears to be exclusively 100
associated with acidic, sulfur oxidising environments and currently has no cultured 101
representatives. The most closely related Xanthomonadaceae sequences to this novel group 102
are from the genus Rhodanobacter, which includes populations that are capable of sulfur 103
oxidation [8], abundant in an anaerobic acid environment [3], and have been previously 104
reported in sewer corrosion environments [16]. The wall-associated Sphingobacteriales spp. 105
were closely related to the Chitinophagaceae [6] which are poorly characterised. The 106
presence of these organisms at high abundance in MICC layers suggests that they are 107
contributing to the corrosion process and warrants further investigation. 108
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The use of universal primers has meant that, for the first time in this environment, the relative 109
abundance of lineages from all three domains can be examined. A wide range of eukaryote 110
sequences were present at low abundance, primarily in wall-associated samples. Archaeal 111
sequences were only detected at extremely low levels, with slightly more on the ceiling than 112
the wall. 113
This study demonstrates that the composition of microbial communities associated with 114
MICC layers was similar between these sewer pipes but differed between the pipe walls and 115
ceilings. Given that well-developed MICC layers were present on both the walls and the 116
ceilings, similar corrosion processes are likely to be occurring despite large differences in 117
community composition. Acidithiobacillus spp. were generally present at low abundance, 118
which indicated that they were unlikely to be the main protagonists of MICC in these 119
samples. The most likely protagonists of MICC in our study system were Acidiphilium, 120
Mycobacterium, Burkholderiales, Sphingobacteriales and Xanthomonadales spp., which 121
represented the dominant populations. Many populations closely related to these are capable 122
of oxidising reduced inorganic sulfur compounds other than H2S gas; consequently the focus 123
of corrosion management techniques may need to be modified to account for these processes. 124
Further culture independent studies are required to determine whether these populations are 125
dominant in MICC layers in other sewer systems. It is also important to improve 126
understanding of the dynamics of MICC layer-associated community assembly as this will 127
indicate parameters that could be manipulated to manage MICC in sewers more effectively. 128
Acknowledgements 129
We gratefully acknowledge funding from the Australian Research Council (Industry Linkage 130
Project: Sewer Corrosion and Odour Research (SCORe) LP0882016) as well as input from 131
research partners and key members of the Australian water industry (for more details see 132
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www.score.org.au). In addition, we are grateful to Dr. Frances Slater and Prof. Zhiguo Yuan 133
for their helpful discussions and Jose Gonzales and colleagues for assisting with the sample 134
collection. 135
Figure Legends 136
Figure 1 Principal Component Analysis ordination summarizing variation in the composition 137
of bacterial communities associated with MICC layers. Operational taxonomic units (OTU) 138
are represented by crosses and the taxonomic affiliation of those that discriminate groups of 139
samples are labelled. 140
Figure 2 Heatmap summarising the percent relative abundances of bacteria (each row 141
representing an OTU) that were present at more than 1% in MICC layer samples from walls 142
and ceilings of the two pipes (1 and 2). The relative similarity of each sample in terms of 143
community composition as determined by complete linkage cluster analysis of OTU 144
abundances is represented at the top of the heatmap. 145
References 146
1. Cho, K.S. and Mori, T., 1995. A newly isolated fungus participates in the corrosion 147 of concrete sewer pipes. Water Science and Technology, 31(7): 263-271. 148
2. Dopson, M. and Johnson, D.B., 2012. Biodiversity, metabolism and applications of 149 acidophilic sulfur-metabolizing microorganisms. Environmental Microbiology. 150
3. Green, S.J., Prakash, O., Jasrotia, P., Overholt, W.A., Cardenas, E., Hubbard, 151 D., Tiedje, J.M., Watson, D.B., Schadt, C.W., Brooks, S.C., and Kostka, J.E., 152 2012. Denitrifying bacteria from the genus Rhodanobacter dominate bacterial 153 communities in the highly contaminated subsurface of a nuclear legacy waste site. 154 Applied and Environmental Microbiology, 78(4): 1039-47. 155
4. Hvitved-Jacobsen, T., Sewer processes microbial and chemical process engineering 156 of sewer networks. . 2002, Boca Raton, FL, USA: CRC Press. 157
5. Islander, R.L., Devinny, J.S., Mansfeld, F., Postyn, A., and Hong, S., 1991. 158 Microbial Ecology of Crown Corrosion in Sewers. Journal of Environmental 159 Engineering-Asce, 117(6): 751-770. 160
6. Kampfer, P., Lodders, N., and Falsen, E., 2011. Hydrotalea flava gen. nov., sp. 161 nov., a new member of the phylum Bacteroidetes and allocation of the genera 162 Chitinophaga, Sediminibacterium, Lacibacter, Flavihumibacter, Flavisolibacter, 163
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Niabella, Niastella, Segetibacter, Parasegetibacter, Terrimonas, Ferruginibacter, 164 Filimonas and Hydrotalea to the family Chitinophagaceae fam. nov. International 165 Journal of Systematic and Evolutionary Microbiology, 61(Pt 3): 518-23. 166
7. Kusumi, A., Li, X.S., and Katayama, Y., 2011. Mycobacteria isolated from angkor 167 monument sandstones grow chemolithoautotrophically by oxidizing elemental sulfur. 168 Frontiers in microbiology, 2: 104. 169
8. Lee, C.S., Kim, K.K., Aslam, Z., and Lee, S.T., 2007. Rhodanobacter thiooxydans 170 sp nov., isolated from a biofilm on sulfur particles used in an autotrophic 171 denitrification process. International Journal of Systematic and Evolutionary 172 Microbiology, 57: 1775-1779. 173
9. Moreira, D. and Amils, R., 1997. Phylogeny of Thiobacillus cuprinus and other 174 mixotrophic thiobacilli: proposal for Thiomonas gen. nov. International Journal of 175 Systematic Bacteriology, 47(2): 522-8. 176
10. Nica, D., Davis, J.L., Kirby, L., Zuo, G., and Roberts, D.J., 2000. Isolation and 177 characterization of microorganisms involved in the biodeterioration of concrete in 178 sewers. International Biodeterioration & Biodegradation, 46(1): 61-68. 179
11. Okabe, S., Odagiri, M., Ito, T., and Satoh, H., 2007. Succession of sulfur-oxidizing 180 bacteria in the microbial community on corroding concrete in sewer systems. Applied 181 and Environmental Microbiology, 73(3): 971-980. 182
12. Parker, C.D., 1945. The corrosion of concrete 1. The isolation of a species of 183 bacterium associated with the corrosion of concrete exposed to atmospheres 184 containing hydrogen sulphide. Australian Journal of Experimental Biology and 185 Medical Science, 23(2): 81-90. 186
13. Parker, C.D., 1947. Species of Sulphur Bacteria Associated with the Corrosion of 187 Concrete. Nature, 159(4039): 439-440. 188
14. Roberts, D.J., Nica, D., Zuo, G., and Davis, J.L., 2002. Quantifying microbially 189 induced deterioration of concrete: initial studies. International Biodeterioration & 190 Biodegradation, 49(4): 227-234. 191
15. Sand, W. and Bock, E., 1984. Concrete Corrosion in the Hamburg Sewer System. 192 Environmental Technology Letters, 5(12): 517-528. 193
16. Santo Domingo, J.W., Revetta, R.P., Iker, B., Gomez-Alvarez, V., Garcia, J., 194 Sullivan, J., and Weast, J., 2011. Molecular survey of concrete sewer biofilm 195 microbial communities. Biofouling, 27(9): 993-1001. 196
17. Vincke, E., Boon, N., and Verstraete, W., 2001. Analysis of the microbial 197 communities on corroded concrete sewer pipes - a case study. Applied Microbiology 198 and Biotechnology, 57(5-6): 776-785. 199
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