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

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