Process Biochemistry 49 (2014) 1345–1351

Contents lists available at ScienceDirect

Process Biochemistry

jo urnal homepage: www.elsevier.com/locate/procbio

The bacterial communities of bioelectrochemical systems associated

with the sulfate removal under different pHs

a,b b a,∗ a,b b

Yue Zheng , Yong Xiao , Zhao-Hui Yang , Song Wu , Hui-Juan Xu ,

b b,∗

Fang-Yuan Liang , Feng Zhao

College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Ministry of

Education, Changsha, 410082, China

Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China

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

Article history: Sulfate contamination in ecosystems has been a serious problem. Among various technologies, bioelectro-

Received 12 February 2014

chemical systems (BESs) show the advantage of no-pollution and low-cost for removing sulfate. In order

Received in revised form 16 April 2014

to further expound the biological process of sulfate removal in BESs, 454 pyrosequencing was applied

Accepted 26 April 2014

to analyze the bacterial communities under different pH conditions. The bacterial community proﬁles

Available online 9 May 2014

were analyzed from three aspects: (a) the ␣-diversity and ␤-diversity of bacterial communities, (b) the

distribution of bacterial phylotypes, and (c) the characterizations of dominant operational taxonomic

Keywords:

units (OTUs). We demonstrated that the indexes of phylotype richness and phylogenetic diversity were

Sulfate removal

positively correlated across the pH gradient in the BESs. Among the dominant OTUs, the OTUs which were

Bioelectrochemical systems

Pyrosequencing highly similar to Desulfatirhabdium butyrativorans, Desulfovibrio marrakechensis and Desulfomicrobium sp.

Microbial community might participate in removing sulfate. Standing on genus level, Desulfomicrobium and Sulfuricurvum play

conducing and adverse roles for sulfate removal in alkaline condition, respectively. Desulfovibrio con-

tributed to removing sulfate in the neutral and acidic conditions, while Thiomonas mainly weakened the

performance of sulfate removal in neutral pH condition. These results further clariﬁed how pH condi-

tion directly affected the bacterial communities, which consequently affected the performance of sulfate

pollutant treatment using BESs.

1. Introduction performance of BES technology, which coupled electrochemical

process and biological process [10]. During biological process, the

Wastewater from animal husbandry, mining and food microbial community of electrode bioﬁlm plays a key role in BESs.

processing is usually highly polluted with sulfate, and high The electrode bioﬁlm, an extremely complex community, con-

concentration sulfate-based compounds raise serious health sists of eukaryotes, bacteria, archaea and viruses, in which bacteria

risks and warnings of environmental deterioration [1]. Therefore, account for dominant population [11]. Nowadays, comprehensive

various technologies such as reverse osmosis [2], electrochemical understanding of the microbial community is impeded by the low

method [3] and biotechnology [4–6] have been applied to control sequencing depth of traditional techniques, such as denaturing gra-

sulfate pollutants. dient gel electrophoresis [12] and terminal restriction fragment

Bioelectrochemical system (BES), as a kind of effective method length polymorphism [13]. The traditional techniques only detect a

for sulfate removal, has outstanding advantages in simultaneously snapshot of the dominant species and can hardly represent the taxa

transforming toxic sulfate into value-added elemental sulfur and with low abundances [14]. If the bacterial communities of the BESs

producing electric power. Most of previous studies [7–9] show that under different pH condition could be clariﬁed in depth, more sup-

the BESs suitably remove sulfate, however they mainly focus on the porting basis will catalyze the development of the BESs for sulfate

electrochemical process of BESs. The pH condition has been con- removal.

sidered a signiﬁcant environmental factor on the overall process In this study, the bioﬁlm of sulfate removal BESs was explored

using high-throughput pyrosequencing, which provides enough

sequencing depth to sufﬁciently reﬂect the vast genetic diver-

∗ sity. With the aid of open source softwares and scripts written

Corresponding authors. Tel.: +86 5926190766; fax: +86 592 6190766.

by ourselves, the pyrosequencing data of bacterial communities

E-mail addresses: [email protected] (Z.-H. Yang), [email protected] (F. Zhao).

http://dx.doi.org/10.1016/j.procbio.2014.04.019

1346 Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351

were analyzed from three aspects: (a) comparing different taxonomic according to the Greengenes database. In order to control error as much

as possible, we randomly selected 9000 sequences per sample to explore the ␣-

samples using the ␣-diversity indexes and the UniFrac ␤-diversity

diversity in each sample and compare the ␤-diversity between samples. To reﬂect

matrix, (b) discussing the distribution of bacteria phylotypes, and

the diversity and structure of bacterial community in each sample, Chao1 index

and phylogenetic diversity index were calculated from each sample. In light of the

nant operational taxonomic units (OTUs). With the above analyses, UniFrac matrix, we performed principal coordinate analysis and cluster analysis in

we revealed how pH condition directly affected the bacterial com- R v.2.15.0. The pyrosequencing sequences have been deposited into the NCBI with

accession number SRP028827.

munities of the BESs, which indirectly affected the performance of

sulfate pollutant treatment.

2.4. Statistical analysis

2. Materials and methods

Based on the information of bacteria related to sulfate metabolism [19], we

constructed a functional database containing the sulfate metabolism bacteria. By

2.1. BESs construction and DNA extraction

comparing the data of OTUs with the functional database, a group of sulfate

metabolism bacteria were selected from samples. In order to explore the function

The BESs were built for simultaneously removing sulfates and generating elec-

of electricity production, all OTUs were aligned with the known electrochemically

tricity with organics consumption. The BESs consisted of a working electrode

active bacteria [20] by BioEdit vesion 7.2.0 [21].

(activated carbon cloth, 10 × 10 cm), a counter electrode (activated carbon cloth,

Thirty dominant OTUs, as the representative research cells, were selected, which

10 × 10 cm) and an Ag/AgCl reference electrode (+197 mV vs. standard hydrogen

covered the top 10 abundant OTUs in all samples and the top 12 abundant OTUs of

electrode, CHI111, Chenhua, China) in one chamber. The simulated wastewater

each sample. Then, the representative sequences of the 30 OTUs were aligned with

contained the following components (per liter of deionized water): 1.0 g Na2SO4,

sequences listed in the GenBank database by Basic Local Alignment Search Tool

0.06 g MgSO4·7H2O, 0.5 g KH2PO4, 1.0 g NH4Cl, 0.03 g CaCl2, 0.3 g sodium citrate,

(BLAST). A phylogenetic tree of 30 OTUs was constructed using MEGA 4.0 program

0.1 g ascorbic acid and 1 ml absolute ethyl alcohol (24.58 mmol). The potential of

with the neighbor-joining method. The representative sequences of 30 OTUs were

the BESs was held at +0.2 V (vs. Ag/AgCl). A Series 4300 Battery Tested System (Mac-

deposited in GenBank under accession numbers KF264507–KF264536.

cor Inc., USA) was used to monitor the current of the BESs. After 20 days of stable

operation, microbial samples were taken from the top, middle, and bottom sections

of the working electrode. After evenly mixing, the samples were ﬂushed gently with

3. Results and discussion

50 mM phosphate buffer (pH 7) and centrifuged at 6000 × g to collect bacteria. In

order to make the taking of samples representative for the BESs under different

pH, two samples were taken from the BESs under each pH as reduplications. For 3.1. Enhance sulfate removal using BESs

each sample, genomic DNA was extracted and puriﬁed using a previously reported

protocol [15]. The puriﬁed genomic DNA was quantiﬁed by Micro-Ultraviolet Spec-

◦ The BESs were built for simultaneously removing sulfates and

trophotometer (Nanodrop Inc., USA) and stored at −20 C for further use.

generating electricity with organics consumption. Considering the

capital cost, treatment effect and secondary pollution, pH 4.5

2.2. Bacterial 16S rRNA ampliﬁcation and high-throughput pyrosequencing

was the optimum condition for sulfate removal from wastewater

Before pyrosequencing, the puriﬁed DNA was PCR ampliﬁed with a set of primers (Fig. 1). The performance of sulfate removal declined with pH, but

targeting the V1–V3 hypervariable regions of bacterial 16S rRNAs. The forward

the electrogenesis capacity show a opposite trend [22]. The samples

primer was 5 -AGAGTTTGATCCTGGCTCAG-3 (27F) with the Roche 454 ‘B’ adapter,

and corresponding reduplications under three pH were marked as

and the reverse primer was 5 -TTACCGCGGCTGCTGGCAC-3 (533R) containing the

BES4.5-1, BES4.5-2 (pH 4.5), BES6.5-1, BES6.5-2 (pH 6.5), BES8.5-1,

Roche 454 ‘A’ adapter and speciﬁc 10 bp barcode. The Roche 454 ‘A’/‘B’ adapter

located on the 5 -end of each primer, respectively. Each sample was ampliﬁed with BES8.5-2 (pH 8.5).

20 ␮l PCR reaction system following a previously reported study [16]. The PCR prod-

ucts were quantitated by QuantiFluor -ST, and then mixed for pyrosequencing. The

3.2. Overall pyrosequencing information

high-throughput pyrosequencing was processed on Roche GS FLX PLUS System.

2.3. Processing of pyrosequencing data Overall pyrosequencing information about six samples is exhib-

ited in Table 1. In this study, 79,374 valid reads were produced by

Pyrosequencing data 16S rRNA genes was processed using the Quantitative

Roche GS FLX PLUS System. The optimization procedure of data

Insights Into Microbial Ecology (QIIME, version 1.6) pipeline [17]. Before the sta-

generated a total of 60,589 high-quality sequences with quality

tistical analysis of data, QIIME was used to (a) check the completeness of the

barcodes and the primer sequencing, (b) remove reads shorter than 200 bp and qual- score above 25, length over 200 bp and without chimeras. The

ity score under 25, and (c) remove reads comprising chimera. Next, the sequences average length of sequences was 477.2 bp which was enough to

of different samples were exactly assigned using the unique 10 bp barcodes, and

perform the identiﬁcation of bacterial 16S rRNA genes. Six 16S rRNA

then the barcodes were removed. Only the 97% identity of the effective sequences

gene libraries were constructed from BES4.5-1, BES4.5-2, BES6.5-1,

were divided into OTUs for further analysis, and the most abundant sequence from

BES6.5-2, BES8.5-1 and BES8.5-2. As shown in Fig. S1, the num-

each OTU was selected as the representative sequence. After the representative

sequences were assigned by PyNAST [18], they were used for the classiﬁcation of ber of OTUs of BES4.5 and BES6.5 began to reach a plateau at 9000

Fig. 1. The conﬁguration and performance of bioelectrochemical systems for sulfate removal. (The reference electrode denotes to Ag/AgCl; the two bars in Fig. 1b represents

parallel test under different pHs).

Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351 1347

Table 1

Overall pyrosequencing information from the six samples.

Samples Reads Chimeras Effective sequences Per 9000 sequences

Raw valid reads Effective reads Chao1 Phylogenetic diversity

BES4.5-1 14,114 11,262 353 10,909 873 39.49

BES4.5-2 13,767 10,627 363 10,264 864 35.03

BES6.5-1 13,703 10,603 341 10,262 2038 71.70

BES6.5-2 11,703 9557 249 9308 2166 73.86

BES8.5-1 14,093 10,891 462 10,429 7544 175.26

BES8.5-2 11,994 9784 367 9417 7134 172.99

sequences, while new bacterial phylotypes continued to emerge in Firstly, un-weighted PCoA analysis reﬂected the relationship of

BES8.5. To ensure the fair comparison of different samples at same community diversity. The contribution rates of un-weighted prin-

sequencing depth, 9000 sequences was extracted from each sam- cipal components 1 and 2 accounted for 44.39% and 25.60% of the

ple, randomly. The good’s coverages were 97.2% (BES4.5-1), 97.3% total community variations, respectively. Secondly, the structure of

(BES4.5-2), 93.5% (BES6.5-1), 93.3% (BES6.5-2), 79.0% (BES8.5-1) bacterial community, including community diversity and richness,

and 79.8% (BES8.5-2) using random selections of 9000 sequences was reﬂected by weighted PCoA. There were relatively few vari-

per sample, suggesting that the 9000 sequences was enough to ances in the distribution of samples between un-weighted PCoA

reﬂect the characteristic of the bacterial communities. and weighted PCoA, while weighted principal component 1 soared

to 94.17% and weighted principal component 2 shifted to 4.76%.

The result indicated that weighted principal component 1 was

˛ ˇ

3.3. The -diversity and -diversity of bacterial community more inﬂuential to community structure than community diver-

sity. As pH was the dominated environment variable, pH might

Chao1 index and phylogenetic diversity were calculated using be represented by weighted principal components 1. Considering

random selections of 9000 sequences per sample (Table 1). The two the values of principal components, the distance between BES4.5

␣

-diversity indexes were used for estimating and comparing the and BES6.5 was the shortest in weighted PCoA, suggesting that the

richness and diversity of the bacterial communities. In addition, the community structure of BES4.5 and BES6.5 exhibited the highest

rarefaction curves of Chao1 index and phylogenetic diversity were similarity. This result was also supported by the cluster analysis

constructed at different pHs (Fig. S2). Phylotype richness, measured (Fig. 2) where BES4.5 and BES6.5 were separated from BES8.5.

as Chao1 index, was positively correlated with the pH condition

(r = 0.9477, P = 0.0040). The result was consistent with phylogenetic

diversity which also increased across the pH gradient (r = 0.9632, 3.4. The distribution of bacteria phylotypes

P = 0.0020). The observation of ␣-diversity indicated that the sam-

ple with higher pH represented greater richness and diversity of In order to compare the difference of bacterial distribution

bacterial community in this study. among BES4.5, BES6.5 and BES8.5, the most abundant 500 OTUs

Principal coordinated analysis (PCoA) was used to evaluate (3% distance) could be classiﬁed into three groups (I, II, III) based

the difference and similarity of bacterial communities by two on the cluster analysis of OTUs (Fig. 2). Three OTU groups I,

different forms of un-weighted PCoA and weighted PCoA (Fig. S3). II, III represented relatively high abundance in BES8.5, BES6.5

Fig. 2. Cluster analysis of bacterial communities of the bioelectrochemical systems. The 500 most abundant OTUs (3% distance) were clustered in two forms: OTUs and

samples. The relative abundance of OTUs is reﬂected by the color of scale. Three OTU groups I, II, III represented relatively high abundance in BES8.5, BES6.5 and BES4.5,

respectively. The three OTU groups were assigned to taxonomies at phylum and class level. (For interpretation of the references to colour in this ﬁgure legend, the reader is

referred to the web version of this article.)

1348 Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351

and BES4.5, respectively. These OTUs were assigned to known

taxonomies at phylum and class level, and there were 6 main

phyla and 8 main classes (the relative abundances were more than

1%). The sum of proportion of Proteobacteria, Chloroﬂexi and Bac-

teroidetes accounted for 72.62% in group I, 88.19% in group II and

85.45% in group III.

The relative abundance of Proteobacteria was signiﬁcantly cor-

related across the pH gradient, although in the opposite direction.

At least 33 species of electrochemically active bacteria were con-

ﬁned to the Proteobacteria [20]. Proteobacteria, a major phylum of

bacteria, included not only a number of electrochemically active

bacteria but also a wide variety of bacteria involving in sulfur

metabolism. The Proteobacteria are divided into ﬁve sections,

referred to Alphaproteobacteria, Betaproteobacteria, Deltapro-

teobacteria, Gammaproteobacteria and Epsilonproteobacteria. The

ﬁve classes also mainly existed in the bacterial communities of our

BESs.

The ﬁlamentous Chloroﬂexi bacteria have been detected grow-

ing speciﬁcally on BES anodes [23], and a previous study revealed

the robust coexistence of sulfate-reducing bacteria and Chloroﬂexi

in the sediment [24]. In these sulfate removal BESs, Chloroﬂexi

abounded in group I, but it rarely existed in groups II and III.

In this study, ethanol was selected as the sole carbon source

in the BESs, because ethanol was a preferred electronic donor for

sulfur-reducing bacteria [25]. Similar with our system, a previous

study constructed the thermophilic BESs for distillery wastewater

treatment, which achieved high efﬁciency for electricity genera-

tion and also reduced sulfate along with oxidizing complex organic

substrates [22]. These studies all demonstrated that Bacteroidetes

bacteria were abundant in the sulfate-ethanol BESs.

Pyrosequencing detected 128 bacterial genera in all samples,

and the genera related to sulfate metabolism were selected. There

were mainly four types of bacteria which were involved in sulfate

metabolism: sulfate-reducing bacteria, sulfur-reducing bacteria,

Fig. 3. The schematic of the pathways about sulfate metabolism (a) and the relative

sulfur-oxidizing bacteria and the disproportionation bacteria.

abundances of four types of bacteria (b). (A: sulfate-reducing bacteria; B: sulfur-

Fig. 3a was drawn according to how the bacteria contributed to

reducing bacteria; C: sulfur-oxidizing bacteria; D: disproportionation bacteria) in

removing sulfate. different bioelectrochemical systems. The abundance is presented in terms of an

Sulfate-reducing bacteria played a conducing role in the sulfate average percentage of the two samples in each system, classiﬁed at a conﬁdence

threshold of 97%. (Values in each cell represent percent abundance of each bacteria

metabolism; on the contrary, sulfur-reducing bacteria exerted an

type and red colors indicates more abundant, blue colors indicates less abundant).

adverse inﬂuence on the generation of sulfur, and sulfur-oxidizing

(For interpretation of the references to color in this ﬁgure legend, the reader is

bacteria weakened on the accumulation of sulfur under the con-

referred to the web version of this article.)

dition of constant voltage +0.2 V (vs. Ag/AgCl); disproportionation

bacteria was like a double-edged sword in the sulfate metabolism.

the abundant populations in BES8.5 which was widely reported

Since Fig. S4 showed good reproducibility between the samples and

for producing electricity. Geobacter metallireducens, Geobacter sul-

corresponding reduplications, we averaged the relative abundance

furreducens and Geobacter bremensis were isolated from the anode

of the samples and the corresponding reduplications for convenient

of BESs [26–28]. Besides, G. metallireducens, G. sulfurreducens and

comparison. As illustrated in Fig. 3b, BES4.5 and BES6.5 exhib-

Geobacter lovleyi were also detected in the cathode of BESs [29,30].

ited similar bacterial community composition and both highly

Geobacter might play an important role in producing electricity at

abounded with the genus of Desulfovibrio and Thiomonas. Desul-

pH 8.5. In addition, disproportionation bacteria were only detected

fovibrio, as a sulfate-reducing bacterium, was more easily detected

in BES8.5.

in BES4.5 (2.14%) and BES6.5 (2.16%) than in BES8.5 (0.43%), sug-

gesting that Desulfovibrio mainly contributed to removing sulfate

in the neutral and acidic conditions. As a sulfur-oxidizing bac- 3.5. The characterizations of dominant OTUs

terium, the Thiomonas in BES6.5 was about 1.69 times more than

␣ ␤

that in BES4.5, reﬂected in the fact that Thiomonas exerted greater The -diversity and -diversity of bacterial community had

adverse inﬂuence on removing sulfate in the neutral pH system. been analyzed from a macroscopic perspective. The relative

Unlike BES4.5 and BES6.5, BES8.5 was enriched with Desulfomicro- identiﬁcations and abundances of the dominant OTUs were micro-

bium (2.55%) and sulfuricurvum (8.24%), which could be respectively scopically illustrated in Fig. 4. By pyrosequencing, 6071 different

classiﬁed as sulfate-reducing bacteria and sulfur-oxidizing bacte- OTUs were detected, while 30 dominant OTUs abundantly existed

ria. There were 2, 6 and 7 genera belonging to sulfur-oxidizing in the samples. The relative abundances of the 30 OTUs summed

bacteria in BES4.5, BES6.5 and BES8.5, respectively, indicating that up to 63.62% in all samples, suggesting that the 30 OTUs cov-

the diversities of sulfur-oxidizing bacteria in BES6.5 and BES8.5 ered the majority of effective sequences. According to the results

were greater than that in BES4.5. Based on the above analysis and of BLAST and references, the identiﬁcations of 30 OTUs were dis-

the performance of sulfate removal, the higher diversity of sulfur- played in Fig. 4 for discussing their possible function. There were

oxidizing bacteria, the performance of sulfate removal more likely 12 GenBank matches belonging to cultured bacteria, and the oth-

was weakened. Within the sulfur-reducing bacteria, Geobacter was ers were uncultured bacteria. Considering the function of the

Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351 1349

Fig. 4. Relative abundances and identiﬁcations of the dominant OTUs. (Asterisk in each GenBank match represent cultured bacteria, others represent uncultured bacteria.

“SRSs” is short for “sulfate reduction systems”.).

BESs, we discussed the possible function of OTUs from the two homology (over 97%) with Desulfatirhabdium butyrativorans [37],

perspectives of sulfate removal and power generation. As illus- Desulfovibrio marrakechensis [38] and Desulfomicrobium sp. [39],

trated in the phylogenetic tree (Fig. S5), the 30 OTUs mainly respectively. All of them might make a contribution to removing

fell into Alphaproteobacteria (13.33%), Anaerolineae (16.67%), sulfate or might be involved in sulfate metabolism. There were

Bacteroidia (10.00%), Betaproteobacteria (26.67%), Deltaproteobac- another two OTUs (OTU-2332, OTU-5876) belonging to sulfur-

teria (16.67%) and Gammaproteobacteria (6.67%) at class level. oxidizing bacteria except for the above OTU-6798. They could

Among the 30 dominant OTUs, OTU-2921 and OTU-6798 reduce part of sulfur compounds (such as sulﬁde, elemental sulfur

showed salient abundance. OTU-2921, as the most abundant OTU, and thiosulfate) to form sulfate, which undermined the removal

was the dominant population in BES4.5 (38.02%) and BES6.5 efﬁciency of sulfate in the BESs.

(44.38%), but rarely appeared in BES8.5 (0.01%). The results indi- Electrochemically active bacteria were an indispensable portion

cated that OTU-2921 had higher adaptability in the neutral and of the BESs we constructed. Among 30 dominant OTUs, OTU-

acidic conditions. OTU-2921 was phylogenetically related (97% 2238, OTU-676 and OTU-5199 [40], showed similarities of 99% to

identity) to Paludibacter spp. Previous studies demonstrated that uncultured species detected in microbial fuel cells. These OTUs

Paludibacter spp., as a fermentative bacteria, had appeared in might make a contribution to the electricity production. However,

enrichments of sulfate reduction systems in acidic condition [31], part of electrochemically active bacteria might represent relatively

and Paludibacter spp. was often accompanied by the sulfate- less abundance in the BESs. Accordingly aligned with the known

reducing bacteria in sulfate reduction systems [32–34]. However, electrochemically active bacteria, 56 OTUs showed relative high

there was no valid evidence to prove that Paludibacter spp. can homology (over 96%) with the known electrochemically active

directly reduce sulfate. OTU-6798 was the second most OTU. OTU- bacteria (Fig. 5). There existed eight kinds of electrochemically

6798 had a similarity of 99% with Thiomonas sp., which was a sulfur active bacteria, which were Burkholderia cepacia [41], Pseudomonas

oxidizer [35]. OTU-6798 showed relatively more enrichment in aeruginosa [42], Acinetobacter calcoaceticus [43], Brevundimonas

BES6.5 (18.34%) than that in BES4.5 (8.44%), and the proportion of diminuta [41], Rhodopseudomonas palustris [44], G. lovleyi [30], G.

OTU-6798 in BES8.5 was only 0.76%. The results suggested that the sulfurreducens [27] and Desulfobulbus propionicus [45]. Among 56

function of sulfate removal was weakened by OTU6798 in BES6.5 OTUs, 21 OTUs (37.5%) were identiﬁed as B. cepacia, and were pri-

at a large extent, but the situation could be better in BES4.5 and marily detected in BES4.5. The other OTUs abounded in BES8.5,

BES8.5. The result contributed to clarifying the reason why the which were deﬁned as R. palustris, G. lovleyi, G. sulfurreducens and

performance of sulfate removal in BES4.5 was better than that in D. propionicus. As previous study reported [45], D. propionicus was

BES6.5. not only an electrochemically active bacterium but also a sulfate-

Except for the above OTU-2921, there were 8 other OTUs reducing bacterium.

(OTU-5839, OTU-230 [36], OTU-312, OTU-3813, OTU-7213, OTU- In this study, the bacterial community was analyzed from

2761 [25], OTU-6555, OTU-4254) had been detected in sulfate ␣, ␤-diversity, phylotypes and dominant OTUs. Though part of

reduction systems, three of which could be identiﬁed as sulfate- OTU’s function remained unknown and required further study

reducing bacteria. OTU-5839, OTU-3813 and OTU-6555 had high for conﬁrmation, community analysis demonstrated a range of

1350 Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351

Fig. 5. Relative abundances and identiﬁcations of the OTUs which were homology with the known electrochemically active bacteria. (The percentage in brackets represent

the similarity between the OTU and the known electrochemically active bacteria. Values in each cell represent percent abundance of each OTU and red colors indicates more

abundant, blue colors indicates less abundant). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

interesting phylotypes and OTUs. For example, Paludibacter spp. Acknowledgements

was accompanied by the sulfate-reducing bacteria. Consequently,

it is helpful to purposefully chose study subjects for further phys- This study was sponsored by the main Direction Program of

iological and biochemical research. Based on the information, the Knowledge Innovation (KZCXZ-EW-402), National Natural Science

microorganisms agent, such as inoculant of Desulfovibrio bacteria, Foundation of China (21322703, 51208490). Thanks to Valerie Gail

could be produced to enhance the performance of sulfate removal Gibson for the aid of polishing the language.

under acidic environment.

Appendix A. Supplementary data

4. Conclusions

Supplementary material related to this article can be found, in

We comprehensively explored the bacterial communities of

the online version, at doi:10.1016/j.procbio.2014.04.019.

bioelectrochemical systems under three pHs using 454 pyrose-

quencing. Our study demonstrated that the indexes of phylotype

richness and phylogenetic diversity were positively correlated References

across the pH gradient in the BESs. Among dominant OTUs, the

OTUs which were highly similar to D. butyrativorans, Desulfovib- [1] Lens PNL, Hulshoff Pol L. Environmental technologies to treat sulfur pollution.

London: International Water Association; 2000.

rio marrakechensis and Desulfomicrobium sp. might participate in

[2] Amjad Z. Applications of antiscalants to control calcium sulfate scaling in

removing sulfate. Based on genus level, Desulfomicrobium and Sul-

reverse osmosis systems. Desalination 1985;54:263–76.

furicurvum play conducing and adverse roles for sulfate removal [3] Panayotova M, Panayotov V. An electrochemical method for decreasing the con-

centration of sulfate and molybdenum ions in industrial wastewater. J Environ

in alkaline condition, respectively. Desulfovibrio contributed to

Sci Health A: Tox Hazard Subst Environ Eng 2004;39:173–83.

removing sulfate in the neutral and acidic condition, while

[4] Lens PNL, Visser A, Janssen AJH, Hulshoff Pol LW, Lettinga G. Biotechno-

Thiomonas mainly weakened the performance of sulfate removal logical treatment of sulfate-rich wastewaters. Crit Rev Environ Sci Technol

1998;28:41–88.

in neutral pH condition. These results further clariﬁed how pH

[5] Silva A, Varesche M, Foresti E, Zaiat M. Sulphate removal from indus-

condition directly affected the bacterial communities, which con-

trial wastewater using a packed-bed anaerobic reactor. Process Biochem

sequently affected the performance of sulfate pollutant treatment. 2002;37:927–35.

Y. Zheng et al. / Process Biochemistry 49 (2014) 1345–1351 1351

[6] Sarti A, Pozzi E, Chinalia FA, Ono A, Foresti E. Microbial processes and bacte- coupling the complete oxidation of organic compounds to the reduction of iron

rial populations associated to anaerobic treatment of sulfate-rich wastewater. and other metals. Arch Microbiol 1993;159:336–44.

Process Biochem 2010;45:164–70. [27] Bond DR, Lovley DR. Electricity production by Geobacter sulfurreducens attached

[7] Habermann W, Pommer EH. Biological fuel cells with sulphide storage capacity. to electrodes. Appl Environ Microbiol 2003;69:1548–55.

Appl Microbiol Biotechnol 1991;35:128–33. [28] Nercessian O, Parot S, Délia ML, Bergel A, Achouak W. Harvesting

[8] Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy electricity with Geobacter bremensis isolated from compost. PLoS ONE

generation. Trends Biotechnol 2005;23:291–8. 2012;7:e34216.

[9] Zhao F, Rahunen N, Varcoe JR, Chandra A, Avignone-Rossa C, Thumser AE, et al. [29] Gregory KB, Bond DR, Lovley DR. Graphite electrodes as electron donors for

Activated carbon cloth as anode for sulfate removal in a microbial fuel cell. anaerobic respiration. Environ Microbiol 2004;6:596–604.

Environ Sci Technol 2008;42:4971–6. [30] Strycharz SM, Woodard TL, Johnson JP, Nevin KP, Sanford RA, Löfﬂer FE, et al.

[10] Raghavulu SV, Mohan SV, Goud RK, Sarma P. Effect of anodic pH microenviron- Graphite electrode as a sole electron donor for reductive dechlorination of

ment on microbial fuel cell (MFC) performance in concurrence with aerated tetrachlorethene by Geobacter lovleyi. Appl Environ Microbiol 2008;74:5943–7.

and ferricyanide catholytes. Electrochem Commun 2009;11:371–5. [31] Sánchez-Andrea I, Stams AJ, Amils R, Sanz JL. Enrichment and iso-

[11] Freguia S, Rabaey K, Yuan Z, Keller Jr. Syntrophic processes drive the conversion lation of acidophilic sulfate-reducing bacteria from Tinto River

of glucose in microbial fuel cell anodes. Environ Sci Technol 2008;42:7937–43. sediments. Environ Microbiol Rep 2013;5(October (5)):672–8,

[12] Muyzer G, De Waal EC, Uitterlinden AG. Proﬁling of complex microbial popu- http://dx.doi.org/10.1111/1758-2229.12066.

lations by denaturing gradient gel electrophoresis analysis of polymerase

[32] Zhang JX, Zhang YB, Quan X, Chen S, Afzal S. Enhanced anaerobic digestion of

chain reaction-ampliﬁed genes coding for 16S rRNA. Appl Environ Microbiol

organic contaminants containing diverse microbial population by combined

1993;59:695–700.

microbial electrolysis cell (MEC) and anaerobic reactor under Fe (III) reducing

[13] Liu WT, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversity

conditions. Bioresour Technol 2013;136:273–80.

by determining terminal restriction fragment length polymorphisms of genes

[33] Lindsay MB, Wakeman KD, Rowe OF, Grail BM, Ptacek CJ, Blowes DW, et al.

encoding 16S rRNA. Appl Environ Microbiol 1997;63:4516–22.

Microbiology and geochemistry of mine tailings amended with organic carbon

[14] Yates MD, Kiely PD, Call DF, Rismani-Yazdi H, Bibby K, Peccia J, et al. Convergent

for passive treatment of pore water. Geomicrobiol J 2011;28:229–41.

development of anodic bacterial communities in microbial fuel cells. ISME J

[34] Bijmans MF, De Vries E, Yang CH, Buisman N, Cees J, Lens PNL, et al. Sulfate

2012;6:2002–13.

reduction at pH 4.0 for treatment of process and wastewaters. Biotechnol Prog

[15] Yang ZH, Xiao Y, Zeng GM, Xu ZY, Liu YS. Comparison of methods for total 2010;26:1029–37.

community DNA extraction and puriﬁcation from compost. Appl Microbiol

[35] Arsène-Ploetze F, Koechler S, Marchal M, Coppée JY, Chandler M, Bonnefoy V,

Biotechnol 2007;74:918–25.

et al. Structure, function, and evolution of the Thiomonas spp genome. PLoS

[16] Wang ZJ, Zheng Y, Xiao Y, Wu S, Wu YC, Yang ZH, et al. Analysis of oxygen

Genet 2010;6:e1000859.

reduction and microbial community of air-diffusion biocathode in microbial

[36] Feng SS, Yang HL, Xin Y, Zhang L, Kang WL, Wang W. Isolation of an extremely

fuel cells. Bioresour Technol 2013;144:74–9.

acidophilic and highly efﬁcient strain Acidithiobacillus sp. for chalcopyrite

[17] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK,

bioleaching. J Ind Microbiol Biotechnol 2012;39:1625–35.

et al. QIIME allows analysis of high-throughput community sequencing data.

[37] Balk M, Altınbas¸ M, Rijpstra WIC, Damsté JSS, Stams AJM. Desulfatirhab-

Nat Methods 2010;7:335–6.

dium butyrativorans gen. nov., sp. nov., a butyrate-oxidizing, sulfate-reducing

[18] Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R.

bacterium isolated from an anaerobic bioreactor. Int J Syst Evol Microbiol

PyNAST: a ﬂexible tool for aligning sequences to a template alignment. Bioin- 2008;58:110–5.

formatics 2010;26:266–7.

[38] Chamkh F, Spröer C, Lemos PC, Besson S, El Asli A-G, Bennisse R, et al. Desulfovib-

[19] Liang FY, Deng H, Zhao F. Sulfur pollutants treatment using microbial fuel cells

rio marrakechensis sp. nov., a 1, 4-tyrosol-oxidizing, sulfate-reducing bacterium

from perspectives of electrochemistry and microbiology. Chin J Anal Chem

isolated from olive mill wastewater. Int J Syst Evol Microbiol 2009;59:936–42.

2013;41:1133–9.

[39] Azabou S, Mechichi T, Patel BKC, Sayadi S. Isolation and characterization of

[20] Xiao Y, Wu S, Yang ZH, Zheng Y, Zhao F. Isolation and identiﬁcation of electro-

a mesophilic heavy-metals-tolerant sulfate-reducing bacterium Desulfomicro-

chemically active microorganisms. Prog Chem 2013;25:1771–80.

bium sp. from an enrichment culture using phosphogypsum as a sulfate source.

[21] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and anal-

J Hazard Mater 2007;140:264–70.

ysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999;41:95–8.

[40] da Rosa AC. Diversity and function of the microbial community on anodes of

[22] Liang FY, Xiao Y, Zhao F. Effect of pH on sulfate removal from wastewater using

sediment microbial fuel cells fueled by root exudates. Marburg University;

a bioelectrochemical system. Chem Eng J 2012;218:147–53.

2010. Ph.D. Dissertation.

[23] Ishii S, Shimoyama T, Hotta Y, Watanabe K. Characterization of a ﬁlamen-

[41] Cournet A, Délia ML, Bergel A, Roques C, Bergé M. Electrochemical reduction

tous bioﬁlm community established in a cellulose-fed microbial fuel cell. BMC

of oxygen catalyzed by a wide range of bacteria including Gram-positive. Elec-

Microbiol 2008;8:6.

trochem Commun 2010;12:505–8.

[24] Koizumi Y, Kojima H, Fukui M. Dominant microbial composition and its vertical

[42] Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W. Biofuel cells select for

distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantita-

microbial consortia that self-mediate electron transfer. Appl Environ Microbiol

tive oligonucleotide probe membrane hybridization. Appl Environ Microbiol 2004;70:5373–82.

2004;70:4930–40.

[43] Freguia S, Tsujimura S, Kano K. Electron transfer pathways in microbial oxygen

[25] Kaksonen AH, Plumb JJ, Franzmann PD, Puhakka JA. Simple organic electron

biocathodes. Electrochim Acta 2010;55:813–8.

donors support diverse sulfate-reducing communities in ﬂuidized-bed reactors

[44] Xing DF, Zuo Y, Cheng SA, Regan JM, Logan BE. Electricity generation by

treating acidic metal- and sulfate-containing wastewater. FEMS Microbiol Ecol

Rhodopseudomonas palustris DX-1. Environ Sci Technol 2008;42:4146–51.

2004;47:279–89.

[45] Holmes DE, Bond DR, Lovley DR. Electron transfer by Desulfobulbus propionicus

[26] Lovley DR, Giovannoni SJ, White DC, Champine JE, Phillips E, Gorby YA,

to Fe (III) and graphite electrodes. Appl Environ Microbiol 2004;70:1234–7.

et al. Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of