Denitrifying microbial community with the ability to bromate reduction in a rotating biofilm-electrode reactor

Accepted Manuscript Title: Denitrifying microbial community with the ability to bromate reduction in a rotating biofilm-electrode reactor Authors: Yu Zhong, Qi Yang, Guangyi Fu, Youze Xu, Yingxiang Cheng, Caili Chen, Renjun Xiang, Tao Wen, Xiaoming Li, Guangming Zeng PII: DOI: Reference:

S0304-3894(17)30611-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.08.019 HAZMAT 18784

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

22-5-2017 7-8-2017 8-8-2017

Please cite this article as: Yu Zhong, Qi Yang, Guangyi Fu, Youze Xu, Yingxiang Cheng, Caili Chen, Renjun Xiang, Tao Wen, Xiaoming Li, Guangming Zeng, Denitrifying microbial community with the ability to bromate reduction in a rotating biofilm-electrode reactor, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Denitrifying microbial community with the ability to bromate reduction in a rotating biofilm-electrode reactor Yu Zhonga,b,c,, Qi Yangb,c,*, Guangyi Fua,*, Youze Xua, Yingxiang Chenga, Caili Chena, Renjun Xianga, Tao Wena, Xiaoming Lib,c, Guangming Zengb,c. a

Key Laboratory of Water Pollution Control Technology, Hunan Research Academy of

Environmental Sciences, Changsha 410004, China b

College of Environmental Science and Engineering, Hunan University, Changsha 410082, China

c

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

Education, Changsha 410082, China

*Corresponding author: E-mail: [email protected] (Q. Yang); [email protected] (G. Fu) Tel.: +86-731-88822829; Fax: +86-731-8882

Graphical abstract

Fx1

Highlights 

Bromate removal was achieved by auto-hydrogenotrophic denitrification reduction

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Bromate reduction was inhibited by the high concentration of nitrate

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The microbial community of biofilm was analyzed at phylum and genus level

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Miseq sequencing analysis showed the bromate-reducing bacteria was phylogenetically diverse 1

Abstract In this study, the microbial community for bromate reduction in a rotating biofilm-electrode reactor (RBER) was investigated. Continuous experiment demonstrated that the bromate reduction by an auto-hydrogenotrophic microbial community was inhibited by high concentration nitrate (50mg/L). The bacterial diversity of RBER were examined through the analyse of 16S rRNA gene sequences of clone libraries. The results showed that the bromate-reducing bacteria were phylogenetically diverse at the phylum level, representing the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The relative abundances of these microbial community represented 99.1% of all phylum in the biofilms when bromate served as the sole electron acceptor. Meanwhile, the Bacillus strains became the largest phylotype and represented about 37% of the total bacteria in the biofilm, indicating that the genus Bacillus played the key role in the autohydrogenotrophic process. Moreover, three new bacterial genera, Exiguobacterium, Arthrobacter and Chlorobium appeared with the respective relative abundance being about 7.37%, 1.81%, and 0.52%, which might be the bromate-specific reducing bacteria.

Keywords: Bromate; Nitrate; Biofilm-electrode reactor; Microbial community; High-throughput sequencing 1. Introduction Bromate (BrO3-) is an disinfection byproduct (DBP) during the chlorination or ozonation of bromide-containing water, which has been classified as a group II carcinogen (as a possible human carcinogen) by the International Agency for Research on Cancer (IARC) [1]. In recent years, the pollution of bromate has become more serious due to the excessive use of ozonation in water 2

purification and the uncontrolled discharge of food additives containing bromate into surface water. Today bromate is not only detected within both surface water [2] and drinking water purification plant [3], but also has been observed in some groundwater with a higher concentration of 1 mg/L due to industrial contamination [4]. In order to protect human health, a maximum contaminant level of bromate in drinking water has been established at 10 µg/L (0.078 µmol/L) by the European Union and the U. S. Environmental Protection Agency (EPA) [5, 6]. Strategies of removing stable bromate from water include adsorption process [7, 8], membrane separation [9, 10], catalytic decomposition [11, 12] and biological reduction [13, 14]. Biological reduction has gained more and more interest in the past decade owing to its capability of reducing bromate to innocuous bromide in a cost-effective way [15]. Microbial bromate reduction has been reported in a variety of reactor systems. Hijnen et al. [16] investigated the bromate removal by a denitrifying fixed-bed bioreactor with the ethanol as electron donor. Liu et al. [17] identified the bromate-reducing isolates in the biological activated carbon filter with sodium acetate (CH3COONa) as exogenous carbon source and electron donor. Downing and Nerenberg [18] designed a new reactor, the hollow-fiber membrane biofilm reactor (MBfR), to determine the kinetics of hydrogenbased bromate reduction under denitrifying conditions. In these researches, the external supply of organic carbon sources or hydrogen to the reactors is necessary, while the extra carbon sources may cause secondary pollution and the explosive nature of hydrogen gas may also bring the safety problems. To overcome the above-mentioned disadvantages, an auto-hydrogenotrophic rotating biofilm electrode reactor (RBER) combining biological and electrochemical process has developed to remove bromate based on the hydrogen autotrophic denitrification. In the RBER, H2 was generated on site by electrolysis of water and completely utilized by the microbial community 3

immobilized on the cathode surface, and avoid the waste of excessive H2 in the case of external addition [19]. Indeed biological reduction of bromate had been realized in many types of bioreactor, the study on the microbial communities capable of bromate reduction was little. Hijnen et al. [20] isolated the bromate-reducing bacteria from a mixed population of denitrifying bacteria, which was identified as Pseudomonas spp. Davidson et al. [14] isolated and characterized several bromate-reducing bacteria, suggesting that the diversity of bromate-reducing bacteria was broad. Meanwhile, Bromate could be catalyzed reduction by the purified nitrate reductase [21], and oxygen and nitrite might inhibit bromate reduction [16]. However, research showed that bromate could be reduced by an anaerobic, mixed microbial community but these bacteria were unable to reduce nitrate [22]. Downing and Nerenberg [18] demonstrated that the denitrifying bacterium such as Ralstonia eutropha (ATCC 17697) was incapable of bromate reduction, indicating that bromate reduction is not a functionally linked trait shared by all denitrifying bacteria. These researches suggest the bromate reduction might depend upon the structure of the microbial community. In this study, the ability of denitrifying bacterial communities to reduce the bromate was investigated in a rotating biofilm-electrode reactor. In addition, the high-throughput sequencing technology was used to reveal the relationship between microbial community structure and bromate removal performance in auto-hydrogenotrophic denitrification process. 2. Materials and methods 2.1. Experimental procedure A schematic diagram of the auto-hydrogenotrophic rotating biofilm-electrode reactor (RBER) used in this work is shown in Fig. S1 (see the Supporting Information), which was described 4

detailedly in our previous study [23]. Synthetic wastewater contained K2HPO4, KH2PO4, MgSO4·7H2O, NaHCO3 and trace elements [24]. A 200 mL anaerobic sludge obtaining from the second municipal wastewater treatment plant (Changsha, China) was used as the inocula. Then the inocula were added into the RBER for microbial development and growth by inflowing 25 mg/L nitrate of synthetic wastewater. When denitrification rate was higher than 75% and a dark grey biofilm formed on the surface of activated carbon fibers, the inoculation stage was accomplished. For simplicity, the inoculation stage was referred to “stage 1”. In order to investigate the bromate reduction in presence of high nitrate concentration, 50 mg/L of nitrate was introduced to the RBER at stage 4. The add process was divided into five successive stages untill the target concentration was achieved and the details are listed in Table 1. Before converting to next stage, each stage needed to reach a steady state and the effluent concentrations of all chemical species were stable for at least 2 days. 2.2. Microbial community resolution procedure 2.2.1 Biofilm sampling and DNA extraction The biofilm samples of each stage were collected from the RBER cathode when each operating stage reached a plateau, i.e., stage 1(15 days), stage 2 (24 days), stage 3 (40 days), stage 4 (55 days), stage 5 (70 days) and stage 6 (80 days). The biofilm sample was cut off a 2-cm piece from the surfaces of the RBER cathode using sterilized blade. Then the sample was immediately put into bead tubes for DNA extraction using the Soil DNA Kit (Omega Bio-Tek, Inc., Norcross, USA), and genomic DNA was extracted following the manufacturer’s instruction. The crude DNA was dissolved in 600 μL TE buffer and stored at -25°C. Figure S2 in the Supporting Information showed that it appears clear and bright band in the agarose gel electrophoresis for six crude DNA samples, 5

suggesting the DNA extracts could be used for the subsequent PCR amplification. 2.2.2 PCR amplification The universal bacteria forward primer 338F (5'- ACTCCTACGGGAGGCAGCAG-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') was used in PCR amplification [25], which were targeting the V3-V6 hypervariable regions. The 50-μL PCR reaction mixture was composed of 5 μL 1× FastPfu Buffer, 4 μL 0.25 mM dNTPs, 1.0 μL 10 μM each of forward and reverse primers, 0.3 μL FastPfu Polymerase (Trans Gen Biotech, China), 10 ng DNA template and added ultrapure water to make up the final volume to 20 μL [26]. The specific conditions were introduced in Figure S3 of the Supporting Information. The PCR products were examined on a 2 % (w/v) agarose gel, and then the amplicons were purified using the AXYGEN gel extraction kit (Axygen, USA). The PCR amplicons were sent out for sequencing on the Illumina MiSeq platform at the Shanghai Majorbio Bio-Pharm Technology Co., Ltd. 2.2.3 Sequence analysis The raw sequence read sets obtained from pyrosequencing were processed and analyzed by the QIIME (Quantitative Insights into Microbial Ecology) software package [27]. The resulting effective sequences were used for the subsequent bioinformatic analysis. Operational taxonomic units (OTUs) with identities of 97% were identified using the Mothur software (http://www.mothur.org), and the diversity indices (Chao 1, ACE, Coverage, Shannon and Simpson) were determined based on the calculated OTUs [28]. The sequence with highest relative abundance in each OTU was selected as the representative sequence to search for similar sequences in the National Center for Biotechnology Information (NCBI) nucleotide non-redundant database [29, 30]. The interrelationships between the microbial communities of the different stage samples were visualized using principal component 6

analysis (PCA), and the classification results were used to calculate relative abundances at the phylum and genus level for comparing the observed microbial community structure. 2.3. Analytical methods The liquid samples were collected daily with 5 mL syringes and filtered immediately through a 0.22 mm membrane filter (LC+PVDF membrane, ANPEL Laboratory Technologies Inc., China). The bromate, bromide, nitrate and nitrite concentrations were assayed using ion chromatography (Dionex ICS-900, USA) with an AS19 column and AG19 pre-column, and an eluent concentration of 9.4 mM Na2CO3 and 1.8 mM NaHCO3 (flow rate 1 mL/min). A HQ-30D DO meter (HACH, USA) was used to measure dissolved oxygen, and the pHS-3C model (Rex Instrument Factory, China) was applied to measure pH value. 3. Results and discussion 3.1. Bromate and nitrate removal in the RBER When the RBER fed with 25 mg N/L nitrate successfully completed the inoculating (stage 1), 150 μg/L bromate was added to the influent nitrate medium. Fig. 1a showed the concentrations of bromate in influent and effluent, along with the effluent concentration of bromide. Fig. 1b showed the concentrations of nitrate in influent and effluent, along with the effluent nitrite. When 150 μg/L bromate was introduced to the RBER in stage 2 (days 16-23), the effluent bromate decreased to below the 10-μg/L detection limit, and the effluent concentration of nitrate eventually reduced to about 1.0 mg /L at day 21. No bromide was detected in effluent in stage 2, suggesting that all of influent bromate was removed by adsorption process instead of biological reduction [23]. In stage 3 (days 24-40), when the influent bromate was up to 400 μg/L, almost all of nitrate and bromate was removed (Fig. 1b), and stoichiometric amounts of bromide was found in effluent (Fig. 1a) which 7

suggested that denitrifying bacteria had adapted the bromate load and bromate reduction by denitrifying bacteria had occupied a predominant position in the RBER. Comparison of stages 4 and 5 showed that nitrate could partially inhibit the bromate reduction, and bromate reduction could be completed when nitrate concentration decreased from 50 mg/L to 25 mg/L. It means that the competition of bromate and nitrate for the electron donor existed in the RBER. When bromate was the only electron acceptor (stage 6), its effluent concentration also reduced from influent 800 μg/L to 100 μg/L, and gradually fell to below 10 μg/L. Sustained bromate reduction in the absence of nitrate suggests that hydrogen oxidizing, bromate-reducing bacteria may exist in the RBER. A analogous conclusion has been drawn by some researchers that bromate could reduce to bromide by denitrifying bacteria with and without a preceding nitrate reduction step in an anaerobically incubated medium [20]. 3.2. Bacterial diversity analysis Since the functions and structures of bacterial community had an impact on the biodegradation performance of the RBER, the high-throughput sequencing was carried out to analyze the bacterial community diversities of the RBER biofilms. A total of 211,812 effective sequences of the 16S rRNA gene were generated from six samples and clustered into 1910 operational taxonomic units (OTUs) based on 97% identity (Table 2). The numbers of OTUs, community richness of Chao 1, and ACE index were gradually increased when the RBER operating from stage 1 to stage 3. In stage 4 (days 41-55), the diversity estimators of OTUs and community richness (Chao 1, ACE) index reached a maximum value when a high NO3 - concentration (50 mg/L) was introduced to the RBER. The Shannon-Wiener diversity index considers both richness and evenness of samples [31]. Fig. 2a showed that the Shannon index was different among six samples of the OTUs. The stage 4 sample 8

had the highest diversity (4.53), while the stage 6 sample had the lowest diversity (3.05). The reads of each sample are large enough (>20,000 tags per sample) to reflect the vast diversity of microbial community information as they approached the plateau from less than 5,000 tags per sample. Rarefaction analysis was used to standardize and compare observed taxon richness between samples [32]. The rarefaction curves based on OTUs at 97% similarity showed a similar tendency for six samples (Fig. 2b), and most rarefaction curves reached saturation with large than 99.7% coverage. The stage 4 sample had the highest microbial richness and evenness These results indicated that the recovered sequences commendably represented the diversity of the microbial community in six samples, and further sampling effort can only reveal the few extent of the diversity of microbial community [33]. To evaluate the distribution of OTUs among the different samples, the Venn diagrams of five biofilm samples except for the inoculation sample (stage 1) are list in Fig. 3a. A total of 1625 OTUs were obtained from five biofilm samples. The shared OTUs were 156 (9.6% of the total OTUs) among the five samples which mainly belonged to Firmicutes and Proteobacteria, indicating that these microorganisms exist in the whole operation process of the RBER. Further, the numbers of special OTUs were five, one, six, seven and three at the samples from the stage of 2, 3, 4, 5 and 6, respectively. They represented a low percentage of sequences. Fig. 3b shows the unweighted PCoA based on the absence or presence of bacteria. It is found that the cumulative percentage variance of species explained by the first axis was 73.37 %, while the second axis explained 15.47% of the variation. The samples for stages 1 and stage 2 grouped together have much higher PC1 values when compared to those for stage 3, stage 4, stage 5, and stage 6. Thus, the PCoA analysis supported that the microbial community enriched with bromate initially, while introducing nitrate had great impact 9

on shaping the microbial community structure. 3.3 Phylum-level taxonomic distribution The pyrosequencing targeting the V3-V6 hypervariable regions was used to analyze the diversity and structure of the RBER microbial communities in the different stage samples. Fig. 4 showed the top eight most abundant phyla at the phylum level for six stage samples. In stage 1 (days 0-15), Proteobacteria and Firmicutes were the dominant species on inoculated sludge with altogether 17009 OTUs (in Table 3), which collectively represented about 79.56% of the total bacteria in stage 1. According to previous researches, phylum Proteobacteria (main classes contained Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) was one of the most abundant groups in activated sludge [34, 35]. Heylen et al. reported that most denitrifiers in activated sludge are from the Alphaproteobacteria and Betaproteobacteria, but they also found denitrifiers representing the Gammaproteobacteria and Firmicutes [36]. The phylum Proteobacteria, Actinobacteria and Chloroflexi were decreased dramatically after bromate (150 μg/L) was input in stage 2, and further decreased when bromate was increased to 400 μg/L in stage 3. It implied that the Proteobacteria, Actinobacteria and Chloroflexi may be inhibited by bromate reduction. Actinobacteria was phylogenetically related to Gram-positive bacteria. Although the relative abundance of Actinobacteria was inhibited by bromate reduction, some researchers reported that Actinobacteria could reduce nitrate and chromate using biodegradable meal box as carbon source and biofilm carriers [37]. Meanwhile, Chloroflexi also exhibited the reducing capacity for nitrate in the biofilm of cathode in microbial fuel cell [38]. In stage 4, when bromate concentration increased along with nitrate concentration, the microbial community structures remained similar to stage 2. However, when nitrate concentration decreased to 10

25 mg/L in stage 5, Firmicutes became the absolutely dominant specie, counting for more than 59.33% in the biofilm, while the abundance of Proteobacteria decreased to 29.80%. The results showed that the abundance of Proteobacteria decreased with the decreasing influent nitrate concentration. Chen et al. [32] found that the auto-hydrogenotrophic denitrifying reactor exhibited excellent denitrification capacity. They demonstrated that the Proteobacteria in these reactor had the ability to use H2 as electron donor and nitrate as electron acceptor for nitrate removal. Previous studies indicated that bromate reduction occurs as a cometabolic reaction with the nitrate reductase [20, 39]. Davidson et al. [14] suggested that a bromate-specific reduction pathway might exist in some bromate-reducing bacteria and the predominant mechanism of bromate reduction (i.e., cometabolic or respiratory) was unclear. When the bromate was the only electron acceptor in stage 6, Firmicutes became the absolutely dominant bacterial genera which represented about 63.36% of the total bacteria. Similar results have also been observed by earlier researchers that the bromate-reducing bacteria were phylogenetically related to Gram-positive bacteria belonging to the phyla of Firmicutes, Proteobacteria and Bacteroidetes, while the Firmicutes played key roles in the drinking water treatment process [14]. 3.4 Genus-level distribution Fig. 5 shows the relative proportions of the most abundant bacterial genera for the six samples. In the inoculation of stage 1, the genus of Bacillus, Pseudomonas and Lactococcus were represented about 0.41%, 0.06% and 0.13%, respectively. It showed clearly that Bacillus, Pseudomonas and Lactococcus were gradually increased with the RBER running from stage 1 to stage 6. When bromate was the sole electron acceptor in stage 6, the Bacillus strains became the largest phylotype, counting for more than 37% in the biofilm. Some researchers discovered that Bacillus strains could 11

reduce nitrate and nitrite, and it was phylogenetically related to Gram-positive bacteria belonging to the phyla Firmicutes [40]. Supposing that the total bromine mass balance was achieved and bromide was the only end product from bromate reduction, the sum of normalized concentration of bromide and residual bromate will be equal to influent bromate. However, it was found that the end product bromide was unequal to influent bromate in stage 6. The 4.1% loss of total bromine (data were not shown) in the auto-hydrogenotrophic process should contribute to be adsorbed to the extracellular products. Assunção et al. [13] reported that the bromate adsorption by extracellular metabolic products could be one of the mechanism for bromate removal in anaerobic bacterial community. The Pseudomona strains were the subdominant genus in stage 6, which belong to the family of Pseudomonadaceae [41]. It has been confirmed that the Pseudomona strain was bromate-reducing bacteria, which could reduce bromate to bromide in an anaerobically incubated medium [20]. Meanwhile, Davidson et al. have isolated bromate reducing bacteria Pseudomonas fluorescens, which belong to denitrifying bacteria [14]. In stage 6, the Lactococcus strain became the third dominant phylotype, with the relative abundance about 11.05%. The Lactococcus strains were currently used in the biotechnology industry which was found to play a role in the flavor of the final product [42]. Compare with the bacteria strains in stage 1, there were some new genera (i.e., Exiguobacterium, Arthrobacter and Chlorobium) appeared in stage 6. These strains were related to Gram-positive bacteria, which might be the bromate-specific reducing bacteria. Shi et al. [43] reported that the Arthrobacter sp.W1 and Pseudomona strains played the most significant role in the coking wastewaters treatment by bioaugmented aerated filter reactor. But additional phylogenetic analysis is needed to reveal the existence of these novel microbial communities in the RBER. 12

4. Conclusions The rotating biofilm-electrode reactor exhibited excellent bromate and nitrate removal performance, and the auto-hydrogenotrophic microbial communities were capable to remove bromate in the absence of nitrate. Sequencing analysis of the biofilm samples showed the bromatereducing bacteria were phylogenetically diverse at the phylum level, such as the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. With bromate as the only electron acceptor, the dominant genera were Bacillus, Pseudomonas and Lactococcus. Meanwhile, there were three new bacterial generas (i.e., Exiguobacterium, Arthrobacter and Chlorobium) appeared in this stage and some of them were never reported as bromate reducing bacteria, which might be the bromate-specific reducing bacteria. Therefore these results are helpful for a systematic understanding of microbial communities in auto-hydrogenotrophic denitrification process. Acknowledgment This work was supported by projects of the National Natural Science Foundation of China (NSFC) (Nos. 51378188, 51478170), the International Science & Technology Cooperation Program of China (Nos. 2013DFG91190), and the Natural Science Foundation of Hunan Province (Nos. 2017JJ3148). Appendix A. Supplementary data Supplementary data associated with this article could be found in Appendix A. References [1] R. Butler, A. Godley, L. Lytton, E. Cartmell, Bromate environmental contamination: Review of impact and possible treatment, Crit. Rev. Environ. Sci. Technol. 35 (2005) 193-217. [2] J.C. Kruithof, R.T. Meijers, Bromate formation by ozonation and advanced oxidation and 13

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18

-

-

BrO3 effluent

Br effluent

1000

800

800

600

600

400

400

200

200

0

0

Stage 1

(b)

Stage 2

Stage 3

Stage 4

Stage 5

Stage 6

80

50

Nitrate concentration (mg/L)

-

influent NO3

effluent NO3

-

effluent NO2

-

40 60 30 40 20 20 10

0 0

10

20

30

40

50 Time (day)

60

70

80

Effluent nitrite concentration (mg/L)

Bromate concentration(μ g/L)

-

BrO3 influent

Effluent bromide concentration(μ g/L)

(a) 1000

0 90

Fig. 1. (a) Bromate and bromide concentration, and (b) nitrate and nitrite concentration from stage 1 to stage 6 in influent and effluent of the RBER.

19

Fig. 2. (a) Shannon-Wiener curves, and (b) Rarefaction curves of the OTUs for six biofilm samples with cut-off threshold of 97% similarity. (a)

(b)

Fig. 3. (a) Venn diagrams of the microbial community at different stage, and (b) principal coordinates analysis (PCoA) using Weighted-UniFrac from pyrosequencing. (OTUs at 3 % distance).

20

Fig. 4. Phylum-level abundance of pyrosequences from different stage samples with cut-off threshold of 97% similarity.

21

Fig. 5. Genus-level abundance of pyrosequences from different stage samples with cut-off threshold of 97% similarity.

Table 1 Experimental conditions for each stage of the RBER system. Experiments a

Operating mode

Performance period (d)

Bromate feed (μg/L)

Nitrate feed (mg/L)

Inoculation

Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6

15 (0-15) 8(16-23) 17(24-40) 15(41-55) 15(56-70) 20(71-90)

0 150 400 800 800 800

25 25 25 50 25 0

Continuously test

a

Other experimental conditions: HRT 12 h; electric current 10 mA; temperature (35±2)℃; initial pH (7.2±0.2); DO

(0.4±0.1) mg/L.

Table 2 The bacterial community richness and diversity estimators of each sample. Samples

Effective Sequences

OTU

Chao

ACE

Coverage

Stage 1 /Inoculation Stage 2 Stage 3 Stage 4 Stage 5 Stage 6

35353 30523 26446 26089 51399 42002

285 351 377 381 306 210

332 376 412 417 350 260

310 380 401 404 354 264

0.998222 0.997895 0.997989 0.997942 0.997193 0.997427

22

Table 3 Distribution of main bacteria in phylum-level from different stage samples Bacterial subdivision

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Stage 6

Firmicutes

8124

7837

10342

7294

12684

13545

Proteobacteria

8885

8785

5980

8679

6370

6511

Bacteroidetes

473

2324

3380

3633

1366

657

Actinobacteria

1612

1085

621

845

293

455

Chloroflexi

853

497

198

338

83

23

Candidate_division_TM7

820

402

323

123

42

11

Candidate_division_WS6

243

95

163

37

130

15

Chlorobi

0

0

0

0

288

111

Others

368

353

371

429

50

122

23