Vertical distribution of microbial community and metabolic pathway in a methanogenic propionate degradation bioreactor

Accepted Manuscript Vertical distribution of microbial community and metabolic pathway in a methanogenic propionate degradation bioreactor Ying Li, Yongming Sun, Gaixiu Yang, Keqin Hu, Pengming Lv, Lianhua Li PII: DOI: Reference:

S0960-8524(17)31576-6 http://dx.doi.org/10.1016/j.biortech.2017.09.028 BITE 18856

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

19 July 2017 30 August 2017 4 September 2017

Please cite this article as: Li, Y., Sun, Y., Yang, G., Hu, K., Lv, P., Li, L., Vertical distribution of microbial community and metabolic pathway in a methanogenic propionate degradation bioreactor, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.09.028

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Vertical distribution of microbial community and metabolic pathway in a

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methanogenic propionate degradation bioreactor

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Ying Li a,b, Yongming Sun a#, Gaixiu Yang a, Keqin Hud, Pengming Lv a, Lianhua Li a,c 

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a

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b

7

d

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Abstract:

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The methanogenic propionate degradation consortia were enriched in a propionate-fed semi-continuous bioreactor.

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The microbial community shift with depth, the microbial network and its correlation with metabolic pathway were

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also investigated. The results demonstrated that the maximum organic loading rate (OLR) of the reactor was 2.5 g

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propionic acid (HPr) L-1d-1 with approximately 1.20 LL-1d-1 of volumetric methane production (VMP). The

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organisms in the enrichment were spanning 36 bacterial phyla and 7 archaeal orders. Syntrophobacter, the main

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Hpr oxidizer in the digester, dominated bacteria with relative abundance changing from 63% to 37% with depth.

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The predominant methanogens shift from hydrogenotrophic Methanoculleus (~60%) at the upper liquid layer to

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acetoclastic Methanothrix (~51%) at the lower sediment layer in the bioreactor. These methanogens syntrophically

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support Syntrophobacter by degrading HPr catabolism by-products (H2 and acetate). Other bacteria could scavenge

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anabolic products (carbohydrate and protein) presumably derived from detrital biomass produced by the

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HPr-degrading community.

Laboratory of Biomass Bio-chemical Conversion, GuangZhou Institute of Energy Conversion, Chinese Academy of Sciences,Guangzhou 510640, PR China

Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, PR China

c

Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, PR China Wuhan Kaidi Electric Power Engineering Co.,Ltd ,Wuhan 430073,PR China

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Keywords: Anaerobic digestion, Propionate degradation, Microbial community, Metabolic pathway

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1. Introduction

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Propionate is a major intermediate of organic matter in the anaerobic digestion, which accounts for 35 % of

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the precursor for methane production (Koch et al., 1983). The syntrophic propionic acid degradation to acetic acid

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and hydrogen by acetogens coupled with acetic acid and hydrogen removal via methanogenesis is the major route

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for its degradation in digesters (Ariesyady et al., 2007). This dominant propionic acid oxidation pathway, however,

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is the most thermodynamic unfavourable reaction among the volatile fatty acids (VFA) degradation, and occurs  #

Corresponding author at: No. 2 Nengyuanlu, Tianhe District, Guangzhou 510640, PR China. E-mail address: [email protected] The first two authors contributed equally to this paper

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only when the partial pressure of hydrogen is low enough (Boone & Xun, 1987). Propionate was often found

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accumulated in anaerobic digesters (Regueiro et al., 2015; Zhang & Banks, 2012), which even causes a failure of

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stable methane production.

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Degradation of propionate and limiting its accumulation are important for improving performance of an

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anaerobic digester. Bioaugmentation could be one of the approaches to meet this challenge, which is the practice of

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adding specific microorganisms to a system to enhance a desired activity (Schauer-Gimenez et al., 2010). As the

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previous studies suggested, adding propionate-utilizing cultures (Li et al., 2017; Schauer-Gimenez et al., 2010; Tale

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et al., 2015) or VFA-degrading culture (Acharya et al., 2015) could reduce propionate accumulation and improve

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digestion.

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Since the pure propionate-oxidizing bacteria are often difficult to be isolated and cultured, several studies

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enriched propionate-utilizing cultures in the completely stirred tank reactor(Shigematsu et al., 2006) or the upflow

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anaerobic sludge bed reactors(Ma et al., 2009) fed with synthetic wastewater containing propionate as the sole

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carbon source. For the propionate-utilizing enrichment, nevertheless, knowledge of the whole microbial community

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structure has not been well characterized because of past technological limitations. Second-generation sequencing

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techniques have revolutionized the microbiome study by producing huge amount of data leading to increased

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coverage depth further permitting identification of even the less abundant community members (Nathani et al.,

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2015),which provides a powerful tool for dissecting microbial community structure.

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In an attempt to obtain the bioaugmentation consortia, this study enriched propionate-degrading cultures, which

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can enhance digestion by accelerating the conversion of acetate and propionate to methane. The microbial

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community structure and its distribution shift with the depth of the reactor were characterized at taxonomic level as

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well as to establish the database for the similar AD system. A more complete understanding of the microbial

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network and its correlation with metabolic pathway of nutrient utilization was also provided.

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2. Materials and methods

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2.1 Propionate-degrading consortia enrichment

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Inoculum: The inoculum for propionate-degrading consortia enrichment was taken from an anaerobic digester

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fed with energy crop (70L, Laboratory of biomass bio-chemical conversion, GuangZhou Institute of Energy

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Conversion, Chinese Academy of Sciences, China).

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Bioreactor: The propionate-degrading consortia were enriched in a BioReactor Simulator (2L, Bioprocess

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Control AB, Sweden).The bioreactor is equipped with a funnel-shaped inlet port and an bend outlet port (Fig.1).

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The pressure from the fresh feed makes the same volume of automatic discharge. There are two gas outlets through

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the stopper. One is used for biogas sampling, the other one collects to the gas flow meter for biogas production

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measurement. The biogas production can be real-time recorded by the computer.

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Digestion procedure: The reactor was maintained at 35 ± 1℃ in an water bath and operated in daily

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fill-and-draw mode with identical hydraulic retention time (HRT) of 20 days by removing appropriate volume of

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reactor content and replacing it same volume of feed once per day. The bioreactor was running without stir during

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the whole enrichment process .The feed comprised a certain amount of sodium propionate and the volume was

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made up with nutrient medium. The nutrient medium contained the following [mg/L]:NH4Cl [400]; MgSO4·6H2O

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[250]; KCl[400]; CaCl2·2H2O [120]; (NH4)2HPO4 [80]; FeCl3·6H2O [55]; CoCl2·6H2O [0.5]; NiCl2·6H2O[0.5] the

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trace metal salts MnCl2·4H2O,CuCl2·2H2O, AlCl3·6H2O, Na2WO4·2H2O, H3BO3, Na2SeO3 and ZnCl2 [each at

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0.5](Tale et al., 2011). OLR started at 0.5 g Hpr L-1d-1 in HRT1 and was then step-wise rising to 3.0 g Hpr L-1d-1 by

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adding the appropriate amount of sodium propionate.

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2.2 Sampling for the analyses of digestion performance and microbial community

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The bioreactor was running without stir during the whole enrichment process, therefore the solid from the inoculum (mainly straw residue) sediment at the bottom of the reactor lead to two layers present. The samples for determination of pH, VFA and alkalinity were taken from the effusion of liquid layer of the bioreactor at certain intervals. For a better understanding of the microbial community distribution with the depth, three samples were taken

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from the bioreactor on day 260 (relative stable and high digestion performance) at the upper (U), middle (M), and

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bottom (B) of the digester with the distance from the top of the digestate surface of 3cm, 8cm and 13cm,

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respectively (Fig 1), representing the liquid layer, the sediment layer and the junction of both layers.

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2.3 Analytical methods for the digestion performance

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Biogas was automatically recorded by the computer. pH was determined using a FE28-Standard meter (Mettler

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-Toledo, Switzerland) with a combination glass electrode calibrated in buffers at pH 7.0 and 9.2. Alkalinity was

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measured by a Titroline 5000 titrator (Julabo,German)with 0.25 N H2SO4 to endpoints of pH 5.7 and 4.3, allowing

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calculation of total alkalinity (TA), partial alkalinity (PA) and intermediate alkalinity (IA). VFA were quantified in a

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Waters e2698 High Performance Liquid Chromatography (Waters, USA) with a Bio-RAD column. Biogas

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composition (CH4 and CO2) was determined using a GC-2014 Gas Chromatograph (Shimadzu, Japan) calibrated

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with 65% (v/v) CH4 and 35% (v/v) CO2.

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2.4 Metagenomic DNA extraction and amplification

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DNA extraction was performed using the fast DNA spin kit for soil (QBIOgene Inc., Carlsbad, CA, USA),

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according to the manufacturer’s instructions. DNA quality was assessed using gel electrophoresis (1% agarose) and

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DNA concentrations were determined using a Qubit Fluorometer (Thermo, USA).

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The extracted DNA sample was then handled according to the protocol of genomic DNA sample preparation kit

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(Illumina). The DNA fragmentation was firstly performed using Covaris S2 Ultrasonicator, and the DNA fragments

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were then processed by end reparation, A-tailing, adapter ligation, DNA size-selection. PCR reaction and products

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purification based on Illumina Miseq2x300 instructions. For Archaea, the 16S rRNA genes were amplified through

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three rounds of PCR. The primers for the first round were 340F (5’-CCCTAYGGGGYGCASCAG-3’) and 1000R

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(5’- GGCCATGCACYWCYTCTC-3’). Then the PCR products were used as templates for a second PCR with

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349F(5’-CCCTACACGACGCTCTTCCGATCTN(barcode)GYGCASCAGKCGMGAAW-3’) and 806R

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(5’-GACTGGAGTTCCTTGGCACCCGAGAATTCCAGGACTA CVSGGGTATCTAAT-3’), and the third round

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PCR amplified with Illumina nested primers. The bacteria 16S rRNA genes were amplified through two rounds of

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PCR. Firstly, using 341F (5’- CCCTACACGACGCTCTTCCGATCTG (barcode) CCTACGGGNGGCWGCAG -3’)

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and 805R (5’-GACTGGAGTTCCTTGGCACCCGAGAATTCCAGACTACHVGGGTATCTAATCC3’).

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The PCR products were then used as templates for a second PCR with Illumina Nested primers.

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2.5 Whole genome pyrosequencing analysis

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Before sequencing, PCR products of different samples were normalized in equimolar amounts in the final mixture,

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which was used to construct the PCR amplicon libraries. Sequencing was carried out on an Illumina HiSeq 2000,

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and the raw sequences have been deposited in the NCBI project, under the accession number PRJNA391398. The

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obtained sequences were phylogenetically allocated down to the phylum, class, and genus level with the RDP

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classifier（http://rdp.cme.msu.edu/misc/resources）.To define the relative abundance of a given phylogenetic group,

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the number of sequences affiliated to that group was divided by the total number of obtained sequences. The results

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were used for the analysis and comparison of microbial community structure differences. QIIME was used for

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network analysis. The species with the abundance of more than 1%.The network layout was made by R language

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igraph package, with significant contact (weight> 100) nodes.

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3. Results and discussion

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3.1 Digestion performance

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The digestion performance in terms of volumetric biogas (methane) production, methane content, VFA

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concentration, pH and alkalinity was shown as Fig 2. During the first 14 HRT (0-280 d), the volumetric methane

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production (VMP) increased from 0.2 L L-1 d-1 to 1.2 L L-1 d-1with the OLR step-wise rising from 0.5 g Hpr L-1 d-1

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to 2.5 g Hpr L-1 d-1 .In this period, the methane percentage remained above 70%.

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However, both VMP and methane content decreased when the OLR increased to 3.0 g Hpr L-1d-1 on day

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281.This was due to the continuous accumulation of propionic acid. In order to relieve propionic acid accumulation,

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the propionic acid was removed in the daily feeding when the propionic acid concentration reached 11.9 g L-1 on

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day 301.After one week (302-309 d) of no carbon source feed, the accumulated propionate was degraded, with a

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concentration change from 11.9 g L-1 to 0.5 g L-1.

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The OLR was then set back to 3.0 g HPr L-1d-1 on day 310, resulting in a sharp accumulation of propionic acid.

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In the meantime, the concentration of acetic acid started to increase. However, VMP showed a significant increase

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in the first 10 days (310-320 d) of 3.0 g HPr L-1d-1. When propionic acid concentration exceeded 10.0 g L-1, VMP

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began to decrease again. At the later stage of the experiment (340-360d), although the concentration of propionic

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acid increased from 10.0 g L-1 to 18.0 g L-1, the VMP remained relatively stable approximately 0.9 L L-1d-1.

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The pH of the digestate was above 7.45 in the whole experimental period even at the VFA accumulation stage,

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this may due to the carbon source (Hpr) was added in the form of sodium propionate. IA: PA was below 0.5 when

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the OLR was not above 2.5 g HPr L-1d-1, while IA: PA was increasing since the OLR was up to 3.0 g HPr L-1d-1. On

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day 350, IA: PA exceeded 1.0 and kept increasing, indicating an unstable fermentation system(Ripley et al., 1986).

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The above results suggest that the maximum organic loading of static semi-continuous bioreactor fed with

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propionate as a sole carbon source was less than 3.0 g HPr L-1d-1. The maximum OLR of the reactor was 2.5 g HPr

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L-1d-1 with approximately 1.20 LL-1d-1 of volumetric methane production.

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3.2 Microbial community shift with depth

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More than 40,000 bacterial reads for each sample were obtained, and the number of operational taxonomic units

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(OTUs) was around 700, while over 20,000 archaea reads identified as 50 OTUs were found for each sample. The

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higher OTU numbers of bacteria compared were consistent with previous studies(Chen et al., 2016) , showing

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bacteria were more diverse than archaea.

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3.2.1 Archaeal community shift with depth

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For Archaeal community structure in the reactor the relative abundance of archaeal 16S rRNA gene at the order and

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genus levels is shown in Fig 3. Methanomicrobiales and Methanosarcinales were two predominant orders among

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all samples tested. Methanomicrobiales, a group of hydrogenotrophic methanogens was dominant at the top area of

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the reactor with the relative abundance of 60.35%, while Methanosarcinales (51.47%), the strict acetoclastic

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methanogens (Kendall & Boone, 2006) was found to be the main order at the bottom area of the reactor (Fig.3A).

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At the genus level, Methanothrix belonging to Methanosarcinales order was the predomiant acetoclastic

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methanogens among all samples, while the diversity of hydrogenotrophic methanogens increased along with the

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depth, which from one dominant genus (Methanoculleus) at the top to three predominant groups (Methanoculleus，

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Methanosphaerula and Methanobacterium) at the bottom (Fig.3B, Table 1). The other methanogenic archaea in the

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bioreactor and their distribution in different depths are also listed Table 1.It shows that aceticlastic methanogens

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dominated over hydrogenotrophic methanogens at the bottom of the bioreactor, while hydrogenotrophic

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methanogens were the main methanogenic archaea at the top area, suggesting that hydrogenotroph might be the

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major methanogensis pathway in the liquid layer of the digestate, in contrast, aceticlastic methanogensis pathway

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was the main pathway in the sediment layer .This might because the concentration of H2 and CO2 (the substrate of

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hydrogenotrophic methanogens) at the top layer was higher than that at the bottom of the bioreactor, while the

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concentration of acetic acid (the substrate of aceticlastic methanogens) at the bottom sediment might be a bit higher

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than that at the top layer since the fermentation of the dead cell biomass (more details see the discussion in 3.3).

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3.2.2 Bacterial community shift with depth

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The taxonomic classification of bacterial sequences by RDP classifier is shown in Fig 4. Proteobacteria

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(41.1~71.3%) and Bacteroidetes (9.2~16.8%) and Synergistetes (6.4~ 10.2 %) were identified as the three most

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dominant phyla in U and M samples, besides Chloroflexi (13.6%) were also dominant in sample B (Fig 4 A). In

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addition, sequences belonging to Firmicutes Thermotogae, Euryarchaeota and 29 other phyla were detected in

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some of the samples, but at low occurrence (below 8% for each sample).

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The abundance of Proteobacteria decreased along with depth. In contrast, Bacteroidetes was increasing from the

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top to the bottom. This might be related with the distribution of the substrate for cell growth in the bioreactor. The

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nutrition became more complicated with depth leading to the diversity of bacteria was increasing. So the

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Proteobacteria population lost its absolute predominance at the bottom sediment, while the members of

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Bacteroidetes were more competitive at the bottom since the relative abundance substrate.

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Deltaproteobacteria, a syntrophic propionate degrader community also correlated with species that use

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hydrogen/formate as main substrate(Felchner-Zwirello et al., 2013), was the dominant class in phylum

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Proteobacteria.. Their dominances were most probably related with the long term feeding with propionate.

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3.3 Microbial network in the propionate-degrading community

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Fig 5 shows the network across the main genus in the propionate-degrading enrichment. In the figure, the size

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of the pie represents the total abundance of each genus in the bioreactor, and the different color areas of the pie

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represent the contribution of the each sample (U, M, B) to the abundance of this genus.

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It is clear that the most abundance genus in the bioreactor was Syntrophobacter with the biggest size of pie(Fig

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5,Table 2), indicating that Syntrophobacter groups, the sulfate reducers are capable of degrading propionate in

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syntrophic association with methanogens, was the main propionate oxidizers in the digester. The role of

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Syntrophobacter was investigated in the past, for instance a study reported a correlation between increase of

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Syntrophobacter spec. and the degradation of propionate (Moertelmaier et al., 2014). In addition, the other

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propionate oxidizing beacteria, i.e. Syntrophorhabdus, Pelotomaculum, Smithella were observed mainly in the

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bottom of the bioreactor. Pelotomaculum and Syntrophobacter have been also reported as the dominant propionate

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oxidizing bacteria in the previous studies (Ban et al., 2013; Li et al., 2014; Shigematsu et al., 2006).

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The second most abundance groups in the whole bioreactor were Thermovirga, which reported as

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amino-acid-degrading and sulfur-reducing bacteria (Dahle & Birkeland, 2006; Goeker et al., 2012), followed by

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Levilinea. Levilinea can ferment sugars and amino acids into hydrogen, acetic and lactic acids, sytrophic growth

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with hydrogenotrophic methanogens (Yamada & Sekiguchi, 2009). Levilinea were also found in the bioreactor to

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generate volatile fatty acids (VFA) with sludge (Maspolim et al., 2015).Moreover, Table 2 shows that the relative

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abundance of Levilinea and Thermovirga at lower area (B) are both higher than that at the upper bioreactor,

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suggesting that the reaction of amino-acid-degrading were more frequently in the bottom of the bioreactor. This

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might due to the sediment of dead microbial biomass in the bottom of the reactor, which provided the nutrient.

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Other bacterial genera related to sugars fermentation and amino acid-degrading are listed in Table 2.

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Most of the microbial dead cells were precipitated at the bottom of the reactor leading to the acidogens

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enriched in this area, which fermented the dead cell biomass to produce acetic acid. This can explain the

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phenomenon discussed above why the aceticlastic methanogens dominated over hydrogenotrophic methanogens at

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the lower area of the bioreactor.

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3.4 Carbon flux niches in the propionate-degrading community

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According to the microbial community structure and the function of each member, a carbon flux in the

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propionate degradation reactor was made as Fig 6. Propionic acid, the sole carbon source of the digester, was used

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for catabolism and anabolism. The holistic carbon flux from Hpr to CH4 and CO2 may require Hpr degraders,

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detritus scavengers and methanogens to form syntrophic networks. The bacterial groups of Syntrophobacter and

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Pelotomaculum may support Hpr degradation to acetate, CO2 and H2, many others bacteria may contribute to

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scavenging death cell detritus through macromolecule hydrolysis, fermentation and chaining degradation of amino

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acids.

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4. Conclusions

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Results show that the maximum OLR of the propionate-fed reactor was 2.5 gHPrL-1d-1 with approximately 1.20

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LL-1d-1 of VMP. The metagenomic sequencing study successfully dissected the detail microbial community

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structure of the methanogenic propionate degradation enrichment. Syntrophobacter was the main HPr degrader in

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the digester. The groups of Methanomicrobiales (hydrogenotrophic) and Methanosarcinales (acetoclastic) were the

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dominant methanogens at the upper and lower of the bioreactor respectively. These methanogens degrade HPr

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catabolism by-products (acetate and H2) and syntrophically support Syntrophobacter. Other bacteria could

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scavenge anabolic products (carbohydrate and protein) presumably derived from detrital biomass produced by the

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HPr-degrading community.

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Acknowledgements

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Thanks are due to National Key Technology Research and Development Program of the Ministry of Science and

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Technology of China（2015BAD21B03）and the Natural Science Foundation for research team of Guangdong

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Province (2016A030312007) for supporting this research.

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References

219

1.

220 221 222

Acharya, S.M., Kundu, K., Sreekrishnan, T.R. 2015. Improved Stability of Anaerobic Digestion through the Use of Selective Acidogenic Culture. J. Environ. Eng. 141, 04015001.

2.

Ariesyady, H.D., Ito, T., Yoshiguchi, K., Okabe, S. 2007. Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge. Appl. Microbiol. Biotechnol., 75, 673-683.

223

3.

Arun, A.B., Chen, W.-M., Lai, W.-A., Chou, J.-H., Shen, F.-T., Rekha, P.D., Young, C.-C. 2009. Lutaonella

224

thermophila gen. nov., sp nov., a moderately thermophilic member of the family Flavobacteriaceae isolated from a

225

coastal hot spring. Int. J. of System.and Evolution. Microbiol., 59, 2069-2073.

226

4.

Ban, Q., Li, J., Zhang, L., Jha, A.K., Zhang, Y. 2013. Quantitative Analysis of Previously Identified

227

Propionate-Oxidizing Bacteria and Methanogens at Different Temperatures in an UASB Reactor Containing

228

Propionate as a Sole Carbon Source. Appl. Biochem. and Biotechnol. 171, 2129-2141.

229

5.

230 231

Boone, D.R., Xun, L.Y. 1987. Effects of pH, temperature ,and nutrients on propionate degardation by a methanogenic enrichment culture. Appl. and Environ. Microbiol., 53, 1589-1592.

6.

Cardinali-Rezende, J., Debarry, R.B., Colturato, L.F.D.B., Carneiro, E.V., Chartone-Souza, E., Nascimento, A.M.A.

232

2009. Molecular identification and dynamics of microbial communities in reactor treating organic household waste.

233

Appl. Microbiol. Biotechnol., 84, 777-789.

234

7.

Chen, H., Wan, J., Chen, K., Luo, G., Fan, J., Clark, J., Zhang, S. 2016. Biogas production from hydrothermal

235

liquefaction wastewater (HTLWW): Focusing on the microbial communities as revealed by high-throughput

236

sequencing of full-length 16S rRNA genes. Water Res., 106, 98-107.

237

8.

238 239

Chen, S.Y., Dong, X.Z. 2005. Proteiniphilum acetatigenes gen. nov., sp nov., from a UASB reactor treating brewery wastewater. Int. J. of System.and Evolution. Microbiol., 55, 2257-2261.

9.

Dahle, H., Birkeland, N.-K. 2006. Thermovirga lienii gen. nov., sp nov., a novel moderately thermophilic, anaerobic,

240

amino-acid-degrading bacterium isolated from a North Sea oil well. Int. J. of System.and Evolution. Microbiol., 56,

241

1539-1545.

242

10. de Bok, F.A.M., Luijten, M., Stams, A.J.M. 2002. Biochemical evidence for formate transfer in syntrophic

243

propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. Appl. and

244

Environ. Microbiol., 68, 4247-4252.

245 246 247 248 249

11. Felchner-Zwirello, M., Winter, J., Gallert, C. 2013. Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer. Appl. Microbiol. Biotechnol., 97, 9193-9205. 12. Garcia, J.-L., Ollivier, B., Whitman, W.B. 2006. The Order Methanomicrobiales. in Dworkin M. In: Falkow S, Rosenberg E, Schleifer, KH, Stackebrandt E (Eds.), The Pro-karyotes. Springer, New York,pp,208-230. 13. Goeker, M., Saunders, E., Lapidus, A., Nolan, M., Lucas, S., Hammon, N., Deshpande, S., Cheng, J.-F., Han, C.,

250

Tapia, R., Goodwin, L.A., Pitluck, S., Liolios, K., Mavromatis, K., Pagani, I., Ivanova, N., Mikhailova, N., Pati, A.,

251

Chen, A., Palaniappan, K., Land, M., Chang, Y.-j., Jeffries, C.D., Brambilla, E.-M., Rohde, M., Spring, S., Detter,

252

J.C., Woyke, T., Bristow, J., Eisen, J.A., Markowitz, V., Hugenholtz, P., Kyrpides, N.C., Klenk, H.-P. 2012. Genome

253

sequence of the moderately thermophilic, amino-acid-degrading and sulfur-reducing bacterium Thermovirga lienii

254

type strain (Cas60314(T)). Standards in Genomic Sciences, 6, 230-239.

255

14. Grabowski, A., Tindall, B.J., Bardin, V., Blanchet, D., Jeanthon, C. 2005. Petrimonas sulfuriphila gen. nov., sp nov.,

256

a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir. Int. J. of System.and Evolution.

257

Microbiol., 55, 1113-1121.

258

15. Honda, T., Fujita, T., Tonouchi, A. 2013. Aminivibrio pyruvatiphilus gen. nov., sp nov., an anaerobic,

259

amino-acid-degrading bacterium from soil of a Japanese rice field. Int. J. of System.and Evolution. Microbiol., 63,

260

3679-3686.

261

16. Iino, T., Mori, K., Itoh, T., Kudo, T., Suzuki, K.-i., Ohkumal, M. 2014. Description of Mariniphaga anaerophila gen.

262

nov., sp nov., a facultatively aerobic marine bacterium isolated from tidal flat sediment, reclassification of the

263

Draconibacteriaceae as a later heterotypic synonym of the Prolixibacteraceae and description of the family

264

Marinifilaceae fam. nov. Int. J. of System.and Evolution. Microbiol., 64, 3660-3667.

265

17. Imachi, H., Sakai, S., Lipp, J.S., Miyazaki, M., Saito, Y., Yamanaka, Y., Hinrichs, K.-U., Inagaki, F., Takai, K. 2014.

266

Pelolinea submarina gen. nov., sp nov., an anaerobic, filamentous bacterium of the phylum Chloroflexi isolated

267

from subseafloor sediment. Int. J. of System.and Evolution. Microbiol., 64, 812-818.

268

18. Kato, S., Kosaka, T., Watanabe, K. 2009. Substrate-dependent transcriptomic shifts in Pelotomaculum

269

thermopropionicum grown in syntrophic co-culture with Methanothermobacter thermautotrophicus. Microbial

270

Biotechnol., 2, 575-584.

271 272 273 274 275 276 277 278 279

19. Kendall, M.M., Boone, D.R. 2006. The Order Methanosarcinales. in Dworkin M. In: Falkow S, Rosenberg E, Schleifer, KH, Stackebrandt E (Eds.), The Pro-karyotes. Springer, New York, pp. 244-256. 20. Koch, M., Dolfing, J., Wuhrmann, K., Zehnder, A.J.B. 1983. Pathways of propionate degradation by enriched methanogenic cultures. Appl. Microbiol. Biotechnol., 45, 1411-1414. 21. Kroeninger, L., Berger, S., Welte, C., Deppenmeier, U. 2016. Evidence for the involvement of two heterodisulfide reductases in the energy-conserving system of Methanomassiliicoccus luminyensis. Febs Journal, 283(3), 472-483. 22. Li, C., Moertelmaier, C., Winter, J., Gallert, C. 2014. Effect of moisture of municipal biowaste on start-up and efficiency of mesophilic and thermophilic dry anaerobic digestion. Bioresour Technol., 168, 23-32. 23. Li, Y., Zhang, Y., Sun, Y., Wu, S., Kong, X., Yuan, Z., Dong, R. 2017. The performance efficiency of

280

bioaugmentation to prevent anaerobic digestion failure from ammonia and propionate inhibition. Bioresour Technol.,

281

231, 94-100.

282

24. Ma, J., Mungoni, L.J., Verstraete, W., Carballa, M. 2009. Maximum removal rate of propionic acid as a sole carbon

283

source in UASB reactors and the importance of the macro- and micro-nutrients stimulation. Bioresour Technol., 100,

284

3477-82.

285

25. Maus, I., Wibberg, D., Stantscheff, R., Cibis, K., Eikmeyer, F.-G., Koenig, H., Puehler, A., Schlueter, A. 2013.

286

Complete genome sequence of the hydrogenotrophic Archaeon Methanobacterium sp Mb1 isolated from a

287

production-scale biogas plant. J. of Biotechnol., 168, 734-736.

288 289

26. Moertelmaier, C., Li, C., Winter, J., Gallert, C. 2014. Fatty acid metabolism and population dynamics in a wet biowaste digester during re-start after revision. Bioresour Technol., 166, 479-84.

290

27. Nathani, N.M., Patel, A.K., Mootapally, C.S., Reddy, B., Shah, S.V., Lunagaria, P.M., Kothari, R.K., Joshi, C.G.

291

2015. Effect of roughage on rumen microbiota composition in the efficient feed converter and sturdy Indian

292

Jaffrabadi buffalo (Bubalus bubalis). BMC genomics, 16, 1116.

293

28. Nesbo, C.L., Bradnan, D.M., Adebusuyi, A., Dlutek, M., Petrus, A.K., Foght, J., Doolittle, W.F., Noll, K.M. 2012.

294

Mesotoga prima gen. nov., sp nov., the first described mesophilic species of the Thermotogales. Extremophiles, 16,

295

387-393.

296 297 298 299 300 301 302

29. Regueiro, L., Lema, J.M., Carballa, M. 2015. Key microbial communities steering the functioning of anaerobic digesters during hydraulic and organic overloading shocks. Bioresour Technol., 197, 208-216. 30. Ripley, L.E., Boyle, W.C., Converse, J.C. 1986. Improved alkalimetric monitoring for anaerobic-digestion of high-strength wastes. Journal. Water Pollution Control Federation, 58, 406-411. 31. Schauer-Gimenez, A.E., Zitomer, D.H., Maki, J.S., Struble, C.A. 2010. Bioaugmentation for improved recovery of anaerobic digesters after toxicant exposure. Water Res., 44, 3555-3564. 32. Shigematsu, T., Era, S., Mizuno, Y., Ninomiya, K., Kamegawa, Y., Morimura, S., Kida, K. 2006. Microbial

303

community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based

304

on 16S rRNA and acetate kinase genes. Appl.microbiol. and biotechnol., 72, 401-415.

305 306 307 308 309

33. Tale, V.P., Maki, J.S., Struble, C.A., Zitomer, D.H. 2011. Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters. Water Res, 45, 5249-56. 34. Tale, V.P., Maki, J.S., Zitomer, D.H. 2015. Bioaugmentation of overloaded anaerobic digesters restores function and archaeal community. Water Res., 70, 138-147. 35. Yamada, T., Sekiguchi, Y. 2009. Cultivation of Uncultured Chloroflexi Subphyla: Significance and Ecophysiology

310

of Formerly Uncultured Chloroflexi 'Subphylum I' with Natural and Biotechnological Relevance. Microbes and

311

Environ., 24, 205-216.

312

36. Zhang, Y., Banks, C.J. 2012. Co-digestion of the mechanically recovered organic fraction of municipal solid waste

313 314 315

with slaughterhouse wastes. Biochem.Engineering Journal., 68, 129-137.

Figure1

Fig. 1 Schematic diagram of sampling points for microbial community analysis of propionate-degrading enrichment(the depth of the upper (U), middle (M), and bottom (B) sampling points were 3cm, 8cm and 13cm, respectively. The sediment in the bottom was the straw residue from the initial inoculum)

Figure2

Fig.2 The digestion performance of static semi-continuous bioreactor fed with propionate as a sole carbon source

Figure3

Fig.3 Relative abundance of archaeal 16S rRNA gene at the order level (A) and genus level (B) in the reactor with different depth at 3cm (shown as U) , 8cm (shown as M) and 15cm (shown as B )

Figure4

Fig.4 Relative abundance of bacterial 16S rRNA gene at the phylum level (A) and class level (B) in the reactor along the different depth at 3cm (shown as U), 8cm (shown as M) and 15cm (shown as B)

Figure5

Fig.5 Microbial network in the propionate-degrading community at the genus level (Each pie represents a genus.The size of the pie means the total abundance of the genus in the bioreactor.The different color areas of the pie represent the contribution of the each sample,U M and B to the abundance of the genus)

Figure6

Fig.6 Holistic carbon flux from propionate to CH4 with the functional microorganisms

316

Table 1 Distribution of methanogenic archaea in the bioreactor along with the depth Gens

317 318

Order

Relative abundance (%) U

M

B

Methanogenesis type

Methanoculleus

Methanomicrobiales

59.68

47.24

15.88

Hydrogenotrophic(Garcia et al., 2006)

Methanothrix

Methanosarcinales

28.02

27.30

50.69

Acetoclastic(Kendall & Boone, 2006)

Methanobacterium

Methanobacteriales

6.93

3.26

9.36

Hydrogenotrophic(Maus et al., 2013)

Methanosphaerula

Methanomicrobiales

0.32

7.14

10.81

Hydrogenotrophic(Garcia et al ,2006)

Methanomassiliicoccus

Methanomassiliicoccales

0.09

2.39

2.06

Hydrogenotrophic(Kroeninger et al., 2016)

Methanospirillum

Methanomicrobiales

0.33

0.30

0.42

Hydrogenotrophic(Garcia et al ,2006)

Methanosphaera

Methanobacteriales

0.04

0.17

0.19

Hydrogenotrophic(Cardinali-Rezende et al., 2009)

Methanosarcina

Methanosarcinales

0.03

0.06

0.06

Acetoclastic (Kendall & Boone, 2006)

Methanolinea

Methanomicrobiales

0.01

0.01

0.03

Hydrogenotrophic(Garcia et al ,2006)

Methanoregula

Methanomicrobiales

0.00

0.00

0.05

Hydrogenotrophic(Garcia et al ,2006)

Methanofollis

Methanomicrobiales

0.00

0.01

0.01

Hydrogenotrophic(Garcia et al ,2006)

Acetoclastic

31.97

36.67

76.17

Hydrogenotrophic

68.03

63.33

23.83

319

Table 2 The predominant beactiral genera informantion in the bioreactor

Genus

320 321 322

Phylum

Mainly Substrate

Relative abundance （%） U

M

B

Syntrophobacter

Proteobacteria

Propionic acid(de Bok et al., 2002)

62.97

47.25

37.30

Pelotomaculum

Firmicutes

Propionic acid(Kato et al., 2009)

0.56

2.34

4.04

Levilinea

Chloroflexi

Sugars, Amino acid (Yamada & Sekiguchi, 2009).

0.34

3.08

9.36

Mesotoga

Thermotogae

Sugars,proteinaceous compounds (Nesbo et al., 2012)

4.68

4.47

4.10

Ornatilinea

Chloroflexi

Protein /cellulose (Podosokorskaya et al., 2013).

0.23

1.30

2.00

Mariniphaga

Bacteroidetes

Sugars (Iino et al., 2014)

0.56

0.97

1.35

Proteiniphilum

Bacteroidetes

Proteinaceous compounds (Chen & Dong, 2005)

1.17

0.94

0.76

Petrimonas

Bacteroidetes

Sugars(Grabowski et al., 2005)

0.42

0.23

0.17

Pelolinea

Chloroflexi

Sugars(Imachi et al., 2014)

0.18

0.16

0.34

Thermovirga

Synergistetes

Amino acid (Dahle & Birkeland, 2006)

5.80

9.09

9.86

Lutaonella

Bacteroidetes

Amino acid(Arun et al., 2009)

1.84

1.05

0.29

Aminivibrio

Synergistetes

Amino acid(Honda et al., 2013)

0.71

0.83

0.47

The total reads of U, M and B were 40887, 44135 and 61400, respectively

323

1. Syntrophobacter was the main propionate oxidizer in the digester.

324

2. The dominant methanogens shifted from Methanoculleus to Methanothrix with depth.

325

3. Propionic acid was used for catabolism and anabolism.

326 327