Rumen fermentation and acetogen population changes in response to an exogenous acetogen TWA4 strain and Saccharomyces cerevisiae fermentation product

Rumen fermentation and acetogen population changes in response to an exogenous acetogen TWA4 strain and Saccharomyces cerevisiae fermentation product 外源添加产乙酸菌和酿酒酵母发酵物对瘤胃发酵特性及产乙酸菌菌群结构的影响 1 first-author 2 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jiakunwang@zju.edu.cn †E-mail:jiakunwang@zju.edu.cn jiakunwang@zju.edu.cn Institute of Dairy Science, MoE Key Laboratory of Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China Institute of Dairy Science, MoE Key Laboratory of Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China Department of Agricultural, Food & Nutritional Science, Faculty of Agricultural, Life & Environmental Sciences, University of Alberta, Edmonton, AB, T6G 2P5, Canada Department of Agricultural, Food & Nutritional Science, Faculty of Agricultural, Life & Environmental Sciences, University of Alberta, Edmonton, AB, T6G 2P5, Canada The presence of yeast cells could stimulate hydrogen utilization of acetogens and enhance acetogenesis. To understand the roles of acetogens in rumen fermentation, an in vitro rumen fermentation experiment was conducted with addition of acetogen strain (TWA4) and/or Saccharomyces cerevisiae fermentation product (XP). A 2×2 factorial design with two levels of TWA4 (0 or 2×10 7 cells/ml) and XP (0 or 2 g/L) was performed. Volatile fatty acids (VFAs) were increased ( P < 0.05) in XP and TWA4XP, while methane was increased only in TWA4XP ( P < 0.05). The increase rate of microorganisms with formyltetrahydrofolate synthetase, especially acetogens, was higher than that of methanogens under all treatments. Lachnospiraceae was predominant in all acetogen communities, but without close acetyl-CoA synthase (ACS) amino acid sequences from cultured isolates. Low- Acetitomaculum ruminis -like ACS was predominant in all acetogen communities, while four unique phylotypes in XP treatment were all amino acid identified low- Eubacterium limosum -like acetogens. It differs to XP treatment that more low- A. ruminis -like and less low- E. limosum -like sequences were identified in TWA4 and TWA4XP treatments. Enhancing acetogenesis by supplementation with an acetogen strain and/or yeast cells may be an approach to mitigate methane, by targeting proper acetogens such as uncultured low- E. limosum -like acetogens. 目的 2 酵母可以促进产乙酸菌利用氢生成乙酸。为了解产乙酸菌在瘤胃发酵中的作用，本试验研究了产乙酸菌单独添加及其与酿酒酵母发酵物联合添加对瘤胃发酵特性及产乙酸菌菌群结构的影响。 创新点 2 通过瘤胃甲烷生成量与产乙酸菌菌群结构关联，揭示降低瘤胃甲烷生成的新途径。 方法 2 试验采用2×2双因素试验设计，产乙酸菌TWA4的添加量为0和2×10 7 cells/ml，酿酒酵母发酵物XP的添加量为0和2 g/L。 结论 2 瘤胃体外发酵研究发现，单独添加XP以及TWA4和XP联合添加可增加挥发性脂肪酸产量（ P < 0.05），TWA4和XP联合添加显著增加了甲烷生成量（ P < 0.05）。单独添加TWA4、XP以及两者联合添加均增加了含有甲酰四氢叶酸合成酶的微生物的数量，尤其是产乙酸菌的数量，这类微生物的增长幅度高于产甲烷菌。各处理产乙酸菌均以 Lachnospiraceae 为主，但与已培养的微生物的相似性较低。与 Acetitomaculum ruminis 低相似性的一类未知的产乙酸菌在各处理中均占优势，但XP的添加增加了与 Eubacterium limosum 低相似未知产乙酸菌的数量。与XP不同，TWA4及其与XP的联合添加使与 A. ruminis 低相似性的一类未知的产乙酸菌的数量增加，与 E. limosum 低相似未知产乙酸菌的数量降低。因此，添加与 E. limosum 低相似的未知产乙酸菌和（或）酵母可能是降低瘤胃甲烷生成的一种有效途径。 Acetogen Saccharomyces cerevisiae fermentation product Rumen fermentation Methanogen 产乙酸菌 酿酒酵母发酵物 瘤胃发酵 产甲烷菌 Introduction 1 Endogenous rumen microbiota can convert fiber to volatile fatty acid (VFA), and also result in the production of hydrogen and carbon dioxide (Kamra et al., 2012 2012 ). When hydrogen builds up, it inhibits the oxidation of reduced nicotinamide adenine dinucleotide (NADH), leading to the accumulation of lactic acid and reduction of the fermentation (Wolin et al., 1997 1997 ). To maintain a balanced environment for fermentation in the rumen, methanogens can perform methanogenesis, which converts hydrogen and carbon dioxide to methane. However, this process results in 2%–15% loss of feed energy (Johnson and Johnson, 1995 1995 ) and exacerbation of greenhouse effects. Recent studies have shown that hydrogen is less used for methanogenesis in the gastrointestinal tract of termites (Breznak, 1994 1994 ) and Australian marsupials (Ouwerkerk et al., 2009 2009 ) due to the predominant acetogenesis in the gut. Reductive acetogens are a group of bacteria that can produce acetate from hydrogen and carbon dioxides (4H 2 +2CO 2 →CH 3 COOH+2H 2 O) using the acetyl-CoA pathway (Drake et al., 2006 2006 ). This suggests that acetogens may serve as hydrogen sinks in experiments, which seek to lower methane emissions (van Nevel and Demeyer, 1995 1995 ; Morvan et al., 1996 1996 ; Joblin, 1999 1999 ) in cattle. Joblin et al. (1999) has reported that methane production decreased by 97% and 64%, respectively, when a Methanobrevibacter smithii sp. isolated from a grazing sheep was grown on H 2 /CO 2 in vitro in the presence of a rumen acetogen isolate. Although an acetogen originating from sewage-sludge, Peptostreptococcus productus , competed successfully against methanogens in a simulated gastro-intestinal fermenter (Nollet et al., 1997a 1997a ), such competition failed when in vitro ruminal digesta was used (Nollet et al., 1997b 1997b ; 1998 1998 ). Chaucheyras-Durand et al. ( 1995b 1995b ) have shown that the presence of yeast cells could stimulate utilization of hydrogen by acetogens and enhance acetogenesis in an experiment utilizing a co-culture of acetogen and methanogen. Therefore, we hypothesized that the methanogenesis in the rumen is reduced by the enhanced acetogenesis with a supplement of Saccharomyces cerevisiae fermentation product (XP) and exogenous acetogens. TWA4, a novel reductive homoacetogen isolated from forestomach contents of female tammar wallabies, could outcompete M. smithii in high hydrogen or heterotrophic growth on glycerol (with low hydrogen generated during fermentation) (Gagen et al., 2014 2014 ). Therefore, in this study we assessed the effects of TWA4 and XP on methane production, rumen fermentation, methanogen, and acetogen population, as well as acetogenic diversity using the in vitro rumen fermentation system. Materials and methods 1 Acetogen and S. cerevisiae fermentation product 2 TWA4 strain was kindly provided by Dr. Chris MCSWEENEY (Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia) and was revived from anaerobic glycerol medium using the modified AC11.1 medium (Gagen et al., 2010 2010 ) with H 2 and CO 2 in a proportion of 1:3 at 120 kPa. After growing three generations, the cell density was counted using a hemocytometer (Strober, 2001 2001 ). Original XP was supplied by Diamond V (Cedar Rapids, IA, USA). Experimental design 2 The effects of TWA4 and XP supplementation on ruminal fermentation, methane production, methanogen population, abundance, and diversity of rumen acetogens were determined using a 2×2 factorial design using in vitro rumen fermentation system. The four treatments were the Control (without TWA4 or XP), TWA4 (2×10 7 cells/ml TWA4 without XP), XP (2 g/L XP without TWA4), and TWA4XP (2×10 7 cells/ml TWA4 with 2 g/L XP). In vitro rumen fermentation 2 The rumen fluid was collected from three ruminally fistulated lactating Chinese Holstein cattle (raised in Hangzhou Zhengxing Animal Husbandry Co. Ltd., China) fed twice daily with a mixed diet (roughage:concentrate=55:45) before morning feeding and strained through four layers of gauze into a pre-warmed and insulated bottle. The care and use of fistulated cattle was approved by the Animal Care Committee of Zhejiang University (Hangzhou, China). Rumen fluid was processed under continuous flushing with CO 2 . The 120 ml serum bottle containing 0.5 g dry substrates (50:50 (w/w) mixture of Chinese wild rye meal and corn silage), 40 ml buffered medium, and 10 ml rumen fluid was anaerobically incubated at 39 °C using the semi-automated reading pressure technique (Mauricio et al., 1999 1999 ). To get the final concentration of 2 g/L XP for the XP and TWA4XP treatments, 0.1 g XP was added into the serum bottle following the supplement of 0.5 g dry substrates. The 2.5 ml growing TWA4 medium was injected into the bottle for TWA4 and TWA4XP treatments, while 2.5 ml medium without TWA4 inoculation was injected into the control and XP treatments. A pressure transducer, connected with a computer, was used to measure the accumulated head-space gas pressure through the in vitro incubation. Gas pressure was recorded after 6, 12, 24, and 48 h of incubation and was subsequently converted to the volumes of gas production (GP). At the same time intervals, 2 ml of head-space gas was collected with an airtight needle (SGE Analytical Science, Australia) to measure the methane production using gas chromatography (GC-2010, Shimadzu, Kyoto, Japan) equipped with a Flame Ionization detector (Hu et al., 2005 2005 ). For each treatment, triplicate bottles were included and three blanks were included simultaneously to correct the GP values for gas release from endogenous substrates. At the end of incubation (48 h), 3 ml mixed rumen samples were collected from each bottle and three bottles were sampled under oxygen-free CO 2 , and immediately stored at −80 °C to await further determination of the quantity of acetogens and methanogens, the diversity of acetogens, and the measurement of VFAs. VFAs were determined using the procedure described by Hu et al. ( 2005 2005 ). Dry matter degradation (DMD) was measured using the modified nylon bag method (Goering and van Soest, 1970 1970 ). Total DNA extraction and real-time quantitative polymerase chain reaction (PCR) 2 Total DNA was extracted from rumen fluid collected from a 48-h incubation period using the bead-beating method as previously reported (Gagen et al., 2010 2010 ). The primers specific to formyltetrahydrofolate synthetase gene ( ƒhs ) and methyl coenzyme-M reductase A ( mcrA ) genes (Table 1 1 ) were used to enumerate microorganisms with formyltetrahydrofolate synthetase (FTHFS) (Xu et al., 2009 2009 ) and methanogens (Denman et al., 2007 2007 ), respectively. The 16S rRNA gene of total bacteria was amplified with the primers as reported by Denman and McSweeney ( 2006 2006 ) as shown in Table 1 1 , and the copy number of total bacterial 16S rRNA gene was used as the reference to calculate the relative quantification of target. Real-time PCR was performed with SYBR green in ABI 7500 using the program: one cycle of initial denaturation at 95 °C for 10 s, 40 cycles of denaturation at 95 °C for 15 s, and annealing at 60 °C for 1 min. The relative abundance of each marker gene was estimated as: relative quantification of target=2 −( C T target− C T total bacteria) . F: forward; R: reverse; FTHFS: formyltetrahydrofolate synthetase; mcrA : methyl coenzyme-M reductase A; ACS: acetyl-CoA synthase Target Direction Primer sequence (5'→3') Reference 2 Total bacteria F CGGCAACGAGCGCAACCC 2 Denman and McSweeney, 2006 2006 R CCATTGTAGCACGTGTGTAGCC 2 FTHFS F GTWTGGGCWAARGGYGGMGAAGG 2 Xu et al., 2009 2009 R GARGAYGGWTTTGAYATYAC 2 mcrA F TTCGGTGGATCDCARAGRGC 2 Denman et al., 2007 2007 R GBARGTCGWAWCCGTAGAATCC 2 ACS F CTBTGYGGDGCIGTIWSMTGG 2 Gagen et al., 2010 2010 R AARCAWCCRCADGADGTCATIGG Acetyl-CoA synthase gene sequencing 2 To investigate the effect of TWA4 and XP on acetogen diversity, acetyl-CoA synthase (ACS) genes were amplified and sequenced from the triplicate of DNA extracted from a 48-h incubation period as previously described (Gagen et al., 2010 2010 ). The amplicon triplicates from each treatment were then pooled on an equal concentration basis (Checked by Qubit 2.0, Invitrogen, USA) for clone library construction using the pGEM-T Easy® vector (Promega Co., Madison, Wisconsin, USA) and Escherichia coli competent cell DH5α (TaKaRa, Dalian, China) according to manufacturer’s instructions. In total, 96, 96, 94, and 92 clones were randomly sequenced from the control, TWA4, XP, and TWA4XP libraries, respectively, and were then sequenced with T7 primer (Beijing Genomic Institute, China). The Primer Premier 5.0 (PREMIER Biosoft International, CA, USA) was used to translate DNA sequences to amino acid sequences before alignment. Sequence similarity to ACS was determined by BLASTP analysis (Gish and States, 1993 1993 ) using all existing bacterial ACS amino acid sequences in NCBI database. The ACS amino acid sequences were grouped into phylotypes at a distance of 0.01 using Jones-Taylor-Thornton Matrix of MEGA6 (Tamura et al., 2013 2013 ), because Blautia schinkii and Ruminococcus obeum shared 96.3% ACS amino acid identity, while 0.03 and 0.02 distance levels could not be clustered in our ACS amino acid sequences. The richness of ACS amino acid sequences was evaluated by the number of phylotypes and Chao1 index. The evenness was analyzed by Simpon’s diversity and Pielou’s evenness indices, calculated from Shannon’s diversity index (Felsenstein, 1993 1993 ). Relative abundance of the phylotypes was determined as the sequence numbers in the phylotypes/total number of sequences. The Chao1, Simpon’s and Shannon’s diversity indices, Good’s coverage (1− n scp / n total , n scp is the number of single clone phylotypes and n total is the total number of sequences) were estimated using the summary single command in MOTHUR (Schloss et al., 2009 2009 ). Unifrac.weighted in MOTHUR was used to compare the structure of acetogen community based on ACS amino acid sequences, and then to run the principal coordinates analysis (PCoA) with the pcoa command in MOTHUR. Bootstrapped neighbour joining tree of deduced ACS amino acid sequences was constructed with MEGA 6.06 with 100 resamplings. Similar amino acid sequences identified by BLASTP analysis and ACS of TWA4 (AEL12814 and AEL12815) and Methanococcoides methylutens (KGK98586) were selected as references for tree construction. Putative ACS amino acid sequences determined in the present study have been submitted to the GenBank database under accession numbers KR152340 to KR152636. Statistical analyses 2 Data for DMD, methane production, VFA, and abundance of acetogens and methanogens were analyzed by two-way analysis of variance in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) with individual bottles as the experimental unit, TWA4 and XP supplementation as main effects, where the TWA4×XP interaction was significant, and a secondary test was conducted to separate the efficacy of TWA4 within XP (Robinson et al., 2006 2006 ). Multiple comparisons means among treatments were completed by Duncan’s multiple range tests. Significance was declared if P < 0.05. Results 1 Rumen fermentation characteristics 2 Methane production 3 The methane production (ml/g substrate) was not affected ( P > 0.05) by TWA4 or XP alone except for TWA4 at 6 h incubation (Table 2 2 ). When supplemented with TWA4XP, methane production was increased ( P < 0.05) at all time points by 20% to 107% compared with the control. GP: gas production; VFA: volatile fatty acids; DMD: dry matter degradation; TWA4: acetogen; XP: Saccharomyces cerevisiae fermentation product; TWA4XP: co-addition of acetogen and yeast culture; SEM: pooled standard error of the mean ( n =3). For the P -value, ×: interaction; +: with supplementation; −: without supplementation. Data, shown as mean±standard error ( n =3), within a row with different superscripts differ significantly ( P < 0.05). * Data were expressed as proportions 3 Parameter 3 Control 3 TWA4 3 XP 3 TWA4XP 3 SEM 7 P -value 2 TWA4×XP 3 TWA4 3 XP Main +XP −XP Main +TWA4 −TWA4 13 CH 4 (ml/g substrate) 6 h 7.76±0.15 c 9.89±0.71 b 8.20±0.18 c 16.06±0.42 a 0.43 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.48 12 h 27.69±0.91 b 28.30±2.85 b 24.76±0.20 b 38.36±0.10 a 1.50 <0.01 <0.01 <0.01 0.78 <0.05 <0.01 0.2 24 h 53.97±0.75 b 48.55±0.22 b 49.58±2.88 b 64.76±1.46 a 1.66 <0.01 <0.05 <0.01 0.05 <0.01 <0.01 0.1 48 h 60.19±0.20 b 59.88±0.61 b 59.23±0.83 b 88.26±1.03 a 0.74 <0.01 <0.01 <0.01 0.77 <0.01 <0.01 0.38 13 CH 4 (% of GP) 6 h 17.12±0.17 c 20.76±0.003 b 17.70±1.47 c 27.20±0.73 a 0.77 <0.01 <0.01 <0.01 <0.05 <0.01 <0.01 0.59 12 h 31.44±0.75 ab 31.40±2.93 ab 29.12±0.28 b 35.35±0.04 a 1.52 0.07 0.08 0.61 24 h 40.25±0.57 ab 36.11±0.27 c 38.30±1.74 bc 39.84±1.03 ab 1.03 <0.05 0.24 0.32 <0.05 0.42 <0.05 0.22 48 h 35.51±0.19 b 35.57±0.31 b 36.00±0.22 b 43.74±0.47 b 0.32 <0.01 <0.01 <0.01 0.9 <0.01 <0.01 0.31 13 VFA (mmol/L) Total 99.46±0.15 c 100.05±0.49 c 103.16±0.76 b 114.94±1.08 a 0.71 <0.01 <0.01 <0.01 0.58 <0.01 <0.01 <0.01 Acetate (A) 61.40±0.30 c 62.15±0.17 c 64.28±0.41 b 70.82±1.85 a 0.6 <0.01 <0.01 <0.01 0.39 <0.01 <0.01 <0.01 Propionate (P) 23.11±0.15 b 23.14±0.28 b 23.63±0.30 b 27.18±0.23 a 0.25 <0.01 <0.01 <0.01 0.95 <0.01 <0.01 0.18 Butyrate 9.46±0.09 c 9.47±0.08 c 10.04±0.15 b 11.07±0.28 a 0.17 <0.05 <0.05 <0.01 0.96 <0.01 <0.01 <0.05 A:P * 2.66±0.03 ab 2.69±0.03 ab 2.72±0.03 a 2.61±0.03 b 0.028 <0.05 0.17 <0.05 0.46 0.76 0.07 0.14 DMD (%) 62.34±0.26 61.39±2.33 59.88±2.42 62.59±1.67 1.88 0.65 0.74 0.36 Fermentation parameters 3 DMD was not affected ( P > 0.05) by treatments (Table 2 2 ). XP increased ( P < 0.05) total VFA and acetate and butyrate concentrations and TWA4XP increased ( P < 0.05) acetate, propionate, butyrate, and total VFA concentrations compared with the control. TWA4 showed no effect ( P > 0.05) on VFA production. FTHFS and methanogen abundance 2 After a 48-h incubation period, the relative abundances of FTHFS and methanogens were increased ( P < 0.05) with the addition of TWA4, XP, and TWA4XP, though relative abundance of FTHFS was much lower than methanogen abundance (Table 3 3 ). The increase in the abundance of FTHFS was greater than that of methanogens ( P < 0.05) for XP and TWA4XP compared with TWA4 (Table 3 3 ). The increases of FTHFS and methanogens were 382.1% and 209.4% for TWA4, 656.0% and 478.1% for XP, and 679.1% and 421.9% for TWA4XP, respectively, compared with the control. FTHFS: formyltetrahydrofolate synthetase; TWA4: acetogen; XP: Saccharomyces cerevisiae fermentation product; TWA4XP: co-addition of acetogen and yeast culture; SEM: pooled standard error of the mean ( n =3). For the P -value, ×: interaction; +: with supplementation; −: without supplementation. Data, shown as mean±standard error ( n =3), within a row with different superscripts differ significantly ( P < 0.05) 3 Parameter 5 log ratio to total bacterial 16S rDNA 7 P -value 2 Control 2 TWA4 2 XP 2 TWA4XP 2 SEM 2 TWA4×XP 3 TWA4 3 XP Main +XP −XP Main +TWA4 −TWA4  FTHFS −6.88±0.035 c −6.19±0.023 b −6.19±0.023 a −5.98±0.019 a 0.026 <0.01 <0.01 0.72 <0.01 <0.01 <0.01 <0.01  Methanogen −2.57±0.165 c −2.01±0.020 b −1.73±0.037 a −1.78±0.022 a 0.086 <0.01 <0.05 0.73 <0.01 <0.01 0.09 <0.01 Acetogen diversity 2 In total, 73, 70, 74, and 80 ACS amino acid sequences were detected in the control, TWA4, XP, and TWA4XP groups, respectively. Further amino acid similarity (99%) analysis identified 15 phylotypes for the control, 10 phylotypes for TWA4, 12 phylotypes for XP, and 7 phylotypes for TWA4XP with 3 common phylotypes among the four treatments (Table 4 4 ). Coverage analysis and a rarefaction curve indicated that the sequences obtained covered the majority of acetogen communities in all treatments with 89.0% for the control, 94.3% for TWA4, 93.2% for XP, and 95.0% for TWA4XP groups, respectively (Table 4 4 , Fig. 1 1 ). From the results of numbers of phylotypes and Chao1, the richness of deduced ACS amino acid sequences was highest in the control group, followed by XP, TWA4, and TWA4XP. The control and XP had the highest Pielou’s evenness followed by TWA4, while TWA4XP had the highest Simpson indices value. OTUs: operational taxonomic units. Good’s coverage (1− n scp / n total , n scp is the number of single clone phylotypes and n total is the total number of sequences) was estimated using the summary.single command of MOTHUR. The Pielou’s evenness was estimated as Shannon/ln(Chao1). The total number of sequences was 73, 70, 74, and 80 in the control, TWA4, XP, and TWA4XP treatments, respectively Treatment No. of OTUs Chao1 Shannon Pielou’s evenness Simpson Good’s coverage Control 15 22.0 1.89 0.61 0.23 89.0 TWA4 10 13.0 1.48 0.58 0.32 94.3 XP 12 14.5 1.62 0.61 0.30 93.2 TWA4XP 7 13.0 0.96 0.37 0.51 95.0 Eight out of 22 of the phylotypes showed high (98%–100%) similarity to putative ACS amino acid of uncultured bacterium at GenBank uploaded by Gagen et al. ( 2010 2010 ). All these phylotype sequences showed 62%–90% similarity to the nearest valid taxa including Acetitomaculum ruminis , R . sp. CAG:9, R. obeum , Eubacterium limosum , B. schinkii , B. hydrogenotrophica , and B. wexlerae (Table 5 5 ). Only 3 phylotypes were common phylotypes among the four treatments (Fig. 2 2 ), which occupied about 79.1% of the total ACS sequences. Phylotypes 1 and 2 had 86% similarity with A. ruminis , 96% and 98% similarities with uncultured bacterium. Phylotype 4 had 76% similarity with B. schinkii (Table 5 5 ). P is the phylotypes generated at 99% identity cutoff value. GenBank accession numbers of reference sequences are shown after the species names. Bootstap values of ≥50% are shown at the nodes. The low- A. ruminis -like sequences and low- E. limosum -like sequences are marked with solid circles (●) and squares (■), respectively 2 Phylotype 2 Total Clones 2 Control 2 TWA4 2 XP 2 TWA4XP 2 Nearest valid taxa 2 Nearest relative Description Identity (%) Description Identity (%) 1 158 30 35 38 55 A. ruminis , ADR32215 86 Uncultured bacterium, ADR32256 96 2 62 15 19 11 17 A. ruminis , ADR32215 86 Uncultured bacterium, ADR32274 98 3 15 10 3 2 0 A. ruminis , ADR32215 90 Uncultured bacterium, ADR32250 98 4 15 4 4 3 4 B. schinkii , ADR32225 76 B. schinkii , ADR32225 76 5 11 0 0 11 0 E. limosum , ADR32216 70 Uncultured bacterium, ADR32288 96 6 4 1 3 0 0 B. hydrogenotrophica , ADR32222 84 Uncultured bacterium, ADR32235 90 7 4 2 0 2 0 E. limosum , ADR32216 72 Uncultured bacterium, ADR32278 100 8 4 1 2 0 1 E. limosum , ADR32216 62 Uncultured bacterium, AEM54104 100 9 3 2 1 0 0 B. hydrogenotrophica , ADR32222 79 Uncultured bacterium, ADR32267 82 10 3 1 1 1 0 E. limosum , ADR32216 65 Firmicutes bacterium CAG:124, WP_021932507 73 11 3 2 1 0 0 E. limosum , ADR32216 68 Firmicutes bacterium CAG:170, WP_022223268 76 12 3 1 0 1 1 B. wexlerae , WP_026648192 77 Uncultured bacterium, ADR32238 98 13 3 1 0 2 0 R. obeum , WP_044925744 73 Uncultured bacterium, ADR32247 100 14 1 0 0 0 1 B. hydrogenotrophica , ADR32222 66 Uncultured bacterium, AEM54114 100 15 1 0 0 1 0 E. limosum , ADR32216 69 Uncultured bacterium, ADR32278 98 16 1 1 0 0 0 E. limosum , ADR32216 68 Firmicutes bacterium CAG:124, WP_021932507 73 17 1 0 0 1 0 R . sp. CAG:9, WP_022380765 83 R . sp. CAG:9, WP_022380765 83 18 1 0 0 0 1 A. ruminis , ADR32219 73 Firmicutes bacterium CAG:170, WP_022223268 79 19 1 0 1 0 0 A. ruminis , ADR32219 70 Firmicutes bacterium CAG:170, WP_022223268 76 20 1 1 0 0 0 B. wexlerae , WP_029676805 80 Uncultured bacterium, ADR32267 86 21 1 0 0 1 0 E. limosum , ADR32216 73 E. limosum , ADR32216 73 22 1 1 0 0 0 E. limosum , ADR32216 69 Uncultured bacterium, ADR32288 81 Total 297 73 70 74 80 Based on amino acid identified phylotypes, uncultured bacteria sequences were predominant in all acetogen communities. The dominant acetogens were unchanged in the treatments (phylotypes 1 and 2; Table 5 5 and Fig. 3 3 ) while the acetogen communities were significantly different among four treatments (weighted significance < 0.001; Fig. 3b 3b ), whose community changes were reflected in the appearance new phylotypes. For XP treatment, about all the four unique phylotypes were identified as low- E. limosum- like acetogens (Table 5 5 ). There were no TWA4 ACS amino acid sequences actually recovered in the TWA4 and TWA4XP libraries (Fig. 2 2 ). Discussion 1 Ruminal methane production through enteric fermentation is of global concern due to its contribution to greenhouse gas emission as well as accounting for dietary energy loss in animals. Since the 1950s much effort has been expended trying to reduce methane emission from ruminants. Although the use of agents including chemicals and antibiotics has been successful, their use has been limited due to the probability of high quantities of residuals remaining in the animal products and feces (Kobayashi, 2010 2010 ; Patra, 2012 2012 ). Methanogenesis is the primary method of facilitating the consumption of hydrogen in the rumen, followed by fumarate reduction (Asanuma and Hino, 2000 2000 ; Mitsumori and Sun, 2008 2008 ), sulfate reduction (Morvan et al., 1996 1996 ; Sahakian et al., 2010 2010 ), nitrate and nitrite reduction (Sakthivel et al., 2012 2012 ), and reductive acetogenesis (Henderson et al., 2010 2010 ). Evidence from in vitro incubations of rumen contents (Nollet et al., 1997a 1997a ; le Van et al., 1998 1998 ) and methanogen-free lambs (Fonty et al., 2007 2007 ) indicates that acetogens can function as hydrogenotrophs to suppress methanogenesis. Thus, enhancing reductive acetogenesis in the rumen may be an effective strategy to mitigate methane. Lopez et al. ( 1999 1999 ) investigated the ability of six reductive acetogens to prevent the accumulation of methane in vitro and found that only two of them, E. limosum strains ATCC 8486 and Ser 5, decreased methane production by about 5% after a 24-h incubation period, while total VFA was not affected ( P > 0.05) by the addition of the six acetogens. As with Lopez’s study, the addition of TWA4 alone did not change the methane and VFA production in our experiment. The TWA4, XP, and TWA4XP failed to improve the DMD, revealing that no additional hydrogen and nutrients were produced from the substrates in these treatments compared with the control. Yeast, as a feed additive, has been confirmed as being able to provide nutrients and vitamins to microorganisms (Chaucheyras-Durand et al., 1995a 1995a ). Chaucheyras-Durand and Fonty ( 2001 2001 ) reported that the supplementation of S. cerevisiae CNCM I-1077 tended to improve the in sacco degradation of wheat straw, and increased VFA production. From that study, Lascano and Heinrichs ( 2009 2009 ) reported S. cerevisiae yeast culture increased rumen VFA concentration without influencing DMD. They concluded that the increased total VFA concentration with unchanged molar proportions of individual VFA was the result of the increased fermentation rate created by yeast culture supplement. However, reasons for inconsistency between the increased fermentation rate and unchanged DMD were not explained. As with the above study, our results showed that DMD remained unchanged, suggesting that the increased VFA by XP addition or XP and TWA4 co-addition was not a product of fermentable organic matter, such as cellulose or starch. Chaucheyras-Durand et al. ( 1995b 1995b ) showed that the presence of yeast cells stimulated the utilization of hydrogen by acetogens and enhanced acetogenesis, in a co-culture of acetogens and methanogens. In our XP and TWA4XP treatments, the increased VFA was mainly due to the acetate (Table 2 2 ). With the ƒhs primers of Xu et al. ( 2009 2009 ), the rate of acetogen increase was higher than that of methanogens in XP and TWA4XP, suggesting that XP treatments may alter the acetogen communities and increase acetogenesis. However, according to Gagen et al. ( 2010 2010 ), the ƒhs primers used to enumerate acetogens in our experiment could recover partial FTHFS sequence from a wider range of rumen acetogens, although multiple spurious amplicons could be generated from some acetogens and rumen microbial DNA. Until now, no appropriate real-time PCR primers were reported for acetogens, it could only deduce that XP treatments may increase the FTHFS biochemical pathway of rumen microorganisms, especially acetogens, which would cause an increase in VFA production. The diversity of the acetogen community in our experiment showed that Lachnospiraceae was the dominant acetogen in the rumen fermentation system, but without close sequences from cultured isolates, which was similar to the results of Gagen et al. ( 2010 2010 ). However, the species richness of our experiment was lower than that of Gagen et al. ( 2010 2010 ). This difference may be due to the production accumulation in vitro system. It is possible that TWA4 could not survival after a 48-h fermentation period; however, the acetogen community was significantly changed by adding TWA4 alone or with XP, and therefore we posit that future studies are needed to track the fate of TWA4 with the change of fermentation time. Acetogen phylotypes were increased by the addition of XP (Table 5 5 and Fig. 3a 3a ) with four unique phylotypes where amino acid was identified as low- E. limosum -like acetogen (Table 5 5 ). E. limosum and A. ruminis were all isolates from rumen by Sharak Genthner et al. ( 1981 1981 ) and Greening and Leedle ( 1989 1989 ), respectively. These bacteria can utilize H 2 /CO 2 , one-carbon compounds, to produce acetate. However, in contrast to A. ruminis , E. limosum can utilize amino acid to produce acetate and butyrate, and its growth can be stimulated by amino acids (Sharak Genthner et al., 1981 1981 ; Pacaud et al., 1985 1985 ). Amino acids were a substrate for methanogen growth and methanogensis (Mathrani and Boone, 1985 1985 ; Chaucheyras-Durand et al., 1995b 1995b ) as well. Therefore, enhancing acetogenesis by supplement with acetogen strain and/or yeast cells may be a way of mitigating methane, targeting acetogens such as E. limosum which utilize substrates such as amino acids to facilitate growth and acetogenesis. In order to further support our speculation, future experiments should next try isolating the low- E. limosum -like acetogens to test their nutritional characteristics and competition with methanogens. 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All management and experimental procedures were approved by the Animal Care Committee of Zhejiang University, China and were carried out in accordance with the University’s guidelines for animal research. 10.1631/jzus.B1500013 Project supported by the Fundamental Research Funds for the Central Universities (No. 2011XZZX006), the National Natural Science Foundation of China (No. 31172229), and the University of Alberta Research Internship Program Summer 2012, Canada 2015-01-13 2015-07-09 2015-05-15 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 16