Acetogen Communities In The Gut Of Herbivores And Its Role In Methane Emission

Homoacetogenic bacteria have received attention as a hydrogenotrophic population that convert waste gases into important industrial chemicals and biofuels in biofuel industry, and offer a significant energetic advantage to the host animal through competing with gut methanogen for hydrogen utilization. However, there are few reports about the research on acetogen communities in the gastrointestinal tract of herbivores and the role of acetogens in rumen methane mitigation. The aims of this study were to explore the effects of host species, fermentation patteren and sites of digestive tract on acetogen community through researching on the composition of acetogen populations in the rumen of dairy cow, beef and sheep, the forestomach of tammer wallaby, the caecum of rabbit and fece of horse; to evaluate the effect of age develoment on acetogen community through researching on acetogen populations in the caecum of rabbits at different age stage fed with the same diet and raised in the same environment; to explore the hydrogen utilization ability of gut acetogens by enriching acetogen candidates from the rumen of cow and sheep, caecum of rabbit and fece of horse; to find the possibility of enhancing rumen acetogenesis to reduce methane production and regulate rumen fermentation by supplementing the rumen with exogenous acetogens only or together with yeast culture, with exogenous acetogens when methanogenesis was inhibited, with phloroglucinol when methanogenesis was inhibited.1. Effects of host species and organ site on acetogen communities:the rumen content of six late lactation dairy cows and four Hu sheeps aged 4 months were sampled through rumen fistula, the caecum content of six Japanese White rabbits aged 6 months were sampled after slaughtering, the feces of four Australia horses aged 4 years were sampled by casing sampling, then using the method of constructing clone libraries to get an enough acetogen diversity information from them, the acetogen diversity data of the forestomach of tammer wallaby and rumen of beef were got from the Genbank published before. The acetogen communities in the gut of cow, sheep, beef, wallaby, horse and rabbit were rather different drived by the factors of host species, fermentation pattern, and organ site. The OTU numbers in the gut of cow, sheep, beef, wallaby, horse and rabbit were 18,14,23,12,31 and 65 respectively. The acetogen communities in the caecum of rabbit and fece of horse showed a better evenness. Even though the cow, sheep, beef and wallaby were both forestamoch fermentation herbivores, the acetogen communities in the forestamoch of wallabies were rather different from the ones in rumen, no common OTUs were observed between wallaby and ruminants, there were 4 common OTUs observed in the rumen of cow, sheep and beef. When ruminants cow, beef and sheep were compared, there were 9 OTUs shared by cow and sheep, and the dominant one were both OTU 3. When hindgut fermenters rabbit and horse were compared, there were 3 common OTUs shared by them, but the dominant one of horse was OTU 14 while rabbit was OTU 74. When monogastric herbivores wallaby, rabbit and horse were compared, there were 2 common OTUs shared by wallaby and horse, but no common OTU was shared by wallaby and rabbit, and the dominant OTU observed in wallaby was OTU 46, which was different from the dominant ones in rabbit and horse. The phylogenetic results further confirmed the results that different host species and organ site harbored different acetogen communities. The dominant OTU in the rumen of cow and sheep showed the highest similarity to Acetitomaculum ruminis (87% of similarity), the dominant OTUs in the rumen of beef, forestamoch of wallaby, caecum of rabbit and fece of horse showed the highest similarity to Blautia hydrogenotrophica, Blautia hydrogenotrophica, Blautia hydrogenotrophica, and Blautia producta respectively, and the similarity were 80%,77%,90% and 76% respectively. The acetogen communities in the monogastric herbivores (wallaby, horse and rabbit) affliated more broadly among Ruminococcaceae, Lachnospiraceae, Eubacteriaceae and Clostridiaceae family, while the acetogen communities in the ruminants (cow, sheep and beef) mainly affliated among Lachnospiraceae and Clostridiaceae family. The results of acetogen relative popualtion in the gut of herbivores by using qPCR method showed that there were large number of acetogens in the gut of cow, sheep, rabbit and horse, and the relative population of acetogens showed no signifant difference in the gut of cow, sheep, rabbit and horse. The relative population of methanogens, which are important hydrogenotrophic candidates in rumen, was significantly higher in the rumen of cow compared with the gut of rabbit and horse.2. Effects of age development on gut acetogen communities of herbivores:the caecal contents from 24 New Zealand White rabbits (aged 1,2,4,6 months respectively) fed with same diets were sampled. Miseq sequencing and qPCR were used to identify the composition of acetogen populations with age development. The results showed that there were diverse unknown acetogens in the caecum of rabbit across all the age groups, totally 6101 OTU were observed. And the abundance of acetogens showed no significant difference with age develoment. The structure of acetogen communities changed with age develoment, and to be more and more stable. The abundance of 131 OTUs were significantly changed between rabbits aged 1 and 6 months, while no significantly changed OTUs were observed between rabbits aged 4 and 6 months, suggested acetogen community converge to a more homogeneous and stable arrangement with age development. The significantly changed OTUs between young and adult rabbits were placed differently in the phylogenetic tree, perhaps the different acetogen community resulted in their different hydrogenotrophic character.3. Hydrogen utilization ability of gut acetogen enrichments:Anaerobic microorganism culture technology was used to enrich the acetogen populations from the gut of dairy cow, sheep, rabbit and horse to study their ability of tranforming syngas (H2 and CO2) into energy substances such as acetate, propionate and butyrate. The results showed that acetogen enrichment cultures from the rumen of cow and sheep, caecum of rabbit, fece of horse presented good and similar growth and hydrogenotrophic characters, the highest OD600 value were both observed at 48 h of incubation, and acetate was the primary end product, minor propionate, butyrate and isovaleric acid were also produced, and no methane was produced. During 48 h of incubation, the acetogen enrichment cultures from fece of horse, caecum of rabbit, rumen of cow and sheep could consume every 4.75,5.17,5.53 and 5.23 moles of H2 to 1 mole of acetate. Acetogen enrichment cultures from the fece of horse showed the best ability in consuming hydrogen to produce acetate (P<0.05). The hydrogen consumption showed no significant difference between cow and sheep during 48 h of incubation, but the enrichments from sheep produced significantly more acetate compared to cow (P＜0.05). The hydrogen consumption showed no significant difference between rabbit and horse during 48 h of incubation, but the enrichments from horse produced significantly more acetate compared to rabbit (P<0.05). All the enrichments produced butyrate as well, and the enrichment from horse produced significantly more butyrate compared with others during 48 h of incubation (P＜0.05).4. Effects of adding acetogen TWA4 alone or co-addition with yeast culture on rumen fermentation and acetogenesis:A 2×2 factorial design with two levels of TWA4 (0 or 2×107 cells/ml) and XP (0 or 2 g/L) was performed, and the method of in vitro fermentation system was used. The results showed that volatile fatty acids (VFAs) were increased (P＜0.05) in XP and TWA4XP, while methane was increased only in TWA4XP (P＜0.05). From quantifying the functional gene FTHFS of acetogens, the acetogen population increased with addition of TWA4 alone and co-addition with yeast culture, and the increase rate of 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 (86% of similarity) ACS was predominant in all acetogen communities, while four unique phylotypes in XP treatment were all amino acid identified as low-Eubacterium limosum-like (70% of similarity) 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. Supplementation with an acetogen strain and/or yeast cells may be an approach to regulate rumen fermentation and mitigate methane, with proper acetogen targeted.5. Effects of methanogenesis inhibitor-chloroform on rumen fermentation, methane production and rumen microbiota:Two feeding pattern were used (concentrate:roughage= 40:60; only roughage). Eight healthy steers with similar live weight were choosed and the experimental period was 78 days. There were 4 treatments based on the amount of chloroform added:Control, basal diets (concentrate:roughage= 40:60 or only hay) plus cyclodextrin; Low group,1.0 g/100 kg LW of chloroform-cyclodextrin (CD) was supplemented on the basis of control; Mid group,1.6 g/100 kg LW of chloroform-CD was supplemented on the basis of control; High group,2.0 g/100 kg LW (fed with hay) or 2.6 g/100 kg LW (fed with concentrate:roughage= 40:60) of chloroform-CD was supplemented on the basis of control. The results showed that addition of low dose (1.0 g/100 kg LW), mid dose (1.6 g/100 kg LW) and high dose (2.0 or 2.6 g/100 kg LW) of chloroform showed no significant influence on animal dry matter intake with either hay diet or hay and concentrate diet. When animals were fed with hay diet, the concentration of rumen ammonia nitrogen (NH3-N) increased significantly (P＜0.05) after feeding for 3 h with addition of chloroform, and addition of high dose chloroform significantly decreased VFA (volatile fatty acid) production (P＜0.05), while when animals were fed with hay and concentrate diet, the addition of chloroform did not changed these basic rumen fermentation characters (NH3-N and VFA). The addition of mid and high dose of chloroform decreased methane emission (P＜0.01) and increased hydrogen emission (P<0.05) from animals significantly, with significantly dicreased acetate (P＜0.01) and increased propionate production (P<0.05) in the rumen fluid as well. When animals were fed with hay and concentrate diet, the abundance of Fibrobacter succinogenes and protozoa increased significantly (P<0.05) with addition of both three dose of chloroform, the abundance of Methanobrevibacter ruminantium and mcrA gene decreased significantly (P<0.05), while the abundance of rumen cluster C increased significantly (P＜0.05). When the animals were fed with hay diet, the addition of mid and high dose of chloroform increased the abundance of Ruminococcus albus significantly (P＜0.05), and with no effect on the abundance of other microorganisms. The addition of chloroform both changed the rumen microbial composition with hay or hay and concentrate diet, the abundance of some OTUs belong to Fibrobacter, Ruminococcus, Methanobrevibacter genera which relate to rumen cellulolytic activity and methanogenesis significantly changed with the addition of chloroform (P<0.05), and the abundance of some OTUs belong to Succiniclasticum and Butyrivibrio which relate to propionate and butyrate production, OTUs belong to Desulfovibrio which relate hydrogen sink ways changed significantly as well (P＜0.05). The effects of chloroform on rumen microbial community seemed to be persistent, with significantly changed OTUs such as OTUs belong to Ruminococcus and Methanobrevibacter genera observed after chloroform treatmant (P＜0.05).6. Effects of TWA4 and phloroglucinol on rumen fermentation, methane production and acetogen population when methanogenesis was inhibited:Eight healthy steers with similar live weight were choosed and the experimental period was 79 days. Two experimental stages were conducted. There were two treatments in the first experimental stage based on if the phloroglucinol was supplemented:control, basal diet (concentraterroughage= 40:60) plus 1.6 g/100 kg LW of chloroform-CD; phloroglucinol group,75 g/100 kg LW of phloroglucinol was added on the basis of control. There were two treatments in the second experimental stage as well:control, basal diet (concentrate:roughage= 40:60) plus 1.6 g/100 kg LW of chloroform-CD, and plus 750 ml/d of cultures without acetogen TWA4; TWA4 group, basal diet (concentrate:roughage= 40:60) plus 1.6 g/100 kg LW of chloroform-CD, and plus 750 ml/d of TWA4 cultures. The results showed that compared with control group which added chloroform alone, the addition of phloroglucinol further reduced the hydrogen production from animals significantly (P<0.05), and the concentration of acetate in the rumen fluid increase significantly (P<0.05), suggesting phloroglucinol can redirect the excess hydrogen in the rumen towards autotrophic reductive acetogenesis. From the quantifying of FTHFS gene, no significant effect was observed on the rumen acetogen populations with addition of phloroglucinol. Compared with control group which added chloroform alone, the addition of TWA4 had no significant effects on the rumen fermentation, methane production and rumen acetogen populations.Overall, large amounts and diverse acetogens existed in the gastrointestinal tracts of herbivores, and the acetogen communities would be different due to different host species, organ site and age stage. The gut acetogen enrichment cultures can use syngases (H2 and CO2) to synthese energy substances such as acetate, propionate and butyrate, and the hydrogen utilization ability of acetogen enrichment from different kinds of herbivores are different. Perhaps it is not a good way to reduce rumen methane production through supplementing exogenous acetogen, the co-addition of yeast culture can increase the abundance of some acetogen strains and further affect rumen fermentation, phloroglucinol could redirect the hydrogen to synthese acetate when methanogenesis was inhibited.