Methanobrevibacter Research Articles

Functional analysis of microorganisms and metabolites in the cecum of different sheep populations and their effects on production traits.

The purpose of this study was to investigate the effects of intestinal microbiota on the growth and production performance of different groups of sheep, focusing on the role of cecal microbiota in regulating intestinal function, enhancing digestion and absorption, and improving feed utilization. The production performance of MG × STH (Mongolia × Small Tailed Han) F1 hybrids and purebred STH (Small Tailed Han) sheep by measuring various factors, including enzyme activities and VFAs (volatile fatty acids), to analyze changes in cecal fermentation parameters across different sheep groups. Metagenomic and metabolomic sequencing combined with bioinformatics to analyze the cecal contents of the two sheep populations. The study findings indicated that the MG × STH F1 hybrids outperformed the purebred STH in terms of body weight, height, oblique body length, and VFAs (p < 0.05). Additionally, the MG × STH F1 higher levels of protease and cellulase in the cecum compared to the purebred sheep (p < 0.05). Metagenomic analysis identified 4,034 different microorganisms at the species level. Five differential organisms (Akkermansiaceae bacterium, Escherichia coli, unclassified p Firmicutes, Streptococcus equinus, Methanobrevibacter millerae) positively regulated sheep performance. Metabolomics identified 822 differential metabolites indoleacetaldehyde, 2-aminobenzoic acid, phenyl-Alanine, enol-phenylpyruvate and n-acetylserotonin were associated with improved performance of sheep. The combined results from the metagenomic and metabolomic studies suggest a positive correlation between specific microbes and metabolites and the performance of the sheep. In conclusion, the MG × STH F1 hybrids demonstrated superior growth performance compared to the purebred STH sheep. The identified microorganisms and metabolites have promising roles in positively regulating sheep growth and can be considered key targets for enhancing sheep performance.

433 Use of polyclonal antibodies to improve the efficiency of rumen function

Abstract Technologies such as polyclonal antibody preparations (PAP) have been investigated as a tool to improve rumen function, by targeting specific bacteria or molecules. While PAP can be sourced from the serum of immunized mammals, isolating targeted avian-derived antibodies (immunoglobulin Y; IgY) from egg yolk gained popularity due to its reduced impact on hens and greater IgY concentration in yolks. Advantages of avian-derived PAP utilization include cost-effectiveness and convenient production, high stability, and reduced cross-reactivity compared with other PAPs. Furthermore, IgY does not activate the host immune complement system, as observed with PAP derived from mammalian serum. Several proposed strategies explain how PAP might protect the host. It is suggested that PAP can agglutinate bacteria, inhibit bacterial adhesion, suppress bacterial virulence factors, and neutralize toxins. Avian-derived PAP, mainly administered as a direct-fed product in beef and dairy cattle diets, has been studied for its impact on specific rumen microbial populations, especially during high-grain feeding. Research confirms the effectiveness of PAP-IgY in inhibiting the growth of targeted bacteria. In beef steers, feeding PAP-IgY against Streptococcus bovis and Fusobacterium necrophorum resulted in growth inhibition, while IgY against cellulolytic rumen bacteria also showed efficacy in reducing the growth of the targeted strains in vitro. In other studies, PAP-IgY targeting S. bovis and F. necrophorum increased ruminal pH in beef steers and heifers and dairy cows, while also cattle-fed PAP-IgY against F. necrophorum had reduced severity of liver abscesses. When performance was evaluated, PAP-IgY against S. bovis improved feed efficiency of feedlot beef steers, and milk production increased when dairy cows were supplemented with PAP-IgY against lipopolysaccharides, with no observed impact on cow health status. However, not all studies have consistently demonstrated positive effects of PAP-IgY on nutrient digestibility or on mitigating systemic inflammation during high-grain feeding. A recent study revealed that supplementation with PAP-IgY against Methanobrevibacter ruminantium M1 decreased methane output ex-situ and tended to increase molar proportion of propionate in beef steers. In summary, polyclonal antibody preparations, particularly avian-derived IgY, offer a promising alternative to improve rumen function and animal performance. This is evidenced by increased ruminal pH during high grain feeding, inhibition of targeted bacterial growth, and, most recently, methane reduction. However, not all studies demonstrate consistent effects regarding nutrient digestibility. Further research is needed to refine recommendations, including dosage and antibody combinations, and to explore the broader impacts of PAP-IgY supplementation on ruminal and systemic variables to optimize livestock performance.

Relationship between jejunum ATPase activity and antioxidant function on the growth performance, feed conversion efficiency, and jejunum microbiota in Hu sheep (Ovis aries)

BackgroundATPase activity and the antioxidant function of intestinal tissue can reflect intestinal cell metabolic activity and oxidative damage, which might be related to intestinal function. However, the specific influence of intestinal ATPase activity and antioxidant function on growth performance, feed conversion efficiency, and the intestinal microbiota in sheep remains unclear.ResultsThis study analyzed the correlation between ATPase activity and antioxidant function in the jejunum of 92 Hu sheep and their growth performance and feed conversion efficiency. Additionally, individuals with the highest (H group) and lowest (L group) jejunum MDA content and Na+ K+-ATPase activity were further screened, and the effects of jejunum ATPase activity and MDA content on the morphology and microbial community of sheep intestines were analyzed. There was a significant correlation between jejunum ATPase and SOD activity and the initial weight of Hu sheep (P < 0.01). The H-MDA group exhibited significantly higher average daily gain (ADG) from 0 to 80 days old and higher body weight (BW) after 80 days. ATPase and SOD activities, and MDA levels correlated significantly and positively with heart weight. The jejunum crypt depth and circular muscle thickness in the H-ATP group were significantly higher than in the L-ATP group, and the villus length, crypt depth, and longitudinal muscle thickness in the H-MDA group were significantly higher than in the L-MDA group (P < 0.01). High ATPase activity and MDA content significantly reduced the jejunum microbial diversity, as indicated by the Chao1 index and observed species, and affected the relative abundance of specific taxa. Among species, the relative abundance of Olsenella umbonata was significantly higher in the H-MDA group than in the L-MDA group (P < 0.05), while Methanobrevibacter ruminantium abundance was significantly lower than in the L-MDA group (P < 0.05). In vitro culture experiments confirmed that MDA promoted the proliferation of Olsenella umbonata. Thus, ATPase and SOD activities in the jejunum tissues of Hu sheep are predominantly influenced by congenital factors, and lambs with higher birth weights exhibit lower Na+ K+-ATPase, Ca2+ Mg2+-ATPase, and SOD activities.ConclusionsThe ATPase activity and antioxidant performance of intestinal tissue are closely related to growth performance, heart development, and intestinal tissue morphology. High ATPase activity and MDA content reduced the microbial diversity of intestinal tissue and affect the relative abundance of specific taxa, representing a potential interaction between the host and its intestinal microbiota.

THE ROLE OF BACTERIAL AND ARCHAEA IN DETERMINING THE METABOLIC PATHWAY OF BIOGAS FERMENTATION AT LOW TEMPERATURES

The challenge in achieving large-scale biogas production still lies in the biogas fermentation process at low temperatures. Our goal was to delve into the metabolic pathway behind the formation of biogas at these lower temperatures, focusing on the dominant bacterial and archaeal communities. Employing a batch system with activated sludge inoculum at 10°C, we fermented cow manure at 12°C for 150 days. Through genetic sequencing and taxonomic analysis using OTUs from the 16S rDNA gene, we investigated bacterial and archaeal species. Correlation analysis between their abundance was conducted using Pearson correlation and t-tests via IBM SPSS Statistics. Our findings revealed a biogas production of around 0.74 L/day, with CH4 levels surpassing 0.45 L/g VS. Peak efficiency occurred between day 60 and 110, reaching its apex on day 90. Clostridium cellulovorans dominated, ranging from 13.9% to 27%, followed by Terrisporobacter petrolarius, around 16.2% to 23%. Specifically, the formation of biogas (CH4) predominantly occurred through the H2 pathway, led by significant hydrogenotrophic Archaea OTUs like Methanocorpusculum sinense (ranging from 4.95% to 37.10%) and Methanobrevibacter millerae (with relative abundances between 2.00% and 11.20%)

Grazing exclusion alters soil methane flux and methanotrophic and methanogenic communities in alpine meadows on the Qinghai-Tibet Plateau.

Grazing exclusion (GE) is an effective measure for restoring degraded grassland ecosystems. However, the effect of GE on methane (CH4) uptake and production remains unclear in dominant bacterial taxa, main metabolic pathways, and drivers of these pathways. This study aimed to determine CH4 flux in alpine meadow soil using the chamber method. The in situ composition of soil aerobic CH4-oxidizing bacteria (MOB) and CH4-producing archaea (MPA) as well as the relative abundance of their functional genes were analyzed in grazed and nongrazed (6 years) alpine meadows using metagenomic methods. The results revealed that CH4 fluxes in grazed and nongrazed plots were -34.10 and -22.82 μg‧m-2‧h-1, respectively. Overall, 23 and 10 species of Types I and II MOB were identified, respectively. Type II MOB comprised the dominant bacteria involved in CH4 uptake, with Methylocystis constituting the dominant taxa. With regard to MPA, 12 species were identified in grazed meadows and 3 in nongrazed meadows, with Methanobrevibacter constituting the dominant taxa. GE decreased the diversity of MPA but increased the relative abundance of dominated species Methanobrevibacter millerae from 1.47 to 4.69%. The proportions of type I MOB, type II MOB, and MPA that were considerably affected by vegetation and soil factors were 68.42, 21.05, and 10.53%, respectively. Furthermore, the structural equation models revealed that soil factors (available phosphorus, bulk density, and moisture) significantly affected CH4 flux more than vegetation factors (grass species number, grass aboveground biomass, grass root biomass, and litter biomass). CH4 flux was mainly regulated by serine and acetate pathways. The serine pathway was driven by soil factors (0.84, p < 0.001), whereas the acetate pathway was mainly driven by vegetation (-0.39, p < 0.05) and soil factors (0.25, p < 0.05). In conclusion, our findings revealed that alpine meadow soil is a CH4 sink. However, GE reduces the CH4 sink potential by altering vegetation structure and soil properties, especially soil physical properties.

Unlocking gut microbiota potential of dairy cows in varied environmental conditions using shotgun metagenomic approach

Food security and environmental pollution are major concerns for the expanding world population, where farm animals are the largest source of dietary proteins and are responsible for producing anthropogenic gases, including methane, especially by cows. We sampled the fecal microbiomes of cows from varying environmental regions of Pakistan to determine the better-performing microbiomes for higher yields and lower methane emissions by applying the shotgun metagenomic approach. We selected managed dairy farms in the Chakwal, Salt Range, and Patoki regions of Pakistan, and also incorporated animals from local farmers. Milk yield and milk fat, and protein contents were measured and correlated with microbiome diversity and function. The average milk protein content from the Salt Range farms was 2.68%, with an average peak milk yield of 45 litters/head/day, compared to 3.68% in Patoki farms with an average peak milk yield of 18 litters/head/day. Salt-range dairy cows prefer S-adenosyl-L-methionine (SAMe) to S-adenosyl-L-homocysteine (SAH) conversion reactions and are responsible for low milk protein content. It is linked to Bacteroides fragilles which account for 10% of the total Bacteroides, compared to 3% in the Patoki region. The solid Non-Fat in the salt range was 8.29%, whereas that in patoki was 6.34%. Moreover, Lactobacillus plantarum high abundance in Salt Range provided propionate as alternate sink to [H], and overcoming a Methanobrevibacter ruminantium high methane emissions in the Salt Range. Furthermore, our results identified ruminant fecal microbiomes that can be used as fecal microbiota transplants (FMT) to high-methane emitters and low-performing herds to increase farm output and reduce the environmental damage caused by anthropogenic gases emitted by dairy cows.

Specific gastrointestinal microbiota profiles in Chinese Tan sheep are associated with lauric acid content in muscle

The biological mechanisms underlying meat quality remain unclear. Currently, many studies report that the gastrointestinal microbiota is essential for animal growth and performance. However, it is uncertain which bacterial species are specifically associated with the meat quality traits. In this study, 16S rDNA and metagenomic sequencing were performed to explore the composition and function of microbes in various gastrointestinal segments of Tan sheep and Dorper sheep, as well as the relationship between microbiota and meat quality (specifically, the fatty acid content of the muscle). In the ruminal, duodenal, and colonic microbiome, several bacteria were uniquely identified in respective breeds, including Agrobacterium tumefaciens, Bacteroidales bacterium CF, and several members of the family Oscillospiraceae. The annotation of GO, KEGG, and CAZYme revealed that these different bacterial species were linked to the metabolism of glucose, lipids, and amino acids. Additionally, our findings suggested that 16 microbial species may be essential to the content of fatty acids in the muscle, especially C12:0 (lauric acid). 4 bacterial species, including Achromobacter xylosoxidans, Mageeibacillus indolicus, and Mycobacterium dioxanotrophicus, were positively correlated with C12:0, while 13 bacteria, including Methanobrevibacter millerae, Bacteroidales bacterium CF, and Bacteroides coprosuis were negatively correlated with C12:0. In a word, this study provides a basic data for better understanding the interaction between ruminant gastrointestinal microorganisms and the meat quality traits of hosts.

PSXI-5 Impact of Different Vaccine Formulations in Reducing Methane Production in Beef Cattle

Abstract Ruminants are the main contributor to agricultural emissions of the greenhouse gas methane (CH4), which is one of the main greenhouses causing global warming. The objective of this pilot study was to test the effectiveness of 4 different vaccine formulations (VF) on methane abatement in beef cattle. Two weeks before the start of the trial, steers aged between 9 to 10 months with body weights (236.57 ± 20,61 kg) were trained to use the GreenFeed (C-Lock Inc., Rapid City, SD, USA) system for methane measurement. At the end of the adaptation period, animals that best accepted the GreenFeed system were selected (n = 20) for the experiment. Animals were randomly assigned to one of four different formulations of vaccine treatments as well as a control group. Each treatment group had the following number of steers: Control/Untreated (UTD, n = 4), F1/OVA_ adj (n= 4), F2/Ag1_adj (n= 4), F3/pep1_Ag1_adj (n = 4), F4/pep2_Ag1_adj (n = 4). On day 0, vaccine was administered followed by booster injections on day 20 with 2mL injected intramuscularly in the anterior region of the neck both times. Through jugular venipuncture, blood was collected into a 10 mL vacutainer tube (Becton Drive, Franklin Lakes, NJ) to obtain serum on day 34 post-treatment to determine methanogen-specific IgA and IgG concentration. Rumen fluid (14 mL) was collected on pre-treatment (d0), and post treatments (d 20 and d 34) into a glass jar using an esophageal stomach tube connected to a vacuum and occurred pre-feeding to determine the relative abundance of total archaea and Methanobrevibacter ruminantium. Methane production was sampled on individual animals using the GreenFeed system pre-treatment (d -8 to -1) and post treatment (d 22 to d 29). The GreenFeed system estimates daily methane production (DMP, g/day) by measuring gas concentration for 3 to 5 mins on individual animals. Data were analyzed as one-way ANOVA with Tukey’s HSD post-hoc analysis. Preliminary results showed overall mean methane emission pre-treatment (165.853 ± 2.69 g/d) compared with post-treatment (192.164 ± 2.33 g/d) increased by 16%. However, methane emissions between treatment groups showed statistical difference (P &amp;lt; 0.001) specifically between treatment group F2 and F4. Serum concentration of IgA and IgG, ruminal relative abundance of total archaea and Methanobrevibacter ruminantium did not show significant difference (P &amp;gt; 0.10). From the results, vaccine formulation F2 and F4 showed effects in the methane emission between the treatment groups. Future research will consider increasing the vaccine dosage and analyzing more samples. This project was supported by HelixNano.

Rumen microbial degradation of bromoform from red seaweed (Asparagopsis taxiformis) and the impact on rumen fermentation and methanogenic archaea

BackgroundThe red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane (CH4) analogues, primarily bromoform (CHBr3). This study aimed to investigate the degradation process of CHBr3 from A. taxiformis in the rumen and whether this process is diet-dependent. An in vitro batch culture system was used according to a 2 × 2 factorial design, assessing two A. taxiformis inclusion rates [0 (CTL) and 2% DM diet (AT)] and two diets [high-concentrate (HC) and high-forage diet (HF)]. Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0, 0.5, 1, 3, 6, 8, 12, 16, 24, 48 and 72 h to assess the pattern of degradation of CHBr3 into dibromomethane (CH2Br2) and fermentation parameters. Additionally, an in vitro experiment with pure cultures of seven methanogens strains (Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanosarcina barkeri, Methanobrevibacter millerae, Methanothermobacter wolfei and Methanobacterium mobile) was conducted to test the effects of increasing concentrations of CHBr3 (0.4, 2, 10 and 50 µmol/L).ResultsThe addition of AT significantly decreased CH4 production (P = 0.002) and the acetate:propionate ratio (P = 0.003) during a 72-h incubation. The concentrations of CHBr3 showed a rapid decrease with nearly 90% degraded within the first 3 h of incubation. On the contrary, CH2Br2 concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation. Neither CHBr3 degradation nor CH2Br2 synthesis were affected by the type of diet used as substrate, suggesting that the fermentation rate is not a driving factor involved in CHBr3 degradation. The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M. smithii, M. ruminantium, M. stadtmanae, M. barkeri, M. millerae, M. wolfei, and M. mobile.ConclusionsThe present work demonstrated that CHBr3 from A. taxiformis is quickly degraded to CH2Br2 in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr3 degradation.

188 Characterization of the Vagino-Uterine Microbiota in Pregnant and Non-Pregnant Beef Cattle Using 16S Rrna Gene Sequencing and Culturing

Abstract Emerging evidence suggests that urogenital microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. The objectives of this study were to: 1) characterize the vaginal and uterine microbiota between beef cattle that became pregnant via artificial insemination (AI) and those that did not; 2) identify differentially abundant taxa between pregnant and non-pregnant cattle; and 3) characterize the culturable fraction of the vaginal and uterine microbiota using extensive culturing. Vaginal and uterine swab samples were collected from two cohorts of Angus-crossbred cattle: mature cows (n =100) and heifers (n = 72, vaginal swab only) at the time of AI. At 35 days post-AI, pregnancy diagnoses were made via ultrasound. A subset of vaginal and uterine swabs from cows (27 non-pregnant and 31 pregnant) and heifers (26 non-pregnant and 33 pregnant) were selected and processed for microbiota assessment using 16S rRNA gene (V3-V4) sequencing. For culturing, a subset of cryopreserved vaginal and uterine swabs (128) were plated onto three different agar types and cultured, and isolates were identified by near-full length 16S rRNA gene sequencing. Sequencing results revealed 11 taxa that were more abundant in the vaginas of heifers that failed to become pregnant via AI than heifers that did become pregnant. While there was no significant difference in vaginal microbiota community structure (P = 0.21) between pregnant and non-pregnant cows, non-pregnant cows tended to have greater microbial richness and Shannon diversity (P ≤ 0.08) compared with pregnant cows. No differentially abundant taxa were detected in the vaginas of pregnant versus non-pregnant cows. Pregnant cows had a distinct uterine microbiota community structure (R2 = 0.032, P = 0.008) but similar alpha diversity (P &amp;gt; 0.1) compared with non-pregnant cows. Twenty-eight differentially abundant taxa were observed in the uterine microbiota between pregnant and non-pregnant groups, with 11 of them more abundant in pregnant cows. Methanobrevibacter ruminantium (archaea) and Fusobacterium necrophorum were among these 11 positive pregnancy-associated taxa. Although community structure was significantly different between the vagina and uterus, 277 “core taxa” (present in ≥ 80% of all samples) were shared between the two communities. A total of 733 bacterial isolates were recovered from vaginal (512) and uterine (221) swabs under aerobic (n = 363, 83 different species) and anaerobic (n = 370, 69 species) culturing. Potential pathogenic isolates within the Trueperella, Mannheimia and Histophilus genera were most exclusively identified from vaginal swabs. Only two Lactobacillus isolates were recovered. Overall, our results indicate that pregnancy-associated taxonomic signatures are present in the bovine uterine and vaginal microbiota. Among 39 pregnancy status-associated taxa, 41% were unclassified at the genus level, suggesting that shotgun metagenomics and culturomics are needed to identify and characterize the pregnancy-associated taxa in cattle.

Screening macroalgae for mitigation of enteric methane in vitro

This study investigated the effects of 67 species of macroalgae on methanogenesis and rumen fermentation in vitro. Specimens were analyzed for their effect on ruminal fermentation and microbial community profiles. Incubations were carried out in an automated gas production system for 24-h and macroalgae were tested at 2% (feed dry matter basis) inclusion rate. Methane yield was decreased 99% by Asparagopsis taxiformis (AT) when compared with the control. Colpomenia peregrina also decreased methane yield 14% compared with control; no other species influenced methane yield. Total gas production was decreased 14 and 10% by AT and Sargassum horneri compared with control, respectively. Total volatile fatty acid (VFA) concentration was decreased between 5 and 8% by 3 macroalgae, whereas AT reduced it by 10%. Molar proportion of acetate was decreased 9% by AT, along with an increase in propionate by 14%. Asparagopsis taxiformis also increased butyrate and valerate molar proportions by 7 and 24%, respectively, whereas 3 macroalgae species decreased molar proportion of butyrate 3 to 5%. Vertebrata lanosa increased ammonia concentration, whereas 3 other species decreased it. Inclusion of AT decreased relative abundance of Prevotella, Bacteroidales, Firmicutes and Methanobacteriaceae, whereas Clostridium, Anaerovibrio and Methanobrevibacter were increased. Specific gene activities for Methanosphaera stadtmane and Methanobrevibacter ruminantium were decreased by AT inclusion. In this in vitro study, Asparagopsis taxiformis was most effective in decreasing methane concentration and yield, but also decreased total gas production and VFA concentration which indicates overall inhibition of ruminal fermentation. No other macroalgae were identified as potential mitigants of enteric methane.

Bacterial species in the ruminal content of steers fed oilseeds in the diet.

The aim of this study was to evaluate bacterial species and diversity of methanogenic Archaea in the solid fraction of the ruminal content, through the gene sequences of the conserved 16S rDNA region, in response to the following diets: canola, cottonseed, sunflower, soybean, corn silage, and control diet. Six rumen-fistulated crossbred steers, with body weight (BW) of 416.33 ± 93.30kg, were distributed in a 6 × 6 Latin square design. Regardless of the diet provided, amylolytic, proteolytic, and lactic bacteria were identified in the rumen fluid. Cellulolytic bacteria were predominant for all diets, reaching 47.75% of operational taxonomic units (OTUs) in animals fed with the cottonseed diet. Amylolytic bacteria reach 62.51% of OTU in animal fed sunflower diet, while proteolytic bacteria correspond to 65.96% of OTU in this same diet. Also, Megasphaera elsdenii bacterium was identified for all diets, with a greater percentage of OTU in steers fed the cottonseed diet. The diversity analysis of the species identified the methanogenic Archaea Methanobrevibacter ruminantium in all diets. We conclude that the control and corn silage diets have the most similar bacterial flora; diets with oilseeds had 47.5% similarity in rumen flora bacteria species. Animals fed with soybean showed a reduced number of methanogenic Archaea in the rumen content, which could be an alternative feed for cattle due to their low potential for energy losses with the production of methane.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

163 Assessment of the Methanogenic Archaeal Communities Throughout the Gastrointestinal Tract in Beef Cattle with Different Breeds

Abstract Methane mitigation in ruminants is important to reduce greenhouse gas emissions and enhance feed utilization. Methanogenic archaea inhabit the gastrointestinal tract (GIT) of ruminants where they utilize H2 reducing equivalents formed by other microbiota (i.e., bacteria, fungi, and protozoa) to reduce CO2 to methane. This study aimed to assess methanogens in the GIT of beef cattle and evaluate the influence of gut regions and cattle breeds on methanogenic ecology. Digesta were collected from six gut regions (rumen, duodenum, jejunum, ileum, colon, rectum) in forty-two beef steers representing three breeds (Angus n=13, Charolais n=14, Kinsella composite hybrid n=15). Methanogens were characterized using 16S rRNA gene amplicon sequencing with methanogen-specific primers and analyzed using RIM-DB with QIIME2. Total methanogens were quantified using qPCR with universal methanogen primers. Gut region and breed effects were assessed using the non-parametric Kruskal-Wallis test. Estimation of methanogenic populations showed a region effect (P &amp;lt; 0.05), with the highest copy number observed in the rumen and the lowest in the duodenum and jejunum. Ten methanogenic species were identified (with a relative abundance ≥ 0.01% in ≥ 50% of individuals per breed) throughout the GIT with Methanobrevibacter ruminantium (predominant in all regions), Methanobrevibacter gottschalkii, and Methanosphaera sp. ISO3-F5 observed in all regions and in all breeds. Divergent methanogenic compositions were observed among gut regions (P &amp;lt; 0.05) and breed-specific species were identified (e.g., Methanobacterium bryantii in the colon of Charolais). The alpha-diversity index (Chao1) varied among gut regions (P &amp;lt; 0.05), and rumen showed the highest richness over other gut regions. Breed affected the alpha-diversity where Angus was distinct from other breeds (P &amp;lt; 0.05). These results revealed that methanogens in the GIT are region-dependent and breed-specific, highlighting the importance of taking cattle breed and total-tract ecology into consideration to understand the role of methanogens in the GIT.

PSVIII-16 Metagenomic Analyses of Microbial and Carbohydrate-Active Enzymes in the Rumen of Holstein Steers Supplemented with Seeds of Pharbitis nil

Abstract Our previous research revealed the effects of dietary supplementation of Pharbitis nil seeds (PA) in improving the ruminal methane reduction and digestibility in Hanwoo steers. The objective of this study was to further investigate the effects of PA supplementation on rumen microbial taxonomy, Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology functional categories and the diversity of carbohydrate-active enzymes (CAZymes). Five rumen cannulated Holstein steers (744 ± 35 kg) were fed either basal diet (Control) or basal diet supplemented with PA at 5% dry matter intake (PA5) for two consecutive periods of 40 days in pairwise comparison. Rumen fluid was collected on the last day of each period after 4h of morning feeding. Analysis of rumen microbial metagenome revealed that contigs assigned to taxa Methanobrevibacter millerae decreased (p = 0.090) in PA5 group. Functional annotation of contigs using KEGG database revealed that genes involved in starch and sucrose metabolism were enriched (p = 0.058) in PA5 group compared with control. CAZyme analysis identified genes that encode glycoside hydrolase (GH) accounted for 63.18% and 65.66% of all CAZymes in control and PA5, respectively. The GH2 family (enriched in oligosaccharide-degrading enzymes) genes were greater (p = 0.090) in PA5 than control. Likewise, the abundance of GH26 and GH53 (endo-hemicellulases), GH5_4 (cellulases), and GH13_36 and GH13_42 (α-amylase) were increased (p &amp;lt; 0.1) in PA5 relatively compared with control. Most of the genes were mainly produced by unclassified contigs followed by Cupriavidus oxalaticus and Prevotella ruminicola 23. De novo assembly and binning recovered a total of 107 metagenome-assembled genomes taxonomically assigned to different bacterial phyla and one archaeal phyla. Of these genomes, 39 represented the uncultivated bacteria and 1 represented Lactobacillus genera. This study revealed that PA supplemented steers had increased GH activity, which encode enzymes that efficiently degrade plant cell wall polysaccharides in the rumen thereby increasing the fiber digestibility.

Roughage biodegradation by natural co-cultures of rumen fungi and methanogens from Qinghai yaks

Anaerobic fungus–methanogen co-cultures from rumen liquids and faeces can degrade lignocellulose efficiently. In this study, 31 fungus–methanogen co-cultures were first obtained from the rumen of yaks grazing in Qinghai Province, China, using the Hungate roll-tube technique. The fungi were identified according to morphological characteristics and internal transcribed spacer (ITS) sequences. The methanogens associated with each fungus were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequencing. They were five co-culture types: Neocallimastix frontalis + Methanobrevibacter ruminantium, Neocallimastix frontalis + Methanobrevibacter gottschalkii, Orpinomyces joyonii + Methanobrevibacter ruminantium, Caecomyces communis + Methanobrevibacter ruminantium, and Caecomyces communis + Methanobrevibacter millerae. Among the 31 co-cultures, during the 5-day incubation, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded 59.0%–68.1% of the dry matter (DM) and 49.5%–59.7% of the neutral detergent fiber (NDF) of wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce CH4 (3.0–4.6 mmol/g DM) and acetate (7.3–8.6 mmol/g DM) as end-products. Ferulic acid (FA) released at 4.8 mg/g DM on corn stalk and p-coumaric acid (PCA) released at 11.7 mg/g DM on sorghum straw showed the highest values, with the following peak values of enzyme activities: xylanase at 12,910 mU/mL on wheat straw, ferulic acid esterase (FAE) at 10.5 mU/mL on corn stalk, and p-coumaric acid esterase (CAE) at 20.5 mU/mL on sorghum straw. The N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks represents a new efficient combination for lignocellulose biodegradation, performing better than previously reported fungus–methanogen co-cultures from the digestive tract of ruminants.

Cross-reactivity of antibodies to different rumen methanogens demonstrated using immunomagnetic capture technology.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19–198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Stimulating Effect of Trichococcus flocculiformis on a Coculture of Syntrophomonas wolfei and Methanospirillum hungatei.

ABSTRACTSyntrophic anaerobic consortia comprised of fatty acid-degrading bacteria and hydrogen/formate-scavenging methanogenic archaea are of central importance for balanced and resilient natural and manufactured ecosystems: anoxic sediments, soils, and wastewater treatment bioreactors. Previously published studies investigated interaction between the syntrophic bi-cultures, but little information is available on the influence of fermentative bacteria on syntrophic fatty acid oxidation, even though fermentative organisms are always present together with syntrophic partners in the above-mentioned ecosystems. Here, we present experimental observations of stimulated butyrate oxidation and methane generation by a coculture of Syntrophomonas wolfei with any of the following methanogens: Methanospirillum hungatei, Methanobrevibacter arboriphilus, or Methanobacterium formicicum due to the addition of a fermentative Trichococcus flocculiformis strain ES5. The addition of T. flocculiformis ES5 to the syntrophic cocultures led to an increase in the rates of butyrate consumption (120%) and volumetric methane production (150%). Scanning electron microscopy of the most positively affected coculture (S. wolfei, M. hungatei, and T. flocculiformis ES5) revealed a tendency of T. flocculiformis ES5 to aggregate with the syntrophic partners. Analysis of coculture’s proteome with or without addition of the fermentative bacterium points to a potential link with signal transducing systems of M. hungatei, as well as activation of additional butyryl coenzyme A dehydrogenase and an electron transfer flavoprotein in S. wolfei.IMPORTANCE Results from the present study open doors to fascinating research on complex microbial cultures in anaerobic environments (of biotechnological and ecological relevance). Such studies of defined mixed populations are critical to understanding the highly intertwined natural and engineered microbial systems and to developing more reliable and trustable metabolic models. By investigating the existing cultured microbial consortia, like the ones described here, we can acquire knowledge on microbial interactions that go beyond “who feeds whom” relations but yet benefit the parties involved. Transfer of signaling compounds and stimulation of gene expression are examples of indirect influence that members of mixed communities can exert on each other. Understanding such microbial relationships will enable development of new sustainable biotechnologies with mixed microbial cocultures and contribute to the general understanding of the complex natural microbial interactions.

Dietary wheat and reduced methane yield are linked to rumen microbiome changes in dairy cows.

Fermentation of pasture grasses and grains in the rumen of dairy cows and other ruminants produces methane as a by-product, wasting energy and contributing to the atmospheric load of greenhouse gasses. Many feeding trials in farmed ruminants have tested the impact of dietary components on feed efficiency, productivity and methane yield (MeY). Such diets remodel the rumen microbiome, altering bacterial, archaeal, fungal and protozoan populations, with an altered fermentation outcome. In dairy cows, some dietary grains can reduce enteric methane production. This is especially true of wheat, in comparison to corn or barley. Using a feeding trial of cows fed rolled wheat, corn or barley grain, in combination with hay and canola, we identified wheat-associated changes in the ruminal microbiome. Ruminal methane production, pH and VFA concentration data together with 16S rRNA gene amplicon sequences were used to compare ruminal bacterial and archaeal populations across diets. Differential abundance analysis of clustered sequences (OTU) identified members of the bacterial families Lachnospiraceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Selenomonadaceae, Anaerovoracaceae and Fibrobacteraceae having a strong preference for growth in wheat-fed cows. Within the methanogenic archaea, (at >99% 16S rRNA sequence identity) the growth of Methanobrevibacter millerae was favoured by the non-wheat diets, while Methanobrevibacter olleyae was unaffected. From the wheat-preferring bacteria, correlation analysis found OTU strongly linked to reduced MeY, reduced pH and raised propionic acid levels. OTU from the genera Shuttleworthia and Prevotella_7 and especially Selenomonadaceae had high anti-methane correlations. An OTU likely representing (100% sequence identity) the fumarate-reducing, hydrogen-utilising, rumen bacterium Mitsuokella jalaludinii, had an especially high negative correlation coefficient (-0.83) versus MeY and moderate correlation (-0.6) with rumen pH, strongly suggesting much of the MeY suppression is due to reduced hydrogen availablity. Other OTU, representing as yet unknown species from the Selenomonadaceae family and the genera Prevotella_7, Fibrobacter and Syntrophococcus also had high to moderate negative MeY correlations, but low correlation with pH. These latter likely represent bacterial species able to reduce MeY without causing greater ruminal acidity, making them excellent candidates, provided they can be isolated, for development as anti-methane probiotics.