Characteristics of rumen microbiota and Prevotella isolates found in high propionate and low methane-producing dairy cows.

Ruminal methane production is the main sink for metabolic hydrogen generated during rumen fermentation, and is a major contributor to greenhouse gas (GHG) emission. Individual ruminants exhibit varying methane production efficiency; therefore, understanding the microbial characteristics of low-methane-emitting animals could offer opportunities for mitigating enteric methane. Here, we investigated the association between rumen fermentation and rumen microbiota, focusing on methane production, and elucidated the physiological characteristics of bacteria found in low methane-producing cows. Thirteen Holstein cows in the late lactation stage were fed a corn silage-based total mixed ration (TMR), and feed digestion, milk production, rumen fermentation products, methane production, and rumen microbial composition were examined. Cows were classified into two ruminal fermentation groups using Principal component analysis: low and high methane-producing cows (36.9 vs. 43.2 L/DMI digested) with different ruminal short chain fatty acid ratio [(C2+C4)/C3] (3.54 vs. 5.03) and dry matter (DM) digestibility (67.7% vs. 65.3%). However, there were no significant differences in dry matter intake (DMI) and milk production between both groups. Additionally, there were differences in the abundance of OTUs assigned to uncultured Prevotella sp., Succinivibrio, and other 12 bacterial phylotypes between both groups. Specifically, a previously uncultured novel Prevotella sp. with lactate-producing phenotype was detected, with higher abundance in low methane-producing cows. These findings provide evidence that Prevotella may be associated with low methane and high propionate production. However, further research is required to improve the understanding of microbial relationships and metabolic processes involved in the mitigation of enteric methane.

- Invited Review - The role of rumen microbiota in enteric methane mitigation for sustainable ruminant production.

Ruminal methane production functions as the main sink for metabolic hydrogen generated through rumen fermentation and is recognized as a considerable source of greenhouse gas emissions. Methane production is a complex trait affected by dry matter intake, feed composition, rumen microbiota and their fermentation, lactation stage, host genetics, and environmental factors. Various mitigation approaches have been proposed. Because individual ruminants exhibit different methane conversion efficiencies, the microbial characteristics of low-methane-emitting animals can be essential for successful rumen manipulation and environment-friendly methane mitigation. Several bacterial species, including Sharpea, uncharacterized Succinivibrionaceae, and certain Prevotella phylotypes have been listed as key players in low-methane-emitting sheep and cows. The functional characteristics of the unclassified bacteria remain unclear, as they are yet to be cultured. Here, we review ruminal methane production and mitigation strategies, focusing on rumen fermentation and the functional role of rumen microbiota, and describe the phylogenetic and physiological characteristics of a novel Prevotella species recently isolated from low methane-emitting and high propionate-producing cows. This review may help to provide a better understanding of the ruminal digestion process and rumen function to identify holistic and environmentally friendly methane mitigation approaches for sustainable ruminant production.

Effect of rumen microbiota transfaunation on the growth, rumen fermentation, and microbial community of early separated Japanese Black cattle.

This study aimed to investigate the effect of rumen microbiota transfaunation on the growth, rumen fermentation, and the microbial community of Japanese Black cattle that were separated early from their dams. Here, 24 calves were separated from their dams immediately after calving, 12 of which were transfaunated via inoculation with rumen fluid from adult cattle at the age of 2 months while the remaining 12 were kept unfaunated (not-inoculated). Feed efficiency monitoring was performed during 7-10 months of age. Body weight and feed intake were not significantly different between the transfaunated and unfaunated cattle. Transfaunation increased the relative levels of acetate and butyrate but decreased those of propionate, which increased the non-glucogenic/glucogenic short-chain fatty acid ratio. Microbial 16S, 18S, and ITS ribosomal RNA gene amplicon analysis showed that rumen microbial diversity and composition differed between transfaunated and unfaunated cattle; transfaunation increased the abundance of acetate- and butyrate-producing bacteria, and decreased the abundance of bacterial genera associated with propionate production. Transfaunation also increased the abundance of Methanomassiliicoccaceae_group10 (1.94% vs. 0.05%) and Neocallimastix (27.1% vs. 6.8%) but decreased that of Methanomicrobium (<0.01% vs. 0.06%). Our findings indicate that rumen microbiota transfaunation shifts rumen fermentation toward acetate and butyrate production through a change in the rumen microbial composition in Japanese Black cattle.

Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6| |mM) in 48 h, and 1,230.1| |mL L-1 of CO2 and 523.5| |mL L-1 of CH4 were produced. The major isozymes identified by zymograms during the first 12| |h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana-lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Rumen microbial composition associated with the non-glucogenic to glucogenic short-chain fatty acids ratio in Holstein cows.

This study aimed to determine the physiological features and rumen microbial composition associated with the non-glucogenic-to-glucogenic short-chain fatty acids ratio (NGR). Holstein cows were housed in a free-stall barn with an automatic milking system and fed a partially mixed ration. Physiological and microbial analyses were performed on 66 datasets collected from 66 cows (50-250 days in milk). NGR was positively correlated with ruminal pH, relative abundances of protozoa and fungi, methane conversion factor, methane intensity, plasma lipids, parity, and milk fat, and negatively correlated with total short-chain fatty acids. To highlight the differences in bacterial and archaeal compositions between NGRs, low-NGR cows (N = 22) were compared with medium-NGR (N = 22) and high-NGR (N = 22) cows. The low-NGR group was characterized by a lower abundance of Methanobrevibacter and a higher abundance of operational taxonomic units belonging to the lactate-producing, such as Intestinibaculum, Kandleria, and Dialister, and the succinate-producing Prevotella. Our findings indicate that NGR affects the methane conversion factor, methane intensity, and blood and milk compositions. Low NGR is associated with a higher abundance of lactate- and succinate-producing bacteria and lower abundances of protozoa, fungi, and Methanobrevibacter.

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid.

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community's structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions.

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema, Succinivibrio, Anaeroplasma, Succiniclasticum, Ruminococcus, and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.

Exploration of microbial communities contributing to effective methane production from scum under anaerobic digestion.

Scum is formed by the adsorption of long-chain fatty acids (LCFAs) onto biomass surface in anaerobic digestion of oily substrates. Since scum is a recalcitrant substrate to be digested, it is disposed via landfilling or incineration, which results in biomass washout and a decrease in methane yield. The microbes contributing to scum degradation are unclear. This study aimed to investigate the cardinal microorganisms in anaerobic scum digestion. We pre-incubated a sludge with scum to enrich scum-degrading microbes. Using this sludge, a 1.3-times higher methane conversion rate (73%) and a faster LCFA degradation compared with control sludge were attained. Then, we analyzed the cardinal scum-degrading microbes in this pre-incubated sludge by changing the initial scum-loading rates. Increased 16S rRNA copy numbers for the syntrophic fatty-acid degrader Syntrophomonas and hydrogenotrophic methanogens were observed in scum high-loaded samples. 16S rRNA amplicon sequencing indicated that Syntrophomonas was the most abundant genus in all the samples. The amino-acid degrader Aminobacterium and hydrolytic genera such as Defluviitoga and Sporanaerobacter became more dominant as the scum-loading rate increased. Moreover, phylogenic analysis on Syntrophomonas revealed that Syntrophomonas palmitatica, which is capable of degrading LCFAs, related species became more dominant as the scum-loading rate increased. These results indicate that a variety of microorganisms that degrade LCFAs, proteins, and sugars are involved in effective scum degradation.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota.

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

Pretreatment of Lignocellulosic Biomass with Cattle Rumen Fluid for Methane Production: Fate of Added Rumen Microbes and Indigenous Microbes of Methane Seed Sludge.

The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor-1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor-1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.

Preservation of rumen fluid for the pretreatment of waste paper to improve methane production.

It is necessary to preserve rumen fluid for transport from slaughterhouses to the pretreatment facilities for use in treating lignocellulosic biomass. In this study, we investigated how the preservation of rumen fluid at various temperatures affects its use in hydrolysis of waste paper. Rumen fluid was preserved anaerobically at 4, 20, and 35 °C for 7 days. The number of protozoa and fibrolytic enzyme activity after preservation at 4 °C were significantly higher than that after preservation at either 20 or 35 °C. Waste paper was subsequently treated with preserved rumen fluid at 37 °C for 48 h. Preservation at 20 °C remarkedly decreased the hydrolysis of waste paper. Xylanase activity in rumen fluid preserved at 35 °C increased during the treatment, which enhanced the solubilization of waste paper as comparable to the control and preservation at 4 °C. Pretreatment of waste paper with rumen fluid preserved at 4 °C showed that the fluid retained high fibrolytic activity, and reduced the loss of organic carbon as substrate for methanogens. Our results suggest that preservation of rumen fluid at 4 °C is most suitable for efficient pretreatment and methane fermentation of waste paper.

Pretreatment with rumen fluid improves methane production in the anaerobic digestion of paper sludge.

Because paper sludge discharged from the waste paper recycling process contains high levels of lignin and ash, it is not hydrolyzed effectively during anaerobic digestion. In this study, we investigated the effects of pretreatment with rumen fluid on paper sludge and on the methane fermentation process. Paper sludge was pretreated with rumen fluid at 37 °C for 6 h. Following pretreatment, 4.5% of the total solids in paper sludge were degraded and converted, and the dissolved chemical oxygen demand and volatile fatty acid concentration increased. Batch methane fermentation was conducted at 37 °C for 20 days. During methane fermentation, the degradation and hydrolysis of paper sludge were enhanced by pretreatment with rumen fluid. The amounts of total methane production from pretreated paper sludge (excluding methane generated from rumen fluid), rumen fluid and untreated paper sludge were 650.4, 819.9 and 190.8 ml, respectively. The volume of methane gas produced from pretreated paper sludge was 3.4 times larger than that from untreated paper sludge. These results indicate that pretreatment with rumen fluid enhances methane production from paper sludge.