Total Methanogen Population Research Articles

Dynamics of the ruminal microbial ecosystem, and inhibition of methanogenesis and propiogenesis in response to nitrate feeding to Holstein calves.

It is known that nitrate inhibits ruminal methanogenesis, mainly through competition with hydrogenotrophic methanogens for available hydrogen (H2) and also through toxic effects on the methanogens. However, there is limited knowledge about its effects on the others members of ruminal microbiota and their metabolites. In this study, we investigated the effects of dietary nitrate inclusion on enteric methane (CH4) emission, temporal changes in ruminal microbiota, and fermentation in Holstein calves. Eighteen animals were maintained in individual pens for 45 d. Animals were randomly allocated to either a control (CTR) or nitrate (NIT, containing 15 g of calcium nitrate/kg dry matter) diets. Methane emissions were estimated using the sulfur hexafluoride (SF6) tracer method. Ruminal microbiota changes and ruminal fermentation were evaluated at 0, 4, and 8 h post-feeding. In this study, feed dry matter intake (DMI) did not differ between dietary treatments (P > 0.05). Diets containing NIT reduced CH4 emissions by 27% (g/d) and yield by 21% (g/kg DMI) compared to the CTR (P < 0.05). The pH values and total volatile fatty acids (VFA) concentration did not differ between dietary treatments (P > 0.05) but differed with time, and post-feeding (P < 0.05). Increases in the concentrations of ruminal ammonia nitrogen (NH3–N) and acetate were observed, whereas propionate decreased at 4 h post-feeding with the NIT diet (P < 0.05). Feeding the NIT diet reduced the populations of total bacteria, total methanogens, Ruminococcus albus and Ruminococcus flavefaciens, and the abundance of Succiniclasticum, Coprococcus, Treponema, Shuttlewortia, Succinivibrio, Sharpea, Pseudobutyrivibrio, and Selenomona (P < 0.05); whereas, the population of total fungi, protozoa, Fibrobacter succinogenes, Atopobium and Erysipelotrichaceae L7A_E11 increased (P < 0.05). In conclusion, feeding nitrate reduces enteric CH4 emissions and the methanogens population, whereas it decreases the propionate concentration and the abundance of bacteria involved in the succinate and acrylate pathways. Despite the altered fermentation profile and ruminal microbiota, DMI was not influenced by dietary nitrate. These findings suggest that nitrate has a predominantly direct effect on the reduction of methanogenesis and propionate synthesis.

Association between fecal methanogen species with methane production and grazed forage intake of beef heifers classified for residual feed intake under drylot conditions

Reduction in greenhouse gas emission from beef production is essential to the survival of the beef industry from environmental and social-economic perspectives. There are different systems available to measure methane from animals, but they are expensive, not easily accessible, and not suitable for large-scale methane measurements on the farm. Therefore exploring indicator traits, which are easy to measure, cost-effective, and suitable for large-scale measurement, are recommended. The objectives of this study were to examine the diversity of fecal methanogen profile among efficient and inefficient beef heifers on pasture and investigate methanogen profile as a possible proxy to predict methane emission in beef cattle consuming a forage diet. Forty pregnant (1st trimester) heifers previously classified for postweaning residual feed intake adjusted for off-test back fat (RFIfat; 20 high and 20 low) were included in this study. To determine individual pasture grazing intake, heifers were dosed with 1 kg of C32 labeled pellets once per day from Day 0 to Day 12, and fecal samples were collected twice daily from Day 8 to Day 15. Fecal samples from Days 8, 10, and 12 were analyzed for their methanogen profile. Animals were monitored individually for methane and carbon dioxide production using a GreenFeed Emissions Monitoring system. Total methanogen population and methanogenic community diversity of fecal samples were not different (P > 0.1) between low and high RFIfat groups, as measured by quantitative PCR and α- and β-diversity indices. However, both groups had a different methanogen profile; the relative abundance of Methanobrevibacter wolinii and relatives were higher (P < 0.002), while that of Methanosphaera species ISO3-F5 was lower (P < 0.01) in low RFIfat cattle compared to the high RFIfat group. We also demonstrated that fecal methanogen profiles may be a useful proxy in predicting daily methane and carbon dioxide emissions with an adjusted R2 of 0.53 and 0.33, respectively, for low RFIfat heifers and 0.46 and 0.57, respectively, for the high RFIfat group.

Effects of Raw and Fermented Rapeseed Cake on Growth Performance, Methane Production, and Breast Meat Fatty Acid Composition in Broiler Chickens.

Simple SummaryThe addition of raw rapeseed cake (RRC) to broiler feed may avoid the negative effects of soybean meal (SBM), including its genetically modified origin, environmental impact, and nonprospective flexible price. Processing RRC through fermentation has also been shown to be beneficial: adding fermented rapeseed cake (FRC) to the broiler diet is a nutritional approach that addresses both the environmental issue of broiler enteric methane emission and the economic issues of soybean meal. The aim of this study was to assess the effects of partial replacement of soybean meal by a 15% addition of RRC or FRC to the diets of broiler chickens on methane emission, nitrogen retention, ether extract digestibility, growth performance, and breast muscle fatty acid composition. The FRC did not show any unfavorable effects on growth performance or nutrient utilization, and it improved the fatty acid profile of the breast muscle; additionally, both methanogens and methane emission from the fermentation of cecal contents were significantly limited. In conclusion, FRC as a partial substituent may be a valuable alternative to soybean meal in broiler chicken nutrition.The study was conducted to evaluate the effects of partial replacement of soybean meal (SBM) by 15% raw or fermented rapeseed cake (RRC or FRC) to broilers’ diets on growth performance, nutrient utilization, methane emission, and breast muscle fatty acid (FA) composition. A total of 420 one-day-old female Ros 308 broilers were used in three independent experiments (300 birds in the first experiment and 60 in the second and third experiments). In each trial, three treatments were set up: a control group (without rapeseed), and diets replaced soybean meal with 15% addition of RRC or FRC. Birds fed the FRC diet experienced no effect (p > 0.05) on performance or nutrients utilization. Methane emission and total methanogen population in the ceca was decreased (p < 0.05) with the FRC diet. The concentrations of n-3 and n-6 FAs in the breast tissue of fourteen-day-old birds were not affected (p > 0.05) by FRC. However, the n-6/n-3 ratio in the breast muscle of 28-day-old birds was reduced (p < 0.001). In conclusion, the replacement of SBM by FRC in the broiler diets did not show any unfavorable effects on performance or nutrient utilization. Furthermore, the breast meat FA profile was improved, methanogen counts significantly decreased, and methane emission was limited.

Effects of dietary replacement of soybean meal with dried distillers grains with solubles on the microbiota occupying different ecological niches in the rumen of growing Hu lambs

BackgroundDiet has a profound impact on the rumen microbiota, and the impact can vary among the different rumen ecological niches (REN). This study investigated the effects of dietary replacement of soybean meal (SBM) with dried distillers grains with solubles (DDGS) on the rumen microbiota occupying different REN of growing Hu lambs. After a 9-week feeding trial, 6 lambs from each dietary treatment (SBM vs. DDGS-based diets) were slaughtered for sample collection. The microbiota of the rumen solid, liquid, and epithelium fractions was examined using amplicon sequencing analysis of bacterial 16S rRNA gene, functional prediction, and qPCR.ResultsNo interaction of dietary protein source (PS) and REN were detected for virtually all the measurements made in this study. The DDGS substitution resulted in very limited influence on bacterial community structure. However, the metabolic pathways predicted from 16S rRNA gene sequences varied greatly between SBM- and DDGS-based diets. The populations of rumen total bacteria, fungi, sulfate-reducing bacteria (SRB), and methanogens were not influenced by DDGS substitution, but the population of protozoa was reduced. The bacterial communities in rumen solid (RS) and liquid (RL) were similar in taxonomic composition but were different in relative abundance of some taxa. In contrast, the bacterial composition and relative abundance of rumen epithelium (RE) were greatly distinct from those of the RS and the RL. In alignment with the bacterial relative abundance, the metabolic pathways predicted from 16S rRNA genes also varied greatly among the different REN. The populations of total bacteria, protozoa, and methanogens attached to the RE were smaller than those in the RS and RL, and the fungal population on the rumen epithelium was smaller than that in the RS but similar to that in the RL. On the contrary, the SRB population on the RE was greater than that in the RS and RL.ConclusionsSubstitution of SBM with DDGS had greater impact to the protozoa than to the other microbes, and the microbial community structure and functions at different REN are distinct and niche-adapted.

PSIX-1 Associative effect of wet-distillers’ grains plus solubles and tannin-rich peanut skin supplementation on in vitro rumen fermentation, greenhouse gas emissions, and microbiome changes

Abstract The role of tannin-rich peanut skin (PS) and associative effects of different levels of wet distillers’ grains plus solubles (WDGS) on ruminal fermentation, microbial changes, and mitigation of greenhouse gas (GHG) and other emissions in bovine rumen fluid were investigated. All gases were collected using an Ankom in vitro system for methane (CH4), nitrous oxide (N2O), and hydrogen sulfide (H2S) analyses. Fifteen % ground PS against 0, 10, 20, 30, and 40 % DM of WDGS were used. RT-qPCR were conducted to determine microbial diversity. In the absence of PS, total CH4 and H2S, or CH4 and H2S productions per gram of DM substrate, were linearly increased (P &amp;lt; 0.05) with increasing WDGS. However, in the presence of PS, those trends were reversed and CH4 and H2S productions were decreased (P &amp;lt; 0.05), suggesting that a diet with 15% PS and supplementation of 10 and 20% WDGS were able to reduce CH4 and H2S emissions by 12 and 33%, respectively. In the presence of PS, rumen fermentation rate (as a measured by VFA) and acetate/propionate (A/P) ratio was decreased with increasing WDGS, with PS x WDGS interactions (P &amp;lt; 0.01). In the presence of PS, there was a decreased (P &amp;lt; 0.05) the average population of Bacteroidetes, total methanogens, Methanobrevibacter sp. AbM4, and total protozoa populations at 40% WDGS, with PS x WDGS interactions (P &amp;lt; 0.01). The population of total methanogens (R2 = 0.57; P &amp;lt; 0.01), Firmicutes populations (R2= 0.46: P &amp;lt; 0.05), and F/B ratio (R2 = 0.46; P &amp;lt; 0.03) were strongly correlated with ruminal methane gas production. Therefore, associative effect of tannin-rich PS and WDGS suppressed methanogenesis pathways directly across their antimethanogenic activity and secondarily throughout their modification of protozoa population.

Effects of soybean oil and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls

ABSTRACT The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.

Associative effects of wet distiller's grains plus solubles and tannin-rich peanut skin supplementation on in vitro rumen fermentation, greenhouse gas emissions, and microbial changes1.

Two sets of in vitro rumen fermentation experiments were conducted to determine effects of diets that included wet distiller's grains plus solubles (WDGS) and tannin-rich peanut skin (PS) on the in vitro digestibility, greenhouse gas (GHG) and other gas emissions, fermentation rate, and microbial changes. The objectives were to assess associative effects of various levels of PS or WDGS on the in vitro digestibility, GHG and other gas emissions, fermentation rate, and microbial changes in the rumen. All gases were collected using an ANKOM Gas Production system for methane (CH4), carbon dioxide (CO2), nitrous oxide (N2O), and hydrogen sulfide (H2S) analyses. Cumulative ruminal gas production was determined using 250 mL ANKOM sampling bottles containing 50 mL of ruminal fluid (pH 5.8), 40 mL of artificial saliva (pH 6.8), and 6 g of mixed diets after a maximum of 24 h of incubation. Fermenters were flushed with CO2 gas and held at 39 °C in a shaking incubator for 24 h. Triplicate quantitative real-time polymerase chain reaction (qPCR) analyses were conducted to determine microbial diversity. When WDGS was supplied in the diet, in the absence of PS, cumulative CH4 production increased (P < 0.05) with 40% WDGS. In the presence of PS, production of CH4 was reduced but the reduction was less at 40% WDGS. In the presence of PS, ruminal lactate, succinate, and acetate/propionate (A/P) ratio tended to be less with a WDGS interaction (P < 0.01). In the presence of PS and with 40% WDGS, average populations of Bacteroidetes, total methanogens, Methanobrevibacter sp. AbM4, and total protozoa were less. The population of total methanogens (R2 = 0.57; P < 0.01), Firmicutes (R2 = 0.46: P < 0.05), and Firmicutes/Bacteroidetes (F/B) ratio (R2 = 0.46; P < 0.03) were strongly correlated with ruminal CH4 production. Therefore, there was an associative effect of tannin-rich PS and WDGS, which suppressed methanogenesis both directly and indirectly by modifying populations of ruminal methanogens.

Effect of different initial low pH conditions on biogas production, composition, and shift in the aceticlastic methanogenic population

Anaerobic digestion (AD) reactors often encounter low pH inhibition during startup and high organic loading periods. The use of a large amount of NaOH in order to raise and buffer the low pH, is reported to be inhibitory to methanogens. In order to address this problem, we acclimatized aceticlastic methanogens to low pH. Methanogens were successfully acclimatized to initial low pH down to 3.5 in a lengthy, five months, acclimatization period. The aceticlastic methanogen, Methanothrix soehngenii which was 96.3% of the total methanogenic population at pH 4.5 and 86.75% at pH 3.5, demonstrated that they were the most tolerant aceticlastic methanogens to low pH. After acclimatization, methane yield at pH 4.5 was comparable to neutral pH. Methanosaeta maintained its dominance over Methanosarcina at an elevated level of acetate (66 mM), and a negative correlation was observed between them. There was a positive correlation between the CH4 content and pH.

Diversity and community of methanogens in the large intestine of finishing pigs

BackgroundMethane emissions from pigs account for 10% of total methane production from livestock in China. Methane emissions not only contribute to global warming, as it has 25 times the global warming potential (GWP) of CO2, but also represent approximately 0.1~3.3% of digestive energy loss. Methanogens also play an important role in maintaining the balance of the gut microbiome. The large intestines are the main habitat for the microbiome in pigs. Thus, to better understand the mechanism of methane production and mitigation, generic-specific and physio-ecological characteristics (including redox potential (Eh), pH and volatile fatty acids (VFAs)) and methanogens in the large intestine of pig were studied in this paper. Thirty DLY finishing pigs with the same diet and feeding conditions were selected for this experiment.ResultA total of 219 clones were examined using the methyl coenzyme reductase subunit A gene (mcrA) and assigned to 43 operational taxonomic units (OTUs) based on a 97% species-level identity criterion. The family Methanobacteriaceae was the dominant methanogen in colonic digesta of finishing pigs, accounting for approximately 70.6% of the identified methanogens, and comprised mainly the genera Methanobrevibacter (57%) and Methanosphaera (14%). The order Methanomassiliicoccales, classified as an uncultured taxonomy, accounted for 15.07%. The methanogenic archaeon WGK1 and unclassified Methanomicrobiales belonging to the order of Methanomicrobiales accounted for 4.57 and 1.37%, respectively. The Eh was negative and within the range − 297.00~423.00 mV and the pH was within the range 5.04~6.97 in the large intestine. The populations of total methanogens and Methanobacteriales were stable in different parts of the large intestine according to real-time PCR.ConclusionThe major methanogen in the large intestine of finishing pigs was Methanobrevibacter. The seventh order Methanomassiliicoccales and species Methanosphaera stadtmanae present in the large intestine of pigs might contribute to the transfer of hydrogen and fewer methane emissions. The redox potential (Eh) was higher in the large intestine of finishing pigs, which had a positive correlation with the population of Methanobacteriale.

Effects of naturally-produced lovastatin on feed digestibility, rumen fermentation, microbiota and methane emissions in goats over a 12-week treatment period.

Twenty male Saanen goats were randomly assigned to four levels of lovastatin supplementation and used to determine the optimal dosage and sustainability of naturally produced lovastatin from fermentation of palm kernel cake (PKC) with Aspergillus terreus on enteric methane (CH4) mitigation. The effects on ruminal microbiota, rumen fermentation, feed digestibility and health of animal were determined over three measuring periods (4-, 8- and 12-weeks) and the accumulation of lovastatin in tissues was determined at the end of the experiment. The diets contained 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated PKC to achieve the target daily intake level of 0 (Control), 2, 4 or 6 mg lovastatin/kg body weight (BW). Enteric CH4 emissions per dry matter intake (DMI), decreased significantly (P<0.05) and equivalent to 11% and 20.4%, respectively, for the 2 and 4 mg/kg BW groups as compared to the Control. No further decrease in CH4 emission thereafter with higher lovastatin supplementation. Lovastatin had no effect on feed digestibility and minor effect on rumen microbiota, and specifically did not reduce the populations of total methanogens and Methanobacteriales (responsible for CH4 production). Similarly, lovastatin had little effect on rumen fermentation characteristics except that the proportion of propionate increased, which led to a decreasing trend (P<0.08) in acetic: propionate ratio with increasing dosage of lovastatin. This suggests a shift in rumen fermentation pathway to favor propionate production which serves as H+ sink, partly explaining the observed CH4 reduction. No adverse physiological effects were noted in the animals except that treated PKC (containing lovastatin) was less palatable at the highest inclusion level. Lovastatin residues were detected in tissues of goats fed 6 mg lovastatin/kg BW at between 0.01 to 0.03 μg/g, which are very low.

Aspergillus terreus treated rice straw suppresses methane production and enhances feed digestibility in goats.

The objectives of this study were to test the efficacy of producing lovastatin in rice straw treated with Aspergillus terreus in larger laboratory scale following the procedure previously reported and to investigate the effectiveness of the treated rice straw containing lovastatin on methane mitigation in goats. The concentration of lovastatin in the treated rice straw was 0.69 ± 0.05g/kg dry matter (DM) rice straw. Our results showed that supplementation of lovastatin at 4.14mg/kg BW reduced methane production by 32% while improving the DM digestibility by 13% (P < 0.05) in goats fed fermented rice straw compared to those fed untreated rice straw. Populations of total methanogens and Methanobacteriales species were significantly reduced (P < 0.05) while the population of total bacteria and Ruminococcus albus were increased in the treatment group (P < 0.05). Our results demonstrated that lovastatin in the treated rice straw acted specifically on the methanogens by inhibiting the activity of HMG-CoA reductase in the methanogens' cell membrane biosynthesis pathway and thus the growth of rumen methanogens as previously reported. This study provides a simple yet practical approach to mitigate enteric methane production particularly in the developing countries which depend heavily on the use of agro-biomass such as rice straw to feed their ruminant animals.

Effects of dietary Greek oregano (Origanum vulgare ssp. hirtum) supplementation on rumen fermentation, enzyme profile and microbial communities in goats.

This study was conducted to examine in vivo long-term effects of dietary dried oregano (Origanum vulgare ssp. hirtum) whole plant on rumen fermentation, enzyme profile and microbial communities. For this purpose, eight healthy, adult, non-lactating Alpine goats were kept in tie stalls equipped for individual feeding and randomly divided into two homogeneous groups: one fed 0.6kg of a concentrate mixture and 0.6kg of wheat straw without any supplementation and served as control group (CON) while the other group (OR) fed the same diet of CON but supplemented with 20g of dried oregano plants (OPs) to provide daily dosage of 1ml of essential oil (EO) per animal. The experimental period lasted 69days and individual rumen fluid samples were obtained every 2weeks at 0 and 4hr after feeding. The results showed that dietary supplementation with OPs increased the protease activity (p<.001) and ammonia concentration (p<.05) in the rumen. Among the studied microbial populations, Peptostreptococcus anaerobius (p=.028) and Clostridium sticklandii (p<.001) were found to be the most sensitive to oregano at the current dosage. Furthermore, the total methanogen population significantly decreased (p<.05). It is concluded that a long-term dietary administration of OPs can suppress specific rumen micro-organisms and modify rumen fermentation favourably at least by means of suppressing methanogens.

The influence of plant polyphenols from oil palm (Elaeis guineensis Jacq.) leaf extract on fermentation characteristics, biohydrogenation of C18 PUFA, and microbial populations in rumen of goats: in vitro study

ABSTRACTOil palm leaf is rich source of secondary metabolites. An in vitro study was conducted to identify the effects of oil palm leaf extract (OPLE) on rumen characteristics of goats by adding 0% (CON), 2.5% (T1), 5% (T2), and 10% (T3)/250 mg DM in 30 mL of buffered rumen fluid for 24 h. Methane gas production (mL/g DM) decreased (P < .05) with increasing levels of OPLE. Acetic to propionic acid ratio was lower (P < .05) in T3 (2.46) compared to the CON (3.12). Real-time PCR analyses indicated that the populations of total methanogens and Methanobacteriaceae in OPLE treatment groups decreased (P < .05) compared to CON, without any significant changes in the population of cellulolytic bacteria. Supplementation of the diet with OPLE significantly (P < .05) decreased the concentration of C18:0 while increased (P < .05) the concentration of C18:1 t-11 and c-9, t-11 CLA after 24 h of incubation.

Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows

This study aimed to investigate the effects of dietary supplementation of different dosages of active dried yeast (ADY) on the fecal methanogenic archaea community of dairy cattle. Twelve multiparous, healthy, mid-lactating Holstein dairy cows (body weight: 584 ± 23.2 kg, milk produced: 26.3 ± 1.22 kg/d) were randomly assigned to one of three treatments (control, ADY2, and ADY4) according to body weight with four replicates per treatment. Cows in the control group were fed conventional rations without ADY supplementation, while cows in the ADY2 and ADY4 group were fed rations supplemented with ADY at 2 or 4 g/d/head. Real-time PCR analysis showed the populations of total methanogens in the feces were significantly decreased (P < 0.05) in the ADY4 group compared with control. High-throughput sequencing technology was applied to examine the differences in methanogenic archaea diversity in the feces of the three treatment groups. A total of 155,609 sequences were recovered (a mean of 12,967 sequences per sample) from the twelve fecal samples, which consisted of a number of operational taxonomic units (OTUs) ranging from 1451 to 1,733, were assigned to two phyla, four classes, five orders, five families and six genera. Bioinformatic analyses illustrated that the natural fecal archaeal community of the control group was predominated by Methanobrevibacter (86.9% of the total sequence reads) and Methanocorpusculum (10.4%), while the relative abundance of the remaining four genera were below 1% with Methanosphaera comprising 0.8%, Thermoplasma composing 0.4%, and the relative abundance of Candidatus Nitrososphaera and Halalkalicoccus being close to zero. At the genus level, the relative abundances of Methanocorpusculum and Thermoplasma were increased (P < 0.05) with increasing dosage of ADY. Conversely, the predominant methanogen genus Methanobrevibacter was decreased with ADY dosage (P < 0.05). Dietary supplementation of ADY had no significant effect (P > 0.05) on the abundances of genera unclassified, Candidatus Nitrososphaera, and Halalkalicoccus. In conclusion, supplementation of ADY to the rations of dairy cattle could alter the population sizes and composition of fecal methanogenic archaea in the feces of dairy cattle. The decrease in Methanobrevibacter happened with a commensurate increase in the genera Methanocorpusculum and Thermoplasma.

Manipulation of Rumen Microbial Fermentation by Polyphenol Rich Solvent Fractions from Papaya Leaf to Reduce Green-House Gas Methane and Biohydrogenation of C18 PUFA

Different solvents (hexane, chloroform, ethyl acetate, butanol, and water) were used to identify the effect of papaya leaf (PL) fractions (PLFs) on ruminal biohydrogenation (BH) and ruminal methanogenesis in an in vitro study. PLFs at a concentration of 0 (control, CON) and 15 mg/250 mg dry matter (DM) were mixed with 30 mL of buffered rumen fluid and were incubated for 24 h. Methane (CH4) production (mL/250 mg DM) was the highest (P < 0.05) for CON (7.65) and lowest for the chloroform fraction (5.41) compared to those of other PLFs at 24 h of incubation. Acetate to propionate ratio was the lowest for PLFs compared to that of CON. Supplementation of the diet with PLFs significantly (P < 0.05) decreased the rate of BH of C18:1n-9 (oleic acid; OA), C18:2n-6 (linoleic acid; LA), and C18:3n-3 (α-linolenic acid; LNA) compared to that of CON after 24 h of incubation. Real time PCR indicated that total protozoa and total methanogen population in PLFs decreased (P < 0.05) compared to those of CON.

Divergence between purified hydrolysable and condensed tannin effects on methane emission, rumen fermentation and microbial population in vitro

This study aimed to investigate the effects of purified hydrolysable (chestnut and sumach) and condensed tannins (mimosa and quebracho) on methane production, rumen fermentation and microbial population structure. The tannins were extracted and purified from their original plant sources, and were characterized for their protein precipitation capacity. The purified tannins were added into 380mg hay:concentrate substrate (70:30w/w) at three different concentrations (0.5, 0.75 and 1.0mg/ml). In vitro incubation was carried out for 24h at 39°C in 100ml calibrated glass syringe containing 30ml of medium (10ml rumen liquor and 20ml double strength buffer). Parameters measured after the incubation were gas production, methane concentration, short chain fatty acids (SCFA: C2, C3, C4, isoC4, C5, isoC5, total SCFA and ratio of C2/C3), in vitro organic matter digestibility (IVOMD) and microbial population structure. The experiment was performed in three runs, represented by two incubation units per run. Results revealed that the protein precipitation capacity of chestnut and sumach tannins was greater than that of mimosa and quebracho tannins. An interaction between different tannins and doses existed with regard to methane concentration (P≤0.05). All the tannins decreased methane concentration either linearly or quadratically, but their magnitudes were different; the magnitude of decrease was greater for the hydrolysable tannins than the condensed ones, and correlated with their protein precipitation capacity. Increasing levels of all tannins decreased IVOMD by following a quadratic pattern (P≤0.05) and there was a tendency that the condensed tannins decreased IVOMD more than the hydrolysable tannins (P≤0.1). All the purified hydrolysable and condensed tannins decreased total methanogen population (P≤0.05) than that of control when added at 1.0mg/ml; the decrease ranged from 22.3 to 36.7% from control. Additions of purified tannins at all levels generally decreased Fibrobacter succinogenes population (P≤0.05). Sumach tannins at all addition levels decreased the population of Ruminococcus flavefaciens (P≤0.05), and the magnitude of decrease was much greater than those of other tannins. It is concluded that hydrolysable tannins had a greater effect in reducing methane emission with less adverse effect on digestibility than those of condensed tannins.

&lt;b&gt;Quantitative analysis of rumen microbial populations by qPCR in heifers fed on &lt;i&gt;Leucaena leucocephala&lt;/i&gt; in the Colombian Tropical Dry Forest

Rumen fermentation and methanogenesis are vital metabolic processes in cattle and are carried out by microbial populations that are affected by dietary factors such as secondary metabolites, nutritional composition and degradability. The aim of this study was to monitor populations of total bacteria, total methanogens and Butirivibrio fibrisolvens in the rumen of Lucerne heifers fed on diets typical of intensive silvopastoral systems (ISS) or of a traditional (control) system. Rumen contents (RC) were collected orally from eight heifers consuming 100% Cynodon plectostachyus (control) and 76% C. plectostachyus + 24% Leucaena leucocephala (ISS) following a crossover design and DNA was extracted and quantified by quantitative PCR. Populations (Log10 (ng g -1 RC)) were 5.6 and 5.8 for total bacteria (p = 0.5343), 3.6 and 3.5 for B. fibrisolvens (p = 0.4742) and 5.0 and 5.3 for total methanogens (p = 0.2661) respectively in control and ISS diets. However, when measured in a separate parallel study, enteric methane emissions (g kg -1 of fermented dry matter) were significantly reduced with the inclusion of L. leucocephala. This fact indicated the importance of investigating the structure, function and interactions of populations beyond quantitative analysis to determine how diet affects rumen microbial populations and their function.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters

Anaerobic digestion (AD) is gaining increasing attention due to the ability to covert organic pollutants into energy-rich biogas and, accordingly, growing interest is paid to the microbial ecology of AD systems. Despite extensive efforts, AD microbial ecology is still limitedly understood, especially due to the lack of quantitative information on the structures and dynamics of AD microbial communities. Such knowledge gap is particularly pronounced in sewage sludge AD processes although treating sewage sludge is among the major practical applications of AD. Therefore, we examined the microbial communities in three full-scale sewage sludge digesters using qualitative and quantitative molecular techniques in combination: denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR). Eight out of eleven bacterial sequences retrieved from the DGGE analysis were not affiliated to any known species while all eleven archaeal sequences were assigned to known methanogen species. Quantitative real-time PCR analysis revealed that, based on the 16S rRNA gene abundance, the hydrogenotrophic order Methanomicrobiales is the most dominant methanogen group (> 94% of the total methanogen population) in all digesters. This corresponds well to the prevailing occurrence of the DGGE bands related to Methanolinea and Methanospirillum, both belonging to the order Methanomicrobiales, in all sludge samples. It is therefore suggested that hydrogenotrophic methanogens, especially Methanomicrobiales strains, are likely the major players responsible for biogas production in the digesters studied. Our observation is contrary to the conventional understanding that aceticlastic methanogens generally dominate methanogen communities in stable AD environments, suggesting the need for further studies on the dominance relationship in various AD systems.

Individuality of ruminal methanogen/protozoa populations in beef cattle fed diets containing dried distillers' grain with solubles

This study investigated the variability of ruminal methanogen/protozoa among beef heifers (n = 4) fed corn- or wheat-dried distillers' grain with solubles (DDGS). Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis and quantitative real-time PCR were used to investigate variation in ruminal methanogenic communities, while protozoal communities were analyzed using light microscopy. Observed methanogen profiles were not affected by DDGS source, but were distinctive within individuals (Global R=0.87, P < 0.01). Populations of total methanogens, total protozoa, Methanobrevibacter sp. AbM4, Entodinium, and Polyplastron varied among heifers and diets, while densities of total Methanosphaera stadtmanae, Isotricha, and Ostracodinium only differed among individual hosts. Responses in the ruminal methanogenic community to DDGS source were host dependent, suggesting that host biology should not be ignored if dietary strategies are used to alter ruminal methanogens to lower enteric methane emissions.