Complete genome sequence of Methanosphaera sp. ISO3-F5, a rumen methylotrophic methanogen.

Methanosphaera spp. are methylotrophic methanogenic archaea and members of the order Methanobacteriales with few cultured representatives. Methanosphaera sp. ISO3-F5 was isolated from sheep rumen contents in New Zealand. Here, we report its complete genome, consisting of a large chromosome and a megaplasmid (GenBank accession numbers CP118753 and CP118754, respectively).

Susceptibility of dairy cows to subacute ruminal acidosis is reflected in both prepartum and postpartum bacteria as well as odd- and branched-chain fatty acids in feces.

BACKGROUND: The transition period is a challenging period for high-producing dairy cattle. Cows in early lactation are considered as a group at risk of subacute ruminal acidosis (SARA). Variability in SARA susceptibility in early lactation is hypothesized to be reflected in fecal characteristics such as fecal pH, dry matter content, volatile and odd- and branched-chain fatty acids (VFA and OBCFA, respectively), as well as fecal microbiota. This was investigated with 38 periparturient dairy cows, which were classified into four groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6. Furthermore, we investigated whether fecal differences were already obvious during a period prior to the SARA risk (prepartum). RESULTS: Variation in reticular pH during a 3-week postpartum period was not associated with differences in fecal pH and VFA concentration. In the postpartum period, the copy number of fecal bacteria and methanogens of unsusceptible (UN) cows was higher than moderately susceptible (MS) or susceptible (SU) cows, while the genera Ruminococcus and Prevotellacea_UCG-001 were proportionally less abundant in UN compared with SU cows. Nevertheless, only a minor reduction was observed in iso-BCFA proportions in fecal fatty acids of SU cows, particularly iso-C15:0 and iso-C16:0, compared with UN cows. Consistent with the bacterial changes postpartum, the lower abundance of Ruminococcus was already observed in the prepartum fecal bacterial communities of UN cows, whereas Lachnospiraceae_UCG-001 was increased. Nevertheless, no differences were observed in the prepartum fecal VFA or OBCFA profiles among the groups. Prepartum fecal bacterial communities of cows were clustered into two distinct clusters with 70% of the SU cows belonging to cluster 1, in which they represented 60% of the animals. CONCLUSIONS: Inter-animal variation in postpartum SARA susceptibility was reflected in post- and prepartum fecal bacterial communities. Differences in prepartum fecal bacterial communities could alert for susceptibility to develop SARA postpartum. Our results generated knowledge on the association between fecal bacteria and SARA development which could be further explored in a prevention strategy.

Performance, Rumen Microbial Community and Immune Status of Goat Kids Fed Leucaena leucocephala Post-weaning as Affected by Prenatal and Early Life Nutritional Interventions.

Leucaena leucocephala represents a local protein source in tropical ruminant diets. However, its full exploitation is impaired by mimosine, unless it is degraded by the rumen microbial community. Recently, the ruminal bacterial communities of newborns were persistently modified through prenatal or postnatal dietary interventions. Such early-life interventions might enhance adaptation of ruminants to Leucaena leucocephala, which was investigated using a 2 × 2 factorial design trial that tested both supplementation of L. leucocephala in the late pregnancy diet of goat does, and supplementation of live yeast to their newborns. The composition of ruminal bacteria, immune status, as well as organic matter digestibility (OMD) and performance of kids were studied during and after the intervention. Ten pregnant goats were divided into two groups: the D+ and D- groups, which either received or did not receive 30 g of L. leucocephala forage meal during the last 7 ± 0.5 weeks of gestation. Twins from each goat were divided into the K+ and K- group (supplemented with or without 0.2 g/d of live yeast from day 3 until weaning at 8 weeks). Rumen samples were collected from 4-, 8-, 14-, and 20-weeks old kids to assess the bacterial community, while immune parameters (white blood cells, immunoglobulin M and G, and chitotriosidase activity) were measured in blood and saliva sampled at 4-, 8-, and 20-weeks. We found a stimulatory effect of the prenatal exposure on the post-weaning dry matter intake of the L. leucocephala supplemented diet, resulting in a higher daily gain and final body weight at 20 weeks in the D+ vs. D- group (406 vs. 370 g DM/d, 85.4 vs. 78.6 g/d, and 15.2 vs. 13.8 kg, respectively). Moreover, Ruminococcus represented a greater proportion of the rumen bacterial community of the D+ vs. D- kids (5.1 vs. 1.6%). Differences in the immune status were relatively small and not thought to be a driving factor of differences in animal performance. Furthermore, postnatal supplementation of live yeast favored maturation of the rumen bacterial community (i.e., greater abundance of Bacteroidetes, in particular Prevotella, and reduced abundance of Firmicutes) and protozoa colonization. Concomitantly, OMD was enhanced post-weaning, suggesting effects of the early-life intervention persisted and could have affected animal performance.

Identifying and exploring biohydrogenating rumen bacteria with emphasis on pathways including trans-10 intermediates.

BACKGROUND: Bacteria involved in ruminal formation of trans-10 intermediates are unclear. Therefore, this study aimed at identifying rumen bacteria that produce trans-10 intermediates from 18-carbon unsaturated fatty acids. RESULTS: Pure cultures of 28 rumen bacterial species were incubated individually in the presence of 40 µg/mL 18:3n-3, 18:2n-6 or trans-11 18:1 under control or lactate-enriched (200 mM Na lactate) conditions for 24 h. Of the 28 strains, Cutibacterium acnes (formerly Propionibacterium acnes) was the only bacterium found to produce trans-10 intermediates from 18:3n-3 and 18:2n-6, irrespective of the growth condition. To further assess the potential importance of this species in the trans-11 to trans-10 shift, different biomass ratios of Butyrivibrio fibrisolvens (as a trans-11 producer) and C. acnes were incubated in different growth media (control, low pH and 22:6n-3 enriched media) containing 40 µg/mL 18:2n-6. Under control conditions, a trans-10 shift, defined in the current study as trans-10/trans-11 ≥ 0.9, occurred when the biomass of C. acnes represented between 90 and 98% of the inoculum. A low pH or addition of 22:6n-3 inhibited cis-9, trans-11 CLA and trans-10, cis-12 CLA formation by B. fibrisolvens and C. acnes, respectively, whereby C. acnes seemed to be more tolerant. This resulted in a decreased biomass of C. acnes required at inoculation to induce a trans-10 shift to 50% (low pH) and 90% (22:6n-3 addition). CONCLUSIONS: Among the bacterial species studied,C. acnes was the only bacterium that have the metabolic ability to produce trans-10 intermediates from 18:3n-3 and 18:2n-6. Nevertheless, this experiment revealed that it is unlikely that C. acnes is the only or predominant species involved in the trans-11 to trans-10 shift in vivo.

Bacterial direct-fed microbials fail to reduce methane emissions in primiparous lactating dairy cows.

Direct-fed microbials (DFM) are considered as a promising technique to improve animal productivity without affecting animal health or harming the environment. The potential of three bacterial DFM to reduce methane (CH4) emissions, modulate ruminal fermentation, milk production and composition of primiparous dairy cows was examined in this study. As previous reports have shown that DFM respond differently to different diets, two contrasting diets were used in this study. Eight lactating primiparous cows were randomly divided into two groups that were fed a corn silage-based, high-starch diet (HSD) or a grass silage-based, high-fiber diet (HFD). Cows in each dietary group were randomly assigned to four treatments in a 4 × 4 Latin square design. The bacterial DFM used were selected for their proven CH4-reducing effect in vitro. Treatments included control (without DFM) and 3 DFM treatments: Propionibacterium freudenreichii 53-W (2.9 × 1010 colony forming units (CFU)/cow per day), Lactobacillus pentosus D31 (3.6 × 1011 CFU/cow per day) and Lactobacillus bulgaricus D1 (4.6 × 1010 CFU/cow per day). Each experimental period included 4 weeks of treatment and 1 week of wash-out, with measures performed in the fourth week of the treatment period. Enteric CH4 emissions were measured during 3 consecutive days using respiration chambers. Rumen samples were collected for ruminal fermentation parameters and quantitative microbial analyses. Milk samples were collected for composition analysis. Body weight of cows were recorded at the end of each treatment period. Irrespective of diet, no mitigating effect of DFM was observed on CH4 emissions in dairy cows. In contrast, Propionibacterium increased CH4 intensity by 27% (g CH4/kg milk) in cows fed HSD. There was no effect of DFM on other fermentation parameters and on bacterial, archaeal and protozoal numbers. Similarly, the effect of DFM on milk fatty acid composition was negligible. Propionibacterium and L. pentosus DFM tended to increase body weight gain with HSD. We conclude that, contrary to the effect previously observed in vitro, bacterial DFM Propionibacterium freudenreichii 53-W, Lactobacillus pentosus D31 and Lactobacillus bulgaricus D1 did not alter ruminal fermentation and failed to reduce CH4 emissions in lactating primiparous cows fed high-starch or high-fiber diets.

Rumen Biohydrogenation and Microbial Community Changes Upon Early Life Supplementation of 22:6n-3 Enriched Microalgae to Goats.

Dietary supplementation of docosahexaenoic acid (DHA)-enriched products inhibits the final step of biohydrogenation in the adult rumen, resulting in the accumulation of 18:1 isomers, particularly of trans(t)-11 18:1. Occasionally, a shift toward the formation of t10 intermediates at the expense of t11 intermediates can be triggered. However, whether similar impact would occur when supplementing DHA-enriched products during pregnancy or early life remains unknown. Therefore, the current in vivo study aimed to investigate the effect of a nutritional intervention with DHA in the early life of goat kids on rumen biohydrogenation and microbial community. Delivery of DHA was achieved by supplementing DHA-enriched microalgae (DHA Gold) either to the maternal diet during pregnancy (prenatal) or to the diet of the young offspring (postnatal). At the age of 12 weeks, rumen fluid was sampled for analysis of long-chain fatty acids and microbial community based on bacterial 16S rRNA amplicon sequencing. Postnatal supplementation with DHA-enriched microalgae inhibited the final biohydrogenation step, as observed in adult animals. This resulted particularly in increased ruminal proportions of t11 18:1 rather than a shift to t10 intermediates, suggesting that both young and adult goats might be less prone to dietary induced shifts toward the formation of t10 intermediates, in comparison with cows. Although Butyrivibrio species have been identified as the most important biohydrogenating bacteria, this genus was more abundant when complete biohydrogenation, i.e. 18:0 formation, was inhibited. Blautia abundance was positively correlated with 18:0 accumulation, whereas Lactobacillus spp. Dialister spp. and Bifidobacterium spp. were more abundant in situations with greater t10 accumulation. Extensive comparisons made between current results and literature data indicate that current associations between biohydrogenation intermediates and rumen bacteria in young goats align with former observations in adult ruminants.

The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales.

Methane emissions from agriculture represent around 9 % of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive.

Biohydrogenation of 22:6n-3 by Butyrivibrio proteoclasticus P18.

BACKGROUND: Rumen microbes metabolize 22:6n-3. However, pathways of 22:6n-3 biohydrogenation and ruminal microbes involved in this process are not known. In this study, we examine the ability of the well-known rumen biohydrogenating bacteria, Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18, to hydrogenate 22:6n-3. RESULTS: Butyrivibrio fibrisolvens D1 failed to hydrogenate 22:6n-3 (0.5 to 32 µg/mL) in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Growth of B. fibrisolvens was delayed at the higher 22:6n-3 concentrations; however, total volatile fatty acid production was not affected. Butyrivibrio proteoclasticus P18 hydrogenated 22:6n-3 in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Biohydrogenation only started when volatile fatty acid production or growth of B. proteoclasticus P18 had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n-3. The amount of 22:6n-3 hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n-3 and 22:0. Formation of neither 22:0 nor 22:6 conjugated fatty acids was observed during 22:6n-3 metabolism. Extensive metabolism was observed at lower initial concentrations of 22:6n-3 (5, 10 and 20 µg/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 µg/mL) inhibited its metabolism. Stearic acid formation (18:0) from 18:2n-6 by B. proteoclasticus P18 was retarded, but not completely inhibited, in the presence of 22:6n-3 and this effect was dependent on 22:6n-3 concentration. CONCLUSIONS: For the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n-3. The gradual appearance of intermediates indicates that biohydrogenation of 22:6n-3 by B. proteoclasticus P18 occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated 22 carbon fatty acids. During the simultaneous presence of 18:2n-6 and 22:6n-3, B. proteoclasticus P18 initiated 22:6n-3 metabolism before converting 18:1 isomers into 18:0.

Complete Genome Sequence of Methanogenic Archaeon ISO4-G1, a Member of the Methanomassiliicoccales, Isolated from a Sheep Rumen.

Methanogenic archaeon ISO4-G1 is a methylotrophic methanogen belonging to the orderMethanomassiliicoccalesthat was isolated from a sheep rumen. Its genome has been sequenced to provide information on the genetic diversity of rumen methanogens in order to develop technologies for ruminant methane mitigation.

Methanogen community structure in the rumens of farmed sheep, cattle and red deer fed different diets.

Development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on knowledge of the methanogens present. We investigated the composition of archaeal communities in the rumens of farmed sheep (Ovis aries), cattle (Bos taurus) and red deer (Cervus elaphus) using denaturing gradient gel electrophoresis (DGGE) to generate fingerprints of archaeal 16S rRNA genes. The total archaeal communities were relatively constant across species and diets, and were less variable and less diverse than bacterial communities. There were diet- and ruminant-species-based differences in archaeal community structure, but the same dominant archaea were present in all rumens. These were members of three coherent clades: species related to Methanobrevibacter ruminantium and Methanobrevibacter olleyae; species related to Methanobrevibacter gottschalkii, Methanobrevibacter thaueri and Methanobrevibacter millerae; and species of the genus Methanosphaera. Members of an archaeal group of unknown physiology, designated rumen cluster C (RCC), were also present. RCC-specific DGGE, clone library analysis and quantitative real-time PCR showed that their 16S rRNA gene sequences were very diverse and made up an average of 26.5% of the total archaea. RCC sequences were not readily detected in the DGGE patterns of total archaeal 16S rRNA genes because no single sequence type was abundant enough to form dominant bands.

Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium.

A new anaerobic medium that mimics the salts composition of rumen fluid was used in conjunction with a dilution method of liquid culture to isolate fermentative bacteria from the rumen of a grass-fed sheep. The aim was to inoculate a large number of culture tubes each with a mean of <1 culturable cell, which should maximize the number of cultures that develop from a single bacterium. This minimizes the effort that has to be put into purifying the resultant cultures. Of 1000 tubes, 139 were growth positive. Of the 93 that were able to be subcultured, 54 (58%) appeared to be pure cultures. The phylogenetic placements of these 54 cultures, together with another 6 cultures obtained from a preliminary study, were determined. Using a criterion of <93% 16S rRNA gene sequence identity to a previously named bacterium as a proxy for defining a new genus, 27 (45%) of the 60 cultures belonged to 14 potentially novel genera. Many of these had 16S rRNA genes that shared >97% sequence identity to genes of uncultured bacteria detected in various gastrointestinal environments. This strategy has therefore allowed us to cultivate many novel rumen bacteria, opening the way to overcoming the lack of cultures of many of the groups detected using cultivation-independent methods.