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

Background Bacteria involved in ruminal formation oftrans-10 intermediates are unclear. Therefore, this study aimed at identifying rumen bacteria that producetrans-10 intermediates from 18-carbon unsaturated fatty acids. Results Pure cultures of 28 rumen bacterial species were incubated individually in the presence of 40 mu g/mL 18:3n-3, 18:2n-6 ortrans-11 18:1 under control or lactate-enriched (200 mM Na lactate) conditions for 24 h. Of the 28 strains,Cutibacterium acnes(formerlyPropionibacterium acnes) was the only bacterium found to producetrans-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 thetrans-11 totrans-10 shift, different biomass ratios ofButyrivibrio fibrisolvens(as atrans-11 producer) andC. acneswere incubated in different growth media (control, low pH and 22:6n-3 enriched media) containing 40 mu g/mL 18:2n-6. Under control conditions, atrans-10 shift, defined in the current study astrans-10/trans-11 >= 0.9, occurred when the biomass ofC. acnesrepresented between 90 and 98% of the inoculum. A low pH or addition of 22:6n-3 inhibitedcis-9,trans-11 CLA andtrans-10,cis-12 CLA formation byB. fibrisolvensandC. acnes, respectively, wherebyC. acnesseemed to be more tolerant. This resulted in a decreased biomass ofC. acnesrequired at inoculation to induce atrans-10 shift to 50% (low pH) and 90% (22:6n-3 addition). Conclusions Among the bacterial species studied,C. acneswas the only bacterium that have the metabolic ability to producetrans-10 intermediates from 18:3n-3 and 18:2n-6. Nevertheless, this experiment revealed that it is unlikely thatC. acnesis the only or predominant species involved in thetrans-11 totrans-10 shift in vivo

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 mu g/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 mu g/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 mu g/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

Investigation of rumen methanogens in New Zealand livestock : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand.

Methane emitted by farmed ruminants contributes 30.3% to New Zealand’s anthropogenic greenhouse gas inventory. Methanogens living in the rumen produce methane from H2 and CO2 as a byproduct of feed fermentation. The use of vaccines and small molecule inhibitors against the methanogens are promising methods to reduce methane emissions from extensively-grazed ruminants in New Zealand. Knowledge of the methanogens present in New Zealand ruminants is an important first step for successful vaccine and inhibitor development to target all methanogens. In this study, the methanogen diversity of farmed ruminants (sheep [Ovis aries], cattle [Bos taurus] and red deer [Cervus elaphus]) was investigated using molecular ecological techniques. Ruminants fed different diets had largely similar rumen methanogen communities. The major methanogen groups identified were from the Methanobrevibacter ruminantium clade (Mbb. ruminantium and closely-related species), Methanobrevibacter gottschalkii clade (Mbb. gottschalkii and closely-related species), Methanosphaera spp., and the putative methanogens belonging to the group designated Rumen Cluster C. A total of 37.5 - 57% of 16S rRNA genes in the rumen of a group of cows originated from members of Rumen Cluster C. Chloroform treatment of cows increased the abundance of Rumen Cluster C to 82% - 93% of archaeal 16S rRNA genes. In parallel, a total of 22% of mcrA genes belonged to an unassigned group of archaea, and chloroform treatment increased the unassigned group of archaea to 92% of all mcrA genes. This suggested that Rumen Cluster C archaea contain the gene mcrA. No members of the Rumen Cluster C group have previously been cultured, and currently there is no reported rumen isolate of Methanosphaera spp. A strain of Methanosphaera sp. was isolated from a sheep rumen and initial characterization suggests that this may be a new species. Three enrichment cultures were obtained containing members of Rumen Cluster C as the only archaea. Initial studies of these enrichment cultures showed that these three isolates were from three different sub-groups of Rumen Cluster C and that they produced methane. The investigation of methanogen diversity in New Zealand farmed ruminants and isolation of previously uncultured rumen methanogens reported here in this thesis will significantly aid the development of methane reduction strategies for farmed ruminants in New Zealand

The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

Concerns about the environmental effect and the economic burden of methane (CH4) emissions from ruminants are driving the search for ways to mitigate rumen methanogenesis. The use of direct-fed microbials (DFM) is one possible option to decrease CH4 emission from ruminants. Direct-fed microbials are already used in ruminants mainly to increase productivity and to improve health, and are readily accepted by producers and consumers alike. However, studies on the use of DFM as rumen CH4 mitigants are scarce. A few studies using Saccharomyces cerevisiae have shown a CH4-decreasing effect but, to date, there has not been a systematic exploration of DFM as modulators of rumen methanogenesis. In this review, we explored biochemical pathways competing with methanogenesis that, potentially, could be modulated by the use of DFM. Pathways involving the redirection of H2 away from methanogenesis and pathways producing less H2 during feed fermentation are the preferred options. Propionate formation is an example of the latter option that in addition to decrease CH4 formation increases the retention of energy from the diet. Homoacetogenesis is a pathway using H2 to produce acetate, however up to now no acetogen has been shown to efficiently compete with methanogens in the rumen. Nitrate and sulphate reduction are pathways competing with methanogenesis, but the availability of these substances in the rumen is limited. Although there were studies using nitrate and sulphate as chemical additives, use of DFM for improving these processes and decrease the accumulation of toxic metabolites needs to be explored more. There are some other pathways such as methanotrophy and capnophily or modes of action such as inhibition of methanogens that theoretically could be provided by DFM and affect methanogenesis. We conclude that DFM is a promising alternative for rumen methane mitigation that should be further explored for their practical usage

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

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

Abstract 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

Subacute ruminal acidosis phenotypes in periparturient dairy cows differ in ruminal and salivary bacteria and in the in vitro fermentative activity of their ruminal microbiota

Both ruminal microbial structure and functionality might play a role in inter-individual variation in susceptibility for subacute rumen acidosis (SARA) observed in dairy cows. The aims of this study were to determine whether differences between cows with distinct SARA susceptibility were reflected in distinct (1) ruminal microbial communities, (2) salivary bacterial communities, and (3) fermentative capacity of ruminal microbiota assessed in vitro. To test this hypothesis, rumen samples were collected via an esophageal tube on 21 d postpartum from 38 multiparous Holstein cows, which were classified into 4 groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6.0. During the 21 d postpartum, all cows within a group fulfilled following criteria: susceptible (S, n = 10; mean or median >= 180 min/d), moderately susceptible (MS, n = 7; 60 min/d < mean time of pH below 6 < 180 min/d, and median time of pH below 6 <180 min/d), moderately unsusceptible (MU, n = 11; 10 min/d < mean < 60 min/d, and median time of pH below 6 <= 30 min/d), or unsusceptible (U, n = 10; median = 0 min/d, and mean <10 min/d). Groups did not differ in total daily dry matter intake nor in total, roughage, or concentrate intake during daily 6-h time intervals. Rumen bacterial alpha-diversity did not differ among groups, but beta-diversity varied and bacterial 16S rRNA gene copy numbers were lower in S compared with U cows. The relative abundance of genera Streptococcus, Sharpea, Prevotellaceae_YAB2003, Succinivibrionaceae_UCG-001, Ruminococcus, and Ruminococcaceae_UCG-001 were higher in S compared with U cows. In contrast, Lachnospiraceae_ND3007 and Oscillospiraceae_V9D2013 were more abundant in U cows. Although pH-associated, inter-animal differences were also observed in the salivary bacteria, common differences in ruminal and salivary bacterial genera were limited. The functionality of the rumen microbiota was evaluated in vitro through exposure of the microbial inoculum of S and U cows to an anaerobic buffer at pH 5.8 and 6.8, in the presence of sterile supernatant of their own and of dry cows' rumen fluid (2 x 2 design). Generally, the S inoculum produced more volatile fatty acids, except at low pH with dry cows' supernatant, where volatile fatty acid production was completely impaired and lactate accumulation was highest. Compared with the microbes of U cows, microbes of S cows showed less fermentative activity in situations with 2 stress factors (low pH and an unfamiliar environment, i.e., rumen fluid supernatant of dry cows)