Modelling thermodynamic feedback on the metabolism of hydrogenotrophic methanogens.

The magnitude of the Gibbs free energy change of the substrate transformation that supports the growth of a microbe is decreased when the concentrations of the substrates are decreased and when the concentrations of the products of metabolism are increased. Microbes require a supply of ATP for cell maintenance and growth, and coupling the transformation of substrates to products with the formation of ATP also decreases the magnitude of the Gibbs free energy change. Here we include these three thermodynamic controllers (substrate and product concentration, and ATP formation) in a model of substrate transformation by hydrogenotrophic methanogens that results in a number of realistic behaviours. First, a threshold for substrate use emerges, below which the methanogen cannot metabolise its substrate. Under this model, microbes that capture more of the Gibbs free energy change from substrate transformation in the form of ATP have greater thresholds for their substrate, in line with observations of actual microbes. Second, an apparent saturation constant emerges that is controlled by the thermodynamics of the reaction. This increases with increasing ATP synthesis per substrate, so that methanogens that conserve more ATP grow faster at higher substrate concentrations, but are less competitive at low substrate concentrations. As a result, simply changing the ATP yield (moles of ATP per mole of substrate) results in methanogens with differing ecological strategies through thermodynamic impacts on their metabolism. Third, end-product inhibition through thermodynamic feedback can limit the growth of microbes, and those that capture more ATP per substrate are limited by smaller product concentrations than those that capture less ATP.

Batch vs. continuous direct compression - a comparison of material processability and final tablet quality.

In this study, an in-depth comparison was made between batch and continuous direct compression using similar compression set-ups. The overall material processability and final tablet quality were compared and evaluated. Correlations between material properties, process parameters and final tablet properties were made via multivariate data analyses. In total, 10 low-dosed (1% w/w) and 10 high-dosed (40% w/w) formulations were processed, using a total of 10 different fillers/filler combinations. The trials indicated that the impact of filler type, drug load or process settings was similar for batch and continuous direct compression. The main differentiator between batch and continuous was the flow dynamics in the operating system, where properties related to flow, compressibility and permeability played a crucial role. The less consistent flow throughout a batch process resulted in a significantly higher variability within the tablet press (σCF) and for the tablet quality responses (σMass, σTS). However, the better controlled blending procedure prior to batch processing was reflected in a more consistent API concentration variability. Overall, the comparison showed the benefits of selecting appropriate excipients and process settings to achieve a specific outcome, keeping in mind some key differentiators between both processes.

Nitrogen metabolism and rumen microbial enumeration in lactating cows with divergent residual feed intake fed high-digestibility pasture.

Dairy cattle selected for negative residual feed intake (n-RFI; efficient) should maintain production while reducing dry matter intake over a lactation because of improvements in feed digestion and efficient use of nutrients. The objective of this study was to measure nitrogen (N) digestibility and rumen microbial community composition over a short period during early lactation in lactating Holstein-Friesian cows selected previously for divergent RFI. It was proposed that n-RFI cows would have greater apparent digestibility of N than the positive RFI (p-RFI; inefficient) animals, to compensate for the lower dry matter intake determined during selection for divergence. Sixteen 3-yr-old rumen-cannulated, lactating cows (56 ± 10d in milk) selected for n-RFI (n = 8) and p-RFI (n = 8) were housed in metabolism stalls and fed fresh vegetative ryegrass (Lolium perenne L.) pasture ad libitum as a sole diet during an 8-d digestibility study. Intake of nutrients and outputs of milk, feces, and urine were determined. Rumen parameters were determined by removing, weighing, and sampling digesta, and by cobalt-EDTA dilution. Intakes of N, dry matter, organic matter, or its components did not differ with RFI. Compared with p-RFI cows, n-RFI cows had a greater apparent N digestibility (77.2 vs. 75.5%), and a tendency toward greater dry matter and organic matter digestibilities. The n-RFI cows had a lower fecal N output (126 vs. 138 g/d) and a lower partition of feed N to fecal N (23.1 vs. 24.7%) compared with p-RFI animals. We found no differences between phenotypes in the partition of N to urinary N or milk crude protein but did observe a trend for n-RFI cows to partition less N to milk casein (16.8 vs. 17.9%). Rumen digesta mass was similar for both groups, despite differences in calculated fractional liquid outflow rates, and most bacterial, archaeal, protozoal, and fungal communities were similar for both phenotype groups. In conclusion, dry matter intake and rumen function were similar for both phenotypes when the animals were fed highly digestible fresh ryegrass, but apparent digestibility of dietary N was higher in the efficient (n-RFI) cows. Future research should measure digestion parameters in cows with divergent RFI when fed diets differing in chemical composition (e.g., divergent crude protein contents).

Temporal fermentation and microbial community dynamics in rumens of sheep grazing a ryegrass-based pasture offered either in the morning or in the afternoon.

Eight ruminally-fistulated wethers were used to examine the temporal effects of afternoon (PM; 1600h) v. morning (AM; 0800 h) allocation of fresh spring herbage from a perennial ryegrass (Lolium perenne L.)-based pasture on fermentation and microbial community dynamics. Herbage chemical composition was minimally affected by time of allocation, but daily mean ammonia concentrations were greater for the PM group. The 24-h pattern of ruminal fermentation (i.e. time of sampling relative to time of allocation), however, varied considerably for all fermentation variables (P⩽0.001). Most notably amongst ruminal fermentation characteristics, ammonia concentrations showed a substantial temporal variation; concentrations of ammonia were 1.7-, 2.0- and 2.2-fold greater in rumens of PM wethers at 4, 6 and 8h after allocation, respectively, compared with AM wethers. The relative abundances of archaeal and ciliate protozoal taxa were similar across allocation groups. In contrast, the relative abundances of members of the rumen bacterial community, like Prevotella 1 (P=0.04), Bacteroidales RF16 group (P=0.005) and Fibrobacter spp. (P=0.008) were greater for the AM group, whereas the relative abundance of Kandleria spp. was greater (P=0.04) for the PM group. Of these taxa, only Prevotella 1 (P=0.04) and Kandleria (P<0.001) showed a significant interaction between time of allocation and time of sampling relative to feed allocation. Relative abundances of Prevotella 1 were greater at 2h (P=0.05), 4h (P=0.003) and 6h (P=0.01) after AM allocation of new herbage, whereas relative abundances of Kandleria were greater at 2h (P=0.003) and 4h (P<0.001) after PM allocation. The early post-allocation rise in ammonia concentrations in PM rumens occurred simultaneously with sharp increases in the relative abundance of Kandleria spp. and with a decline in the relative abundance of Prevotella. All measures of fermentation and most microbial community composition data showed highly dynamic changes in concentrations and genus abundances, respectively, with substantial temporal changes occurring within the first 8h of allocating a new strip of herbage. The dynamic changes in the relative abundances of certain bacterial groups, in synchrony with a substantial diurnal variation in ammonia concentrations, has potential effects on the efficiency by which N is utilised by the grazing ruminant.

Microbial degradation of natural and of new synthetic polymers.

In landfills, deposited waste material is usually faced with strictly anoxic conditions. This means that the design of new biodegradable polymers must take into consideration that degradation should be possible especially in the absence of molecular oxygen. Poly-beta-hydroxybutyrate is depolymerized by the anaerobic fermenting bacterium Ilyobacter delafieldii through an extracellular hydrolase. Monomers are degraded inside the cells through classical beta-oxidation. Polyalkanoates containing odd-numbered or branched-chain acid monomers should he degraded in an analogous manner; in most cases the final mineralization of these residues requires special pathways. A comparison of the chemistry of natural polymer biodegradation leads to the conclusion that synthetic biodegradable polymers should be designed in the future to contain linkages which can be cleaved by extracellular hydrolytic enzymes. Recent findings on aerobic and anaerobic bacterial degradation of synthetic polyethers suggest that natural evolution of new depolymerizing enzymes, perhaps from existing hydrolases, could be possible in a reasonable amount of time, provided that the monomers are likely energy sources for a broad variety of microbes.

Methane emissions changed nonlinearly with graded substitution of alfalfa silage with corn silage and corn grain in the diet of sheep and relation with rumen fermentation characteristics in vivo and in vitro.

Feeding grain and corn silage have been proposed as practices to reduce enteric methane (CH) emissions per unit of intake from ruminants, but the inclusion level required in the diet is normally not specified. The objectives of the current study were to determine the CH emission factor (g/kg DMI) of sheep fed alfalfa silage substituted with increasing levels of corn silage or corn grain at a fixed DMI level (2% of BW) and determine its relationship with rumen fermentation characteristics and microbial community composition and with in vitro fermentation characteristics of the same diets incubated using a standard laboratory method. Romney ewe hoggets (approximately 14 mo old; = 64) were randomly allocated to 8 dietary treatments, which included chaffed alfalfa silage alone or substituted with either 25, 50, 75 or 100% corn silage or 25, 50 or 65% rolled corn grain on a DM basis. After acclimatization to the diet, DMI and CH emissions were measured from individual sheep for 2 consecutive days in open-circuit respiration chambers and a rumen sample was collected at 3 h after feeding. The same diets were also incubated in an automated in vitro gas production system for 48 h using rumen liquid of fistulated nonlactating dairy cows grazing pasture. Increasing the substitution of alfalfa silage with corn silage or corn grain in the diet of sheep resulted in a quadratic response ( < 0.01) in CH emissions per unit of DMI (CH/DMI) with either supplement. For both supplements, CH/DMI increased in mixtures of up to 50% supplement inclusion and then decreased with greater supplement inclusion, especially with corn grain inclusion, but the level did not fall below that for 100% alfalfa silage. The ratio of acetate + butyrate to propionate + valerate and the propionate proportion alone in rumen liquid were the strongest single predictors for CH/DMI in the overall data set and explained 37.1 and 32.5%, respectively, of the variation in CH/DMI. Methanogens of (21.1% of total methanogens; = 0.247) and (10.7% of total methanogens; = -0.411) clades had weak to moderate correlations with in vivo CH/DMI. There was a weak quadratic relationship ( < 0.35) between in vivo CH/DMI and the in vitro parameters of gas and CH production and total VFA, whereas there was a moderate relationship ( = -0.50) between in vivo CH/estimated rumen degradable carbohydrates and in vitro CH/DM. In conclusion, CH/DMI changed in a nonlinear fashion with increasing supplement inclusion in the alfalfa forage diet when fed at 2% of BW to sheep; however, implications on predicting its influence on greenhouse gas emissions per unit of animal product, for whole farm emissions in life cycle analysis or total national emissions in the national inventories, should be determined.

Sampling procedure for the measurement of dissolved hydrogen and volatile fatty acids in the rumen of dairy cows.

Dissolved hydrogen (dH) influences the pathways of VFA production and is a precursor of methane formation in the rumen. Measurements of dH in rumen fluid taken at the same time as measuring other rumen fermentation end products would improve our quantitative understanding of the role of dH as a controller of rumen fermentation. Sample collections though a rumen cannula and using oral stomach tubing were compared for measurements of dissolved gases and fermentation end products in the rumen fluid of 4 ruminally cannulated dairy cows fed a total mixed ration of corn silage and concentrate. Rumen fluid was collected at 0, 2.5, and 6 h after morning feeding through the cannula from cranial dorsal rumen, cranial ventral rumen, central rumen, caudal dorsal rumen, and caudal ventral rumen and in parallel by oral stomach tubing at 2 insertion depths of 180 cm (sampling the central rumen) and 200 cm (sampling the caudal dorsal rumen). The cranial dorsal rumen had the greatest pH and smallest VFA concentration among 5 sites sampled. Samples collected by oral stomach tubing had greater ( < 0.001) rumen pH and less ( < 0.001) dissolved methane (dCH) and lower VFA concentration than that collected through rumen cannula. The dH concentrations were positively correlated ( > 0.8) in rumen samples collected by the 2 sampling techniques, with a concordance correlation coefficient larger than 0.8 and scale shift being about 0.1 away from unity. The variations in the measurement of dH, dCH, pH, and VFA in samples collected by oral stomach tubing are most likely the result of saliva contamination. The time of sampling relative to feeding had significant influence ( < 0.01) on dissolved gases and fermentation end products, with the greatest concentrations of dH, dCH, and VFA measured 2.5 h after morning feeding. The dH was correlated positively ( > 0.58) with dCH and negatively ( < -0.65) with the estimated net H production relative to the amount of VFA produced. This indicated that greater dH enhanced rumen CH production and also led to fermentation pathways that produce less H, such as enhanced propionate and butyrate production. In summary, oral stomach tubing could be a feasible method to measure ruminal dH in intact animals, but caution should be taken to minimize saliva contamination. Measurements made using both techniques yield similar conclusions for the effects of dH on fermentation pathways and CH generation.

Using the W&H integrated uncertainty analysis framework with non-initiated experts.

The W&H (Walker and Harremoës) integrated uncertainty analysis framework was put forward with the aim of providing a conceptual basis for the systematic treatment of uncertainty in model-based decision support activities such as policy analysis, integrated assessment and risk assessment. It provides a heuristic tool that can be applied in decision support exercises to classify and report the various dimensions of uncertainty. The intention is to stimulate better communication among analysts as well as between them and policymakers and stakeholders. The framework successfully articulates diverse scholarly understandings of 'uncertainty', 'ignorance', and 'quality' in science for policy, Nevertheless, experience with the W&H framework has revealed that many of the concepts put forward are relatively unfamiliar--and perhaps somewhat controversial--to experts practising decision support. Thus, efforts are required to communicate the W&H framework to experts in such a way that their knowledge of uncertainty is elicited adequately, without them being overly intimidated or confused by the novelty of the concepts presented to them. After introducing the W&H conceptual framework, this paper presents the methodology that was used in applying the W&H framework in expert elicitations on uncertainty in the risk assessment of genetically modified crops. Experiences with the use of this methodology are discussed and recommendations for further improvement are given.

A guidance for assessing and communicating uncertainties.

In the daily practice of science for policy, as experienced by governmental agencies which inform the policy and the public on the state and outlook of the environment, there is a pressing need for guidance in assessing and communicating uncertainties. This need extends beyond the quantitative assessment of uncertainties in model results, and focuses on the entire process of environmental assessment, running from problem framing towards reporting the results of the study. Using the Netherlands Environmental Assessment Agency (RIVM/MNP) as a case, the development, structure and content of such a guidance system is highlighted. Conditions for a successful implementation of the guidance system are discussed, and some prospects for future work are outlined.

A modified version of the Molly rumen model to quantify methane emissions from sheep.

We modified the rumen submodel of the Molly dairy cow model to simulate the rumen of a sheep and predict its methane emissions. We introduced a rumen hydrogen (H2) pool as a dynamic variable, which (together with the microbial pool in Molly) was used to predict methane production, to facilitate future consideration of thermodynamic control of methanogenesis. The new model corrected a misspecification of the equation of microbial H2 utilization in Molly95, which could potentially give rise to unrealistic predictions under conditions of low intake rates. The new model included a function to correct biases in the estimation of net H2 production based on the default stoichiometric relationships in Molly95, with this function specified in terms of level of intake. Model parameters for H2 and methane production were fitted to experimental data that included fresh temperate forages offered to sheep at a wide range of intake levels and then tested against independent data. The new model provided reasonable estimates relative to the calibration data set, but a different parameterization was needed to improve its predicted ability relative to the validation data set. Our results indicate that, although feedback inhibition on H2 production and methanogen activity increased with feeding level, other feedback effects that vary with diet composition need to be considered in future work on modeling rumen digestion in Molly.

RUMINANT NUTRITION SYMPOSIUM: Use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis.

Globally, methane (CH4) emissions account for 40% to 45% of greenhouse gas emissions from ruminant livestock, with over 90% of these emissions arising from enteric fermentation. Reduction of carbon dioxide to CH4 is critical for efficient ruminal fermentation because it prevents the accumulation of reducing equivalents in the rumen. Methanogens exist in a symbiotic relationship with rumen protozoa and fungi and within biofilms associated with feed and the rumen wall. Genomics and transcriptomics are playing an increasingly important role in defining the ecology of ruminal methanogenesis and identifying avenues for its mitigation. Metagenomic approaches have provided information on changes in abundances as well as the species composition of the methanogen community among ruminants that vary naturally in their CH4 emissions, their feed efficiency, and their response to CH4 mitigators. Sequencing the genomes of rumen methanogens has provided insight into surface proteins that may prove useful in the development of vaccines and has allowed assembly of biochemical pathways for use in chemogenomic approaches to lowering ruminal CH4 emissions. Metagenomics and metatranscriptomic analysis of entire rumen microbial communities are providing new perspectives on how methanogens interact with other members of this ecosystem and how these relationships may be altered to reduce methanogenesis. Identification of community members that produce antimethanogen agents that either inhibit or kill methanogens could lead to the identification of new mitigation approaches. Discovery of a lytic archaeophage that specifically lyses methanogens is 1 such example. Efforts in using genomic data to alter methanogenesis have been hampered by a lack of sequence information that is specific to the microbial community of the rumen. Programs such as Hungate1000 and the Global Rumen Census are increasing the breadth and depth of our understanding of global ruminal microbial communities, steps that are key to using these tools to further define the science of ruminal methanogenesis.

Heterotrophic sulfur reduction by Thermotoga sp. strain FjSS3.B1.

Thermotoga sp. strain FjSS3.B1 was able to reduce sulfur to sulfide when grown on a mineral medium with glucose as the sole carbon and energy source. There was no increase in specific growth yield coupled to sulfur reduction, but the specific growth rate, final growth yield, and tolerance of H2 were all increased in the presence of sulfur. At dissolved H2 concentrations, of 550 to 600 mumol/l (at 77 degrees C) growth was not possible unless sulfur was added. Glucose was fermented via the Embden-Meyerhof-Parnas pathway to lactate, acetate, H2 and CO2 (and other unidentified minor products). The thermodynamic problems associated with the relatively high redox potential electrons from the 1,3-bisphosphoglycerate/glyceraldehyde 3-phosphate couple (E'0 = -350 mV) are overcome by reducing sulfur to sulfide (E'0 = -270 mV) rather than the energetically unfavourable production of H2 (E'0 = -414 mV). Under high hydrogen partial pressures there was increased production of lactate as an alternative electron sink. The results indicate that sulfur reduction operates primarily as an electron sink rather than as a detoxification reaction or energy-generating mechanism.

Phylogenetic analysis by 16S ribosomal DNA sequence comparison reveals two unrelated groups of species within the genus Ruminococcus.

Phylogenetic analysis of the genus Ruminococcus based on 16S rDNA sequence data showed the genus to be phylogenetically heterogenous. Ruminococcus species fall within the radiation of the Bacillus/Clostridium subphylum of the Gram-positive line of descent. Two distinct and unrelated clusters are recovered. One group contains R. flavefaciens, R. albus, R. bromii, and R. callidus. The second group constitutes R. gnavus, R. hansenii, R. lactaris, R. obeum, R. productus and R. torques which together with some Clostridium species may constitute a new genus.

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions.

A mixed culture, WoAct, growing on acetone, consisted of two dominant morphotypes: a rod-shaped acetone-fermenting bacterium producing acetate, and an acetate-utilizing Methanosaeta species. Dense cell suspensions, largely free of the aceticlastic methanogen and supplemented with bromoethanesulfonate, were able to degrade acetone and grow in small volumes in membrane-separated culture devices in which the acetate produced could diffuse into a large volume of medium. Acetone degradation and growth halted when the acetate concentration reached about 10 to 12 mM. Cell suspensions were able to degrade acetone in the absence of active methanogenesis, but the addition of 10 mM acetate inhibited acetone metabolism. Addition of an active culture of Methanosaeta sp. greatly stimulated the rate of acetone degradation. The results show that acetate removal in the mixed culture is not a prerequisite for growth and acetone degradation by the acetone-fermenting bacterium.

Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium.

A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.

Rumen microbial (meta)genomics and its application to ruminant production.

Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome-host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

Progress in the development of vaccines against rumen methanogens.

Vaccination against rumen methanogens offers a practical approach to reduce methane emissions in livestock, particularly ruminants grazing on pasture. Although successful vaccination strategies have been reported for reducing the activity of the rumen-dwelling organism Streptococcus bovis in sheep and S. bovis and Lactobacillus spp. in cattle, earlier approaches using vaccines based on whole methanogen cells to reduce methane production in sheep have produced less promising results. An anti-methanogen vaccine will need to have broad specificity against methanogens commonly found in the rumen and induce antibody in saliva resulting in delivery of sufficiently high levels of antibodies to the rumen to reduce methanogen activity. Our approach has focussed on identifying surface and membrane-associated proteins that are conserved across a range of rumen methanogens. The identification of potential vaccine antigens has been assisted by recent advances in the knowledge of rumen methanogen genomes. Methanogen surface proteins have been shown to be immunogenic in ruminants and vaccination of sheep with these proteins induced specific antibody responses in saliva and rumen contents. Current studies are directed towards identifying key candidate antigens and investigating the level and types of salivary antibodies produced in sheep and cattle vaccinated with methanogen proteins, stability of antibodies in the rumen and their impact on rumen microbial populations. In addition, there is a need to identify adjuvants that stimulate high levels of salivary antibody and are suitable for formulating with protein antigens to produce a low-cost and effective vaccine.

Development of a vaccine to mitigate greenhouse gas emissions in agriculture: vaccination of sheep with methanogen fractions induces antibodies that block methane production in vitro.

AIM: To develop an understanding of the immune responses of ruminants to methanogens, and to provide proof of a concept that harnessing the immune system of ruminants is a potentially viable approach to mitigate greenhouse gas emissions from agriculture. METHODS: Four subcellular fractions, namely cytoplasmic, two cell-wall preparations, and cell wall-derived proteins were prepared from Methanobrevibacter ruminantium M1. Twenty sheep (10 months of age) were vaccinated with these fractions or with whole cells (n=4 per group). Sheep were re-vaccinated once after 3 weeks, and antibody responses to M. ruminantium M1 antigens in sera and saliva measured using ELISA at 2 weeks after the second vaccination. Antigens recognised by the antisera were visualised using Western blotting. The antisera were tested in vitro for their impact on M. ruminantium M1, measuring the effect on cell growth, methane production, and ability to induce agglutination. RESULTS: Basal levels (pre-vaccination) of antibodies against M. ruminantium M1 antigens were low. Vaccination with the antigenic fractions induced strong antibody responses in serum. Both IgG and IgA responses to methanogen antigens were detected in saliva following vaccination. Western blot analysis of the antisera indicated reactivity of antibodies, and a wide range of proteins was present in the different methanogen fractions. Antisera against the various fractions agglutinated methanogens in an in-vitro assay. In addition, these antisera decreased the growth of a pure culture of a methanogen and production of methane in vitro. CONCLUSIONS: Antigens from methanogens are immunogenic in ruminants, and antisera from sheep vaccinated with fractions of methanogens have a significant impact on these organisms, inducing cell agglutination, and decreasing growth of methanogens and production of methane. Only antisera to selected methanogen fractions were able to achieve these effects. The results demonstrate the feasibility of a vaccination strategy to mitigate emission of methane.

Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the 'Alphaproteobacteria' from activated sludge.

A novel Gram-negative coccus/coccobacillus, strain Ben 114(T), growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the 'Alphaproteobacteria', with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-beta-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the 'Alphaproteobacteria', namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the 'Alphaproteobacteria', the D. vanus subgroup. The type strain is Ben 114(T) (=NCIMB 13612(T)=CIP 107350(T)).

Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species.

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting "Campylobacter" sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G + C of DNA isolated from both strains was 36-37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.