Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture.

AIMS: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. METHODS AND RESULTS: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. CONCLUSIONS: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan-lignin complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.

Degradation of forage chicory by ruminal fibrolytic bacteria.

AIMS: Determine the susceptibility of forage chicory (Cichorium intybus L.) to degradation by ruminal fibrolytic bacteria and measure the effects on cell-wall pectic polysaccharides. METHODS AND RESULTS: Large segments of fresh forage chicory were degraded in vitro by Lachnospira multiparus and Fibrobacter succinogenes, but not by Ruminococcus flavefaciens or Butyrivibrio hungatei. Cell-wall pectins were degraded extensively (95%) and rapidly by L. multiparus with a simultaneous release of uronic acids and the pectin-derived neutral monosaccharides arabinose, galactose and rhamnose. Fibrobacter succinogenes also degraded cell-wall pectins extensively, but at a slower rate than L. multiparus. Immunofluorescence microscopy using monoclonal antibodies revealed that, after incubation, homogalacturonans with both low and high degrees of methyl esterification were almost completely lost from walls of all cell types and from the middle lamella between cells. CONCLUSIONS: Only two of the four ruminal bacteria with pectinolytic activity degraded fresh chicory leaves, and each showed a different pattern of pectin breakdown. Degradation was greatest for F. succinogenes which also had cellulolytic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of extensive removal of pectic polysaccharides from the middle lamella and the consequent decrease in particle size may explain the decreased rumination and the increased intake observed in ruminants grazing forage chicory.

Coenzyme B-12-dependent reactions. Part IV. Observations on the purification of ethanolamine ammonia-lyase.

Purification of ethanolamine ammonia-lyase (EC 4.3.1.7) from a Clostridium sp. grown at the University of Sussex, U.K. and the National Institutes of Health, U.S.A., has been compared and an improved isotopic assay for the enzyme has been developed. Successful purification of this enzyme from Sussex-grown cells requires modification of the published procedure (Kaplan and Stadtman (1968) J. Biol, Chem. 243, 1787-1793) principally a 70% decrease in volume during precipitation with 0.4 M NaCl. This modification also increases the yield from N.I.H.-grown cells. Purified enzyme, resolved of inactive cobalamins, has the same high specific activity from both sources and behaves in the same way on disc gel electrophoresis. Sussex enzyme, before resolution, has less than 20% of the specific activity of unresolved N.I.H. enzyme and contains over 50% more inactive cobalamin. The bound cobalamin from both preparations has been identified as a "base-on" Co11 psi-cobalamin.

A precursor in the photolytic cleavage of the Co (III)-C bond of coenzyme B-12 unobservable by electron paramagnetic resonance.

Photolysis of a frozen (80--200 K) anaerobic solution of 5'-deoxyadenosyl-cobalamin in aqueous propan-1,2-diol produces only a small Co(II) signal detectable by electron paramagnetic resonance (EPR). Upon warming to room temperature and refreezing without further irradiation the Co(II) signal increases many-fold. The interpretation is that at low temperature there is an EPR-undetectable "incipient" homolysis of the Co-C bond of the coenzyme which is revealed at higher temperature. The possible implications of this observation for the coenzyme B-12-dependent enzymes are noted.

Effect of Methanobrevibacter smithii on Xylanolytic Activity of Anaerobic Ruminal Fungi.

Three different ruminal anaerobic fungi, Neocallimastix frontalis PNK2, Sphaeromonas communis B7, and Piromonas communis B19, were grown axenically or in coculture with Methanobrevibacter smithii on xylan. N. frontalis and S. communis in monoculture and coculture accumulated xylobiose, xylose, and arabinose in the growth medium; arabinose was not metabolized, but xylobiose and xylose were subsequently used. The transient accumulation of xylose was much less evident in cocultures. Both the rate and extent of xylan utilization were increased by coculturing, and metabolite profiles became acetogenic as a result of interspecies hydrogen transfer; more acetate and less lactate were formed, while formate and hydrogen did not accumulate. For each of the three fungi, there were marked increases in the specific activities of extracellular xylanase (up to fivefold), alpha-l-arabinofuranosidase (up to fivefold), and beta-d-xylosidase (up to sevenfold) upon coculturing. The stimulating effect on fungal enzymes from coculturing with M. smithii was independent of the growth substrate, and the magnitude of the stimulation varied according to the enzymes and the incubation time. For an N. frontalis-M. smithii coculture, the positive stimulation was maintained during an extended (18-day) incubation period, and this affected not only hemicellulolytic enzymes but also polysaccharidase and glycoside hydrolase enzymes that were not involved in xylan breakdown. The specific activity of cell-bound endopeptidase was not increased under the coculture conditions used in this study. The higher enzyme activities in cocultures are discussed in relation to catabolite repression.

Hypergastrinaemia, abomasal bacterial population densities and pH in sheep infected with Ostertagia circumcincta.

Serum gastrin and pepsinogen concentrations, food intake, abomasal pH and abomasal aerotolerant and anaerobic bacterial populations were measured in sheep infected with Ostertagia circumcincta to search for links between hypergastrinaemia, food intake and changes in the abomasal environment. Abomasal pH and serum gastrin and pepsinogen concentrations were elevated in each of five sheep infected via abomasal cannulae with 150000 exsheathed larval stage three, followed 11 days later by 100000 sheathed larvae given intraruminally. Unparasitised abomasa contained aerotolerant bacterial population densities of between 10(3) and 10(6) cells ml(-1) and these did not change significantly following parasitism. In contrast, anaerobic bacterial population densities increased markedly by about 10(4)-fold following parasitism. Anaerobic numbers changed rapidly when abomasal pH increased from 2.5 to 3.5. At pH 4 and above, anaerobic bacterial numbers approached levels expected in rumen contents but parameters other than pH did not relate to bacterial numbers. Brief periods when serum gastrin was lower than expected, coinciding with raised abomasal pH, were not explicable by increased bacterial numbers. Food intake, which decreased for a variable period from around Day 5 p.i., correlated poorly with serum gastrin concentration, suggesting hypergastrinaemia is not the sole cause of anorexia in parasitised animals. The survival of substantial numbers of rumen bacteria in the abomasum at only slightly raised pH may significantly lower the bacterial protein available to the sheep.

Hydration of linoleic acid by bacteria isolated from ruminants.

Two strains of Enterococcus faecalis isolated from the ovine rumen and known to hydrate oleic acid were shown to transform linoleic acid by hydration into two products. The products, identified as 10-hydroxy-12-octadecenoic acid and 13-hydroxy-9-octadecenoic acid, were formed during stationary phase in yields of 13% and 6% respectively. Yields increased to 22% and 14% when culture conditions were optimised. To our knowledge, this is the first report of 13-hydroxy-9-octadecenoic acid production by bacteria. During a search for further linoleic-acid-hydrating bacteria, a strain of Streptococcus bovis isolated from bovine faeces and the ruminal strain S. bovis JB1 were found to hydrate linoleic acid. Both strains formed only one product and the most rapid appearance occurred during exponential growth. The S. bovis product, identified as 13-hydroxy-9-octadecenoic acid, formed in a yield of 28%. This study provides the first information on linoleic acid hydration by ruminal bacteria.

Movement of nutrient and non-nutrient elements in the liquid phase in sheep rumen.

Changes in the concentrations of B, Ca, Fe, K, Mg, Mn, Na, P, S, Sr and Zn in the liquid phase in sheep rumen were determined to obtain information on their rate of disappearance from the soluble pool after feeding. Sheep were fed alfalfa (Medicago sativa) hay hourly. The concentration of infused Cr(III)-EDTA into the rumen (over a period of 4 d) was used to establish steady-state conditions, after which feeding was stopped and the rapidly changing elemental concentrations in 30,000 x g ruminal supernatant fluid were determined by plasma emission spectrometry. The concentrations of all elements, apart from Fe, Na and P, decreased in a first-order manner over 5 h after feeding. For one sheep, these concentrations (microM) were: B, 199 to 126; Cr, 184 to 111; Ca, 4,150 to 1,130; K, 51,000 to 28,800; Mn, 11 to 4.2; Mg, 4,190 to 1,810 and Zn, 14 to 7.8. For S, first-order decay was observed over only the 0- to 2-h period (2230 microM to 865 microM). Phosphorus increased from 10.4 to 13.1 mM over the 5-h period, whereas Na remained constant (85 mM); this was attributed to saliva input. Mean half-lives for B (8.8 h), K (7.5 h) and Zn (5.3 h) were similar to those of Cr (6.3 +/- .3 h), the water flow marker. Mean half-lives of Ca (2.6 h), Mg (4.0 h), Mn (3.3 h), Sr (3.3 h) and S (1.8 h) were shorter (P less than .01) than that of the Cr marker.(ABSTRACT TRUNCATED AT 250 WORDS)

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen.

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.

Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes.

Anaerobic phycomycetous fungi have been isolated from rumen fluid by using the Hungate roll tube technique. Cultures on solid substrates in roll tubes can be stored for long periods, and fungal zoospores can be enumerated in roll tubes.

Hydration of oleic acid by a Selenomonas strain from the ovine rumen.

A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.

Conversion of oleic acid to 10-hydroxystearic acid by two species of ruminal bacteria.

Bacteria able to convert oleic acid to 10-hydroxystearic acid were isolated from the ovine rumen. The solid hydroxy fatty acid produced from bacterial fermentations containing oleic acid was recovered by filtration, extraction into ether and crystallisation. The identity of the product was confirmed by HPLC and gas chromatography/mass spectrometry. One 10-hydroxystearic-acid-producing bacterial group was represented by two strains of an anaerobic gram-negative curved rod with tufts of flagella on the concave surface of the cell. The morphology and other characteristics enabled the strains to be tentatively identified as Selenomonas ruminantium. Another bacterium capable of the same transformation, represented by two strains of a facultatively anaerobic gram positive chain-forming coccus, was identified as Enterococcus faecalis. Since unsaturated fatty acids entering the rumen are normally hydrogenated, hydration of oleic acid represents an alternative fate of unknown significance in vivo.

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis.

A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10-hydroxystearic acid.

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows.

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.

Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen.

AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation. METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan. From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei. During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid. Unlike previously studied ruminal biohydrogenating bacteria, B. hungatei Su6 was able to convert alpha-linolenic acid to stearic acid. Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates. Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid. An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid. CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B. hungatei plays an important role in unsaturated fatty acid transformations in the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years. Access to B. hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.

Isolation and identification of ruminal methanogens from grazing cattle.

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H(2)/CO(2) and formate, and optimally at 38 degrees C and pH 6.5-7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H(2)/CO(2), acetate, and methyl-containing compounds, with optimal growth at 40 degrees C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management.