Biomedical subjects

P J Weimer

Publications and source records attributed to P J Weimer.

At least 19 recordsLinked to original sources

PubMed2003

Solid residues from Ruminococcus cellulose fermentations as components of wood adhesive formulations.

Residues from the fermentation of cellulose by the anaerobic bacteria Ruminococcus albus (strain 7) or Ruminococcus flavefaciens (strains FD-1 or B34b) containing residual cellulose, bacterial cells and their associated adhesins, were examined for their ability to serve as components of adhesives for plywood fabrication. The residues contained differing amounts of protein (0.4-4.2% of dry weight), but the ratios of monosaccharides recovered following two-stage treatment of the residue with detergent (pH 7) and TFA were similar for all three strains (0.71 glucose:0.18 xylose:0.08 mannose:0.02 galactose), suggesting similarities in exopolysaccharide composition. Three-ply aspen panels prepared with fermentation residues (FR) displayed better shear strength and wood failure under dry conditions than following a vacuum/pressure/soak/dry treatment, but adhesive properties were inferior to those prepared with conventional phenol-formaldehyde (PF) adhesives. However, panels prepared by incorporating the R. albus 7 FR into PF formulation, at 73% by weight of the total adhesive, exhibited shear strength and wood failure similar to that obtained with PF adhesive alone. Use of residues from fermentations by these bacteria as components of adhesives may add value to biomass fermentations aimed primarily at producing ethanol and other chemical products.

Adhesives↗

PubMed2003

The survival of silage inoculant lactic acid bacteria in rumen fluid.

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.

Animals↗

PubMed2002

Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol.

The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.

Cellulose↗

PubMed2001

Initial pH as a determinant of cellulose digestion rate by mixed ruminal microorganisms in vitro.

In vitro fermentations of pure cellulose by mixed ruminal microorganisms were conducted under conditions in which pH declined within ranges similar to those observed in the rumen. At low cellulose concentrations (12.5 g/L), the first-order rate constants (k) of cellulose disappearance were successively lower at initial pH values of 6.86, 6.56, and 6.02, but in each case the value of k was maintained over a pH range of 0.3 to 1.2 units, as the fermentation progressed. Plots of k versus initial pH were linear, and k displayed a relative decrease of approximately 7% per 0.1 unit decrease in pH. At high cellulose concentration (50 g/L) and an initial pH of 6.8, cellulose digestion was initially zero order, the absolute rate of digestion declined with pH and digestion essentially ceased at pH 5.3 after only 30% of the added cellulose was digested. Further incubation resulted in a loss of bound N and P, suggesting that at low pH cells lysed or detached from the undigested fibers. Pure cultures of ruminal cellulolytic bacteria also were able to ferment cellulose to a minimum pH of 5.1 to 5.3, but the extent of fermentation was increased by coculture with noncellulolytic bacteria. A model is proposed in which the first-order rate constant of cellulose digestion is determined by the pH at which the fermentation is initiated, and end product ratios reflect greater activity of the noncellulolytic population as pH declines.

Animals↗

PubMed2000

Fermentation of a bacterial cellulose/xylan composite by mixed ruminal microflora: implications for the role of polysaccharide matrix interactions in plant cell wall biodegradability.

Growth of the cellulose-synthesizing bacterium Acetobacter xylinum ATCC 53524 in media supplemented with 5% (w/v) glucose and 0.2% (w/v) of a water-soluble, nearly linear xylan from tobacco stalks resulted in the synthesis of a highly crystalline composite having a xylose/glucose ratio ranging from 0.06 to 0.24. The digestion of one composite (88% cellulose/12% xylan) by mixed ruminal microflora displayed kinetics of gas production similar to those of an unassociated mixture of the two components added in a xylan/cellulose ratio similar to that of the composite. The data suggest that intimate association of xylan and cellulose, as is typically found in secondary plant cell walls, does not inhibit the rate of digestion of the component polysaccharides.

Acetobacter↗

PubMed1999

Accuracy of Klason lignin and acid detergent lignin methods as assessed by bomb calorimetry.

An accurate method for estimation of lignin concentration is important for prediction of the digestible energy content of livestock feeds. The accuracy of lignin concentration estimates based on the Klason lignin and acid detergent lignin methods was compared. Ten diverse forage samples were analyzed for protein, carbohydrates, lipids, organic acids, ash, lignin (by both methods), and gross energy. The accuracy of the two lignin concentration estimates was examined by comparing the measured forage gross energy to a gross energy value calculated from the compositional analysis. Use of the acid detergent lignin estimate in this gross energy calculation accounted for 68-84% of the forage gross energy compared to 85-97% of the gross energy using Klason lignin. These results indicate that while Klason lignin estimates are substantially higher than acid detergent lignin estimates, Klason lignin is the more accurate lignin method and does not overestimate lignin because gross energy recoveries were less than 100%.

Animal Feed↗

PubMed1999

Effect of diet on populations of three species of ruminal cellulolytic bacteria in lactating dairy cows.

The effects of four contrasting diets were determined on populations of three species of ruminal cellulolytic bacteria (Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes) using oligonucleotide probes to rRNA. Diets based on alfalfa silage or corn silage as the primary fiber source were formulated to contain either 24 or 32% neutral detergent fiber measured after alpha-amylase treatment. The diets were fed twice daily to four ruminally fistulated, lactating Holstein cows in a trial using a Latin square design. The cows fed the alfalfa silage diets had higher dry matter intakes and milk production and smaller pH fluctuations than did cows fed the corn silage diets (0.3 vs. 0.8 units). The total populations of the three cellulolytic species at 3 h after feeding ranged from 0.3 to 3.9% of the bacterial domain; R. albus was generally the most abundant of the three species. The data are in general agreement with population assessments obtained by some traditional methods of culture enumeration. Although diet and individual cows had major effects on ruminal pH and volatile fatty acid concentrations and on milk production and composition, differences in cellulolytic populations that were attributable to individual cows were larger than those attributable to diet, suggesting that each cow maintained a unique assemblage of cellulolytic species.

Animals↗

PubMed1998

Manipulating ruminal fermentation: a microbial ecological perspective.

The essential role of ruminal microflora in ruminant nutrition provides the potential for improvement in animal production via altering the numbers or activities of specific classes of microorganisms. Successful alterations will be facilitated by an understanding of the microbial ecology of the rumen based on its mechanistic underpinnings. Demonstrated improvements in ruminal fermentation can be traced to their consonance with well-established principles of microbial ecology (niche occupancy, selective pressure, adaptation, and interactions) and the thermodynamics and kinetics of substrate utilization. Application of these principles to several proposed alterations of the ruminal bacterial population allows a prediction of their relative feasibility. Improving fiber digestion, decreasing protein degradation, and detoxifying feed components that are present in low concentrations will be difficult to achieve in the rumen and are best approached by altering the feed, either genetically or with postharvest treatment. By contrast, the detoxification of feed components present in high concentration, and redirection of electron disposal away from methanogenesis, are more productive targets for microbiological research.

Animals↗

PubMed1997

Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens.

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.

Animals↗

PubMed1997

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens FD-1.

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H2 in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO2 (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, mu = 0.35/h). H2 was formed by a hydrogenase rather than by cleavage of formate, and 13C-NMR and 14C-exchange reaction data indicated that formate was produced by CO2 reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the expense of H2. This shift suggested that reducing equivalents could be balanced through formate or H2 production without affecting the yields of the major carbon-containing fermentation endproducts.

Acetates↗

PubMed1997

Competition for cellulose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitation is in accord with the former's more rapid adherence to cellulose and its higher affinity for cellodextrin products of cellulose hydrolysis. In batch cocultures of F. succinogenes and R. albus, the populations of the two species were similar. However, under cellulose limitation, F. succinogenes was the predominant strain (approximately 80% of cell mass) in cultures simultaneously coinoculated with R. albus. The results from batch cocultures of R. flavefaciens and R. albus were not consistent within or among trials: some experiments yielded monocultures of R. albus (suggesting production of an inhibitory agent by R. albus), while others contained substantial populations of both species. Under cellulose limitation, R. flavefaciens predominated over R. albus (85 and 15%, respectively), as would be expected by the former's greater adherence to cellulose. The retention of R. albus in the cellulose-limited coculture may result from a combination of its ability to utilize glucose (which is not utilizable by R. flavefaciens), its demonstrated ability to adapt under selective pressure in the chemostat to utilization of lower concentrations of cellobiose, a major product of cellulose hydrolysis, and its possible production of an inhibitory agent.

Animals↗

PubMed1997

Competition for cellobiose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

The ruminal cellulolytic bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 coexisted in substrate-excess coculture with about equal population size, but R. flavefaciens outcompeted F. succinogenes for cellobiose in the substrate-limited cocultures whether the two strains were coinoculated or a steady-state culture of F. succinogenes was challenged by R. flavefaciens. This outcome of competition between these two strains is due to a classical pure and simple competition mechanism based on affinity for cellobiose. Although the population size of F. succinogenes was much higher (> 70%) than that of another cellulolytic species, Ruminococcus albus 7 in substrate-excess coculture, F. succinogenes was replaced by a population of R. albus in the substrate-limited coculture in both coinoculation and challenge experiments. R albus outcompeted F. succinogenes, apparently due to selection in the chemostat of a population of R. albus with a higher affinity for cellobiose. R. albus also outcompeted R. flavefaciens under substrate-limited conditions.

Animals↗

PubMed1996

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.

Animals↗

PubMed1996

Why don't ruminal bacteria digest cellulose faster?

The bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus generally are regarded as the predominant cellulolytic microbes in the rumen. Comparison of available data from the literature reveals that these bacteria are the most actively cellulolytic of all mesophilic organisms described to date from any habitat. In light of numerous proposals to improve microbial cellulose digestion in ruminants, it is instructive to examine the characteristics of these species that contribute to their superior cellulolytic capabilities and to identify the factors that prevent them from digesting cellulose even more rapidly. As a group, these species have extreme nutritional specialization. They are able to utilize cellulose (or in some cases xylan) and its hydrolytic products as their nearly sole energy sources for growth. Moreover, each species apparently has evolved to similar maximum rates of cellulose digestion (first-order rate constants of 0.05 to 0.08 h-1). Active cellulose digestion involves adherence of cells to the fibers via a glycoprotein glycocalyx, which protects cells from protozoal grazing and cellulolytic enzymes from degradation by ruminal proteases while it retains-at least temporarily-the cellodextrin products for use by the cellulolytic bacteria. These properties result in different ecological roles for the adherent and nonadherent populations of each species, but overall provide an enormous selective advantage to these cellulolytic bacteria in the ruminal environment. However, major constraints to cellulose digestion are caused by cell-wall structure of the plant (matrix interactions among wall biopolymers and low substrate surface area) and by limited penetration of the nonmotile cellulolytic microbes into the cell lumen. Because of these constraints and the highly adapted nature of cellulose digestion by the predominant cellulolytic bacteria in the rumen, transfer of cellulolytic capabilities to noncellulolytic ruminal bacteria (e.g., by genetic engineering) that display other desirable properties offers limited opportunities to improve ruminal digestion of cellulose.

Animals↗

PubMed1995

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol.

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose l-1 and 4.4 g ethanol l-1 were converted to 2.6 g butyrate l-1 and 4.6 g caproate l-1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.

Animals↗

PubMed1995

Cellodextrin efflux by the cellulolytic ruminal bacterium Fibrobacter succinogenes and its potential role in the growth of nonadherent bacteria.

When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was grown in a cellobiose-limited chemostat, cellobiose and cellotriose could both be detected, and the ratio of cellotriose to cellobiose was approximately 1 to 4. On the basis of these results, cellodextrin production is an equilibrium (mass action) function and not just an artifact of energy-rich cultural conditions. Cellodextrins could not be detected in low-dilution-rate, cellulose-limited continuous cultures, but these cultures had a large number of nonadherent cells. Because the nonadherent cells had a large reserve of polysaccharide and were observed at all stages of cell division, it appeared that they were utilizing cellodextrins as an energy source for growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014-0.076 h-1) and extracellular pH (6.11-6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentration to response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07-0.11 h-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04-0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determine that maintenance coefficient (0.04-0.06 g cellulose/g cells.h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower mu max on microcrystalline cellulose.

Animals↗

PubMed1993

Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer).

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.

Animals↗