Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.     

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.     

The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum.

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 mumole H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 mumole H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction wiion than that from Cl. pasteurianum.     

Hydrogenase activity and methanogenesis in anaerobic sewage sludge,in rumen liquid,and in freshwater sediments

Summary The applicability of hydrogenase determinations to the evaluation of hydrogen transfer reactions occurring within methanogenic environments was investigated. Enzymatic hydrogen production was determined in digester sludge, river sediments, and rumen liquid using reduced methyl viologen, formate, and pyruvate as hydrogen donors. Hydrogenase determinations turned out not to be inhibited by toxic compounds present in sediments of the polluted river Saar. Comparative kinetic studies of the conversion of acetate and of hydrogen to methane support the assumption that carbon dioxide reduction by hydrogen accounts for the major part of methane formed in river sediments. In rumen liquid and in river sediments similar enzyme patterns were observed which were different from that found in digester sludge. The rates of methanogenesis correlated well with hydrogenase activities in all ecosystems studied: Correlation coefficients ranged from 0.84 to 0.95. Rumen liquid and river sediments exhibited higher hydrogenase activities than digester sludge when compared at identical rates of methane production. According to these results, the hydrogenase determination is applicable to the evaluation of the hydrogen transfer, occurring within the microbial biomass of anaerobic ecosystems.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production.

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: pure-culture studies with cellulose and alkaline peroxide-treated wheat straw.

Specific oligonucleotide probes targeted to sites on the 16S rRNA of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, and Fibrobacter succinogenes S85 and a domain Bacteria probe were used to study bacterial interactions during the fermentation of cellulose and alkaline hydrogen peroxide-treated wheat straw in monocultures, dicultures, and tricultures. Results showed that R. albus 8 inhibited the growth of R. flavefaciens FD-1 when grown as a diculture with cellulose or alkaline hydrogen peroxide-treated wheat straw as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85 grown on cellulose or alkaline hydrogen peroxide-treated wheat straw, competition was not detected. R. flavefaciens FD-1 outcompeted F. succinogenes S85 when cellulose was used as the carbon source. In tricultures with cellulose as the carbon source, R. flavefaciens FD-1 was inhibited, R. albus 8 appeared to dominate during the early phase of degradation (12 to 48 h), while F. succinogenes S85 became predominant during the later phase of degradation (60 to 70 h). When alkaline hydrogen peroxide-treated wheat straw was used as a growth substrate, F. succinogenes S85 showed better growth than either R. albus 8 or R. flavefaciens FD-1. However, R. flavefaciens FD-1 was present in small numbers throughout the incubation period, unlike the growth patterns when cellulose was the carbon source.     

Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.     

Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution.

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.     

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.     

Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Biotechnology is a promising approach for the generation of hydrogen, but is not yet commercially viable. Metabolic engineering is a potential solution, but has largely been limited to native pathway optimisation. To widen opportunities for use of non-native [NiFe] hydrogenases for improved hydrogen production, we introduced a cyanobacterial hydrogen production pathway and associated maturation factors into Escherichia coli. Hydrogen production is observed in vivo in a hydrogenase null host, demonstrating coupling to host electron transfer systems. Hydrogenase activity is also detected in vitro. Hydrogen output is increased when formate production is abolished, showing that the new pathway is distinct from the native formate dependent pathway and supporting the conclusion that it couples cellular NADH and NADPH pools to molecular hydrogen. This work demonstrates non-native hydrogen production in E. coli, showing the wide portability of [NiFe] hydrogenase pathways and the potential for metabolic engineering to improve hydrogen yields.     

Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum.

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Importance of the Isovalerate Carboxylation Pathway of Leucine Biosynthesis in the Rumen

Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C(14) from isovalerate-1-C(14). Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C(14) from isovalerate-1-C(14) into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of beta-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C(14), leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.