Ruminococcus Flavefaciens FD-1 Research Articles

Untreated and delignified cotton stalks as model substrates for degradation and utilization of cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Pure cultures of the defined ruminal bacterial strains Fibrobacter succinogenes S85 and BL2, Ruminococcus flavefaciens FD1, Ruminococcus albus 7 and Butyrivibrio fibrisolvens D1, and the pair combinations of strains S85 + D1, BL2 + D1, FD1 + D1 and 7 + D1, were grown on isolated cell walls (CW) of untreated cotton stalks (CS) or ozone-treated CS (OCS) as the sole added carbohydrate substrate. F. succinogenes S85 was the best CW hydrolyzer of CS, solubilizing 27·9% of CW-glucose polymers, 21·9% of xylan, 24·8% of hemicellulose and 26·9% of total CS-CW polysaccharides. The Ruminococci monocultures and cocultures had very little hydrolytic effect on CS but somewhat better hydrolytic effect on OCS-CW. Since OCS-CW contained less lignin and hemicellulose components and more cellulose and total CW polysaccharides than CS-CW all bacterial cultures and cocultures degraded OCS-CW components better than CS-CW polysaccharides. S85 was the best hydrolyzer of OCS residual CW, solubilizing 52·8% of CW-glucose, 51·2% xylan, 51·5% hemicellulose and 52·6% of total OCS-CW monosaccharides. There was complementary action between B. fibrosolvens D1 and the F. succinogenes strains S85 and BL2 with respect to coculture growth and carbohydrate utilization, but not to the extent of CW hydrolysis, which was determined mainly by the F. succinogenes strains during the 120 h of coculture incubations. Utilization of solubilized cellulose, xylan, hemicellulose sugars and total carbohydrates by S85 monoculture was 94·4%, 80·3%, 61·4% and 84·1%, respectively, with respect to CS components, and 96·7%, 64·8%, 62·1% and 89·6% with respect to OCS-CW carbohydrates. Under scanning electron microscopy (SEM) visualization, S85 formed the most dense layer of bacterial cell mass attached to, and colonized on, OCS-CW stems. CW particles of CS were less crowded with attached bacteria. The cell-surface topology of S85 cells attached to CW particles was specified by a coat of characteristic protuberant structures.

Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.

A segmented gas/liquid delivery system for continuous culture of microorganisms on insoluble substrates and its use for growth of Ruminococcus flavefaciens on cellulose

An anaerobic continuous culture device was constructed that permits accurate delivery of media containing insoluble substrates, even at very low volumetric flow rates (<3 ml/h). The system consisted of: (1) a reservoir in which the medium slurry was mixed well by the combined use of stirring and diffusive gas sparging to suspend a cellulose substrate of small (< 45 μm) particle size; (2) a delivery system that segmented the slurry into small (~ 20μl) discrete liquid segments separated by intervening bubbles of CO2 gas; and (3) a stirred, temperature-controlled 2-l fermentation vessel. The device was used to examine substrate consumption, product formation, and cell yield by the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 under steady-state, cellulose-limited conditions at six different dilution rates (D) ranging from 0.017 to 0.101 h−1 (pH 6.4–6.6). Cellulose consumption decreased from 4.00 g/1 (at D=0.017 h−1) to 2.56 g/1 (at D=0.101 h−1). Increases in D resulted in a progressive shift toward production of more acetate and formate, and less succinate. Redox balance calculations revealed a deficiency in reduced products, probably due to the production of H2, which was not directly measured. Reducing sugar values remained low (0.05–0.10 g/1, as glucose) at all D values. The cellulose fermentation was characterized by a low maintenance coefficient (0.07 g cellulose/g cells per hour) and a high true growth yield (YG = 0.24 g cells/g cellulose, corrected for maintenance). Comparison of the data with literature values suggests that the fermentation of cellulose by this organism gives cell yields at least as great as the yields obtained from the fermentation of soluble sugars.

The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria

The defined ruminal bacterial strains Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, and Bacteroides ruminicola GA33 were grown, in monocultures or as combinations of pair strains, on isolated lucerne cell-walls (CW) as the sole carbohydrate substrate. Fibrobacter succinogenes S85 was the dominant strain determining extent of CW hydrolysis in all combinations with S85. The hydrolysis of cellulose, xylan, hemicellulose side-sugars, and total CW monosaccharides by pure S85 were: 58.8, 47.3, 66.9 and 57.0%, respectively. The strains combination S85 plus D1 comprised the highest complementary effect, increasing significantly the hydrolysis of cellulose and total CW monosaccharides by 16% and 13%, respectively, above the values obtained by pure S85. This complementation was expressed also in growth pattern of bacteria. The monocultures of FD1, D1 and GA33 had very little hydrolytic effect on lucerne cellulose, but higher effects on xylan and hemicellulose side-sugars. The combinations D1 plus GA33 and 7 plus GA33 were complementary in the hydrolysis of all CW polysaccharides. The combinations FD1 plus D1, FD1 plus GA33, and 7 plus D1 were complementary only with respect to hemicellulose hydrolysis. On the other hand, the cellulolytic combinations S85 plus FD1, S85 plus 7 and FD1 plus 7 demonstrated negative interactions in lucerne CW polysaccharides hydrolysis. Under scanning electron microscopy (SEM), S85 comprised the most dense layer of bacterial cell mass attached to and colonized on CW particles. The cell surface topology of the cellulolytic strains S85, FD1 and 7 attached to CW particles was specified by a coat of characteristic protuberant structures.

Degradation of wheat straw and alkaline hydrogen peroxide-treated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1.

Degradation of wheat straw (WS) and alkaline hydrogen peroxide (AHP)-treated wheat straw (AHPWS) by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1 was determined by measuring the growth (OD600) of each bacterium and determining DM disappearance (DMD) of the substrate. Complex medium and defined medium with or without the addition of phenylpropanoic acid (PPA) and phenylacetic acid (PAA) were used. Tubes were incubated at 39 degrees C for 8 d. Both OD600 and DMD indicated that AHPWS was degraded to a much greater extent by either bacterium (R. flavefaciens FD-1, 60.8 +/- 1.8% and R. albus 8, 42.3 +/- 3.5%) vs untreated WS (R. flavefaciens FD-1, 16.5 +/- 1.8% and R. albus 8, 8.6 +/- 6%) in the complex medium. Most degradation occurred between d 1 and 4. With the complex medium, addition of PPA and PAA did not stimulate degradation by either bacterium. When the defined medium was used, the addition of PPA and PAA enhanced (P less than .05) degradation of AHPWS (39.6 +/- 2.6%) vs AHPWS with no added PPA and PAA (24.9 +/- 7.6%) by R. albus 8. There was no synergistic effect on degradation when the two species were co-cultured with either WS or AHPWS as the substrate. No effect of PPA and PAA on disappearance of AHPWS was observed for R. flavefaciens FD-1 or when the two bacteria were grown together. Dry matter disappearance analysis showed that R. flavefaciens FD-1 degraded AHPWS more rapidly (6.1 mg/d) than R. albus 8 did (4.2 mg/d) in complex medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellobiose uptake and metabolism by Ruminococcus flavefaciens

The cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 utilizes cellobiose but not glucose as a substrate for growth. Cellobiose uptake by R. flavefaciens FD-1 was measured under anaerobic conditions (N2), using [G-3H]cellobiose. The rate of cellobiose uptake for early- or late-log-phase cellobiose-grown cells was 9 nmol/min per mg of whole-cell protein. Cellobiose uptake was inhibited by electron transport inhibitors, iron-reactive compounds, proton ionophores, sulfhydryl inhibitors, N,N-dicyclohexylcarbodiimide, and NaF, as well as lasalocid and monensin. The results support the existence of an active transport system for cellobiose. Transport of [U-14C]glucose was not detected with this system. Phosphorylation of cellobiose was not by a phosphoenolpyruvate-dependent system. Cellobiose phosphorylase activity was detected by both a coupled spectrophotometric assay and a discontinuous assay. The enzyme was produced constitutively in cellobiose-grown cells at a specific activity of 329 nmol/min per mg of cell-free extract protein.

Nucleotide sequence of thecelA gene encoding a cellodextrinase ofRuminococcus flavefaciens FD-1

The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) from Ruminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CelA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other organisms. Two lysozyme-type active sites were found in the amino-terminal third of the enzyme. In E. coli the cloned Cel A protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposon phoA mutagenesis experiments indicated that CelA is secreted by a mechanism other than a leader peptide.

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial cell surface structures involved in lucerne cell wall degradation by pure cultures of cellulolytic rumen bacteria

Pure cultures of the cellulolytic rumen bacterial strains Bacteroides succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus 7 were grown on lucerne cell walls (CW) or on cellobiose as the sole added carbohydrate substrate. Scanning electron microscopy visualization using cationized-feritin pretreatment have shown that cell surface topology of these strains grown on and attached to CW particles was specified by a dense coat of characteristic protuberant structures. In contrast, when grown on cellobiose, the surface topology of these bacterial strains was smoother, and contained fewer protuberant structures. The ability of these bacterial strains to attach to cellulose was higher for bacteria previously adapted to lucerne CW compared to cellobiose adaptation. Bacteroides succinogenes S85 was the best digester of lucerne CW (46.5%) and also had the best adhesion capability (65.6%) after adaption to grow on CW.

Improved assay for quantitating adherence of ruminal bacteria to cellulose.

A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose.

Methylcellulose inhibition of exo-beta-1,4-glucanase A from Ruminococcus flavefaciens FD-1.

A homogeneous preparation of exo-beta-1,4-glucanase A from Ruminococcus flavefaciens FD-1 was competitively inhibited by low concentrations (less than 3 mM) of methylcellulose. The enzyme was also sensitive to the surfactant properties of methylcellulose at high methylcellulose concentrations.

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1.

An exo-beta-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-beta-D-cellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (greater than or equal to 300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel filtration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate- and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-beta-1,4-glucanase. Exo A required Ca2+ for maximal activity and had an apparent Km of 3.08 mM for NPC, with a Vmax of 0.298 mumol/min per mg of protein. The enzyme had an Mr of 230,000, as determined by gel filtration chromatography, and was a dimer of 118,000-Mr subunits. The N-terminal amino acid sequence of the enzyme is presented.

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.

Effect of phenolic monomers on ruminal bacteria.

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.