Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

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.     

Competition Between Ruminal Cellulolytic Bacteria for Adhesion to Cellulose

Competition for adhesion to cellulose among the three main ruminal cellulolytic bacterial species was studied using differential radiolabeling (¹⁴C/³H) of cells. When added simultaneously to cellulose, Ruminococcus flavefaciens FD1 and Fibrobacter succinogenes S85 showed some competition; however, both species were surpassed competitively by Ruminococcus albus 20. When R. flavefaciens FD1 and F. succinogenes S85 were already adherent, R. albus 20 adhesion occurred without inhibition but involved R. flavefaciens FD1 detachment. Received: 28 October 1996 / Accepted: 28 January 1997     

Degradation and fermentation of cellulose by the rumen anaerobic fungi in axenic cultures or in association with cellulolytic bacteria

Three rumen anaerobic fungi—Neocallinastix frontalis MCH3,Piromyces (Piromonas) communis FL, andCaecomyces (Sphaeromonas) communis FG10—were cultured on cellulose filter paper alone or in association with one of two rumen cellulolytic bacteria,Ruminococcus flavefaciens 007 andFibrobacter succinogenes S85. Cocultures ofN. frontalis orP. communis andR. flavefaciens were markedly less effective than the fungal monocultures in degrading cellulose but more effective than the bacterial monocultures.R. flavefaciens had an antagonistic effect against both of the fungal species. In contrast, no interaction was observed between the two fungal species andF. succinogenes. Cellulose was more effectively degraded by the cocultureC. communis-R. flavefaciens than by the corresponding fungal and bacterial monocultures. The effectiveness of degradation of the cocultureC. communis-F. succinogenes was comparable to that of the bacterial strains but greater than that of the fungi; no interaction was observed between these two microorganisms.     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

Volatile Fatty Acid Requirements of Cellulolytic Rumen Bacteria

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 μmole of valeric per ml and 0.05 μmole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 μmole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 μmole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 μmole/ml, respectively. About 0.3 μmole/ml of each acid was required for satisfactory growth of C1a.     

Influence of Forage Phenolics on Ruminal Fibrolytic Bacteria and In Vitro Fiber Degradation

In vitro cultures of ruminal microorganisms were used to determine the effect of cinnamic acid and vanillin on the digestibility of cellulose and xylan. Cinnamic acid and vanillin depressed in vitro dry matter disappearance of cellulose 14 and 49%, respectively, when rumen fluid was the inoculum. The number of viable Bacteroides succinogenes cells, the predominant cellulolytic organism, was threefold higher for fermentations which contained vanillin than for control fermentations. When xylan replaced cellulose as the substrate, a 14% decrease in the digestibility of xylan was observed with vanillin added; however, the number of viable xylanolytic bacteria cultured from the batch fermentation was 10-fold greater than that of control fermentations. The doubling time of B. succinogenes was increased from 2.32 to 2.58 h when vanillin was added to cellobiose medium, and absorbance was one-half that of controls after 18 h. The growth rate of Ruminococcus albus and Ruminococcus flavefaciens was inhibited more by p-coumaric acid than by vanillin, although no reduction of final absorbance was observed in their growth cycles. Vanillin, and to a lesser extent cinnamic acid, appeared to prevent the attachment of B. succinogenes cells to cellulose particles, but did not affect dissociation of cells from the particles. B. succinogenes, R. albus, R. flavefaciens, and Butyrivibrio fibrisolvens all modified the parent monomers cinnamic acid, p-coumaric acid, ferulic acid, and vanillin, with B. fibrisolvens causing the most extensive modification. These results suggest that phenolic monomers can inhibit digestibility of cellulose and xylan, possibly by influencing attachment of the fibrolytic microorganisms to fiber particles. The reduced bacterial attachment to structural carbohydrates in the presence of vanillin may generate more free-floating fibrolytic organisms, thus giving a deceptively higher viable count.     

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

Summary 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.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 2599-E, 1989 seriesOffprint requests to: J. Miron     

Rumen Cellulosomics: Divergent Fiber-Degrading Strategies Revealed by Comparative Genome-Wide Analysis of Six Ruminococcal Strains

Background

A complex community of microorganisms is responsible for efficient plant cell wall digestion by many herbivores, notably the ruminants. Understanding the different fibrolytic mechanisms utilized by these bacteria has been of great interest in agricultural and technological fields, reinforced more recently by current efforts to convert cellulosic biomass to biofuels.

Methodology/Principal Findings

Here, we have used a bioinformatics-based approach to explore the cellulosome-related components of six genomes from two of the primary fiber-degrading bacteria in the rumen: Ruminococcus flavefaciens (strains FD-1, 007c and 17) and Ruminococcus albus (strains 7, 8 and SY3). The genomes of two of these strains are reported for the first time herein. The data reveal that the three R. flavefaciens strains encode for an elaborate reservoir of cohesin- and dockerin-containing proteins, whereas the three R. albus strains are cohesin-deficient and encode mainly dockerins and a unique family of cell-anchoring carbohydrate-binding modules (family 37).

Conclusions/Significance

Our comparative genome-wide analysis pinpoints rare and novel strain-specific protein architectures and provides an exhaustive profile of their numerous lignocellulose-degrading enzymes. This work provides blueprints of the divergent cellulolytic systems in these two prominent fibrolytic rumen bacterial species, each of which reflects a distinct mechanistic model for efficient degradation of cellulosic biomass.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Differential Fermentation of Cellulose Allomorphs by Ruminal Cellulolytic Bacteria

In addition to its usual native crystalline form (cellulose I), cellulose can exist in a variety of alternative crystalline forms (allomorphs) which differ in their unit cell dimensions, chain packing schemes, and hydrogen bonding relationships. We prepared, by various chemical treatments, four different alternative allomorphs, along with an amorphous (noncrystalline) cellulose which retained its original molecular weight. We then examined the kinetics of degradation of these materials by two species of ruminal bacteria and by inocula from two bovine rumens. Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 were similar to one another in their relative rates of digestion of the different celluloses, which proceeded in the following order: amorphous > III_I > IV_I > III_II > I > II. Unlike F. succinogenes, R. flavefaciens did not degrade cellulose II, even after an incubation of 3 weeks. Comparisons of the structural features of these allomorphs with their digestion kinetics suggest that degradation is enhanced by skewing of adjacent sheets in the microfibril, but is inhibited by intersheet hydrogen bonding and by antiparallelism in adjacent sheets. Mixed microflora from the bovine rumens showed in vitro digestion rates quite different from one another and from those of both of the two pure bacterial cultures, suggesting that R. flavefaciens and F. succinogenes (purportedly among the most active of the cellulolytic bacteria in the rumen) either behave differently in the ruminal ecosystem from the way they do in pure culture or did not play a major role in cellulose digestion in these ruminal samples.     

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 × 10⁹ and 3.54 × 10⁹ copies per milliliter for F. succinogenes, 5.10 × 10⁷ and 7.40 × 10⁷ copies per millilter for R. Flavefaciens, and 4.00 × 10⁶ and 6.00 × 10⁶ copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 × 10⁹, 4.55 × 10⁷, and 4.56 × 10⁶ copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.     

The Effect of a Methanogen, Methanobrevibacter smithii, on the Growth Rate, Organic Acid Production, and Specific ATP Activity of Three Predominant Ruminal Cellulolytic Bacteria

Three predominant ruminal cellulolytic organisms, Fibrobacter succinogenes S85, Ruminococcus albus 8, and R. flavefaciens FD-1, were cultured with a methanogen, Methanobrevibacter smithii. Growth rates, bacterial protein, organic acids, and methane production were measured. When grown in diculture with the methanogen, a fermentative advantage was observed with F. succinogenes S85 as seen by an increase in specific rate of ATP production and organic acid concentration. The introduction of the methanogen did not improve the growth rate, organic acid yield, or specific rate of ATP production for R. albus 8. The growth rate and amount of organic acid end products increased when R. flavefaciens FD-1 was cultured with the methanogen; however, the specific activity of ATP production did not increase. Received: 3 August 1999 / Accepted: 4 September 1999     

Simple method for determination of patulin production by Penicillium griseofulvum Dierckx.

The growth of several cellulolytic species of ruminal bacteria was measured in media containing either cellobiose or cellulose as the energy source and with or without added 3-phenylpropanoic acid (PPA). With Ruminoccoccus albus 7 and 8, the addition of PPA greatly enhanced the rate of cellulose utilization but had little effect on the rate of growth when cellobiose was the energy source. Comparative rates of growth obtained on either cellobiose or cellulose for Ruminococcus flavefaciens FD1 or C94 and Butyrivibrio fibrisolvens 12, 49, or A38 were similar regardless of the PPA content of the growth medium.     

Cellulase from Ruminococcus albus and Mixed Rumen Microorganisms

Cellulase in the cultural filtrates of Ruminococcus albus and cellulase extracted from mixed rumen microorganisms were investigated with acid-swollen cellulose and carboxymethylcellulose as substrates. Maximal activity occurred at approximately pH 5.8 and 47 C. Apparent Michaelis constants (K_m) varied between 0.53 and 0.02% carboxymethylcellulose, depending on the level of activity and the method of assay. R. albus cellulase has a lower K_m value than the enzyme extracted from mixed rumen microorganisms. Antisera from rabbits immunized with a cellulase preparation from R. albus inhibited the cellulolytic activity of both systems. Based on the relative degree of inhibition, approximately 20% of the cellulase of the mixed rumen microorganisms was immunologically similar to R. albus cellulase. Ratios of activity in different assay techniques showed the two sources of activity to be similar in the mechanisms of degradation. However, glucose is the main product of cellulose degradation by mixed rumen microorganisms, and cellobiose is the product of degradation by R. albus.     

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.     

Biochanin A improves fibre fermentation by cellulolytic bacteria

Aims

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity.

Methods and Results

When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short‐chain fatty acids were produced and pH declined. BCA (30 μg ml^?1) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent.

Conclusions

These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition.

Significance and Impact of the Study

BCA could serve as a feed additive to improve cellulosis when cattle are consuming high‐fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.     

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH₄⁺. B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.     

Effects of nitrate on methane production, fermentation, and microbial populations in in vitro ruminal cultures

The objective of this study was to examine the effects of nitrate on methane production, important fermentation characteristics, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, total bacteria, and methanogens using in vitro ruminal cultures. Potential adaptation of the above microbes and persistency of nitrate to mitigate CH₄ production were also evaluated. Methane production was reduced by 70% at 12 μmol ml⁻¹ and nearly completely at ?24 μmol ml⁻¹ nitrate. Production of volatile fatty acids (VFAs) was affected to different extents at different nitrate concentrations. Over a series of six consecutive cultures receiving 12 μmol ml⁻¹nitrate, production of CH₄ and VFA did not change significantly. R. albus and R. flavefaciens seemed to adapt to nitrate, while F. succinogenes and methanogens did not. Nitrate may be used in achieving persistent mitigation of CH4 production by ruminants.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.