Rate of isolated hemicellulose degradation and utilization by pure cultures of rumen bacteria

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.     

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.     

Esterase activity of pure cultures of rumen bacteria as expressed by the hydrolysis of p-nitrophenylpalmitate

Seventy-four strains of rumen bacteria comprising 20 genera were tested for the ability to hydrolyze p-nitrophenylpalmitate (PNPP-C16). This ability was detectable in all cultures tested, but the level of activity was quite variable. Known lipolytic strains of these bacteria showed generally low levels of activity in this assay, which suggests that the hydrolysis of this artificial substrate indicates a general esterase activity and not a lipase activity, as reported in the literature. The highest activity was found to occur in strains known to be feed-particle-associated digesters of starch, pectin and cellulose. In fractionated rumen contents, p-nitrophenylpalmitase activity was largely associated with feed particles. Although the in vivo role of the enzymes that hydrolyze PNPP-C16 remains obscure, it appears that they are primarily of microbial origin, and may be important in hydrolyzing ester bond-containing compounds from plant material.     

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.     

Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation

The formation of water soluble vitamins (vitamin B₁₂, vitamin B₆, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus , and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B₆. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B₁₂. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B₁₂ production, resulted in an increase of physiologically active vitamin B₁₂. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B₆ in addition to the moulds. The influence of Rhizopus on the vitamin B₁₂ formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.     

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     

Effects of sudden temperature shifts on pure cultures of four strains of freshwater bacteria

Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified asCytophaga johnsonae (C-21),Cytophaga sp. (M-17),Pseudomonas fluorescens (KD), andEnterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. TheC. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereasCytophaga sp. (M-17) shift-up cultures showed a lower andP. fluorescens (KD) a higher growth rate. The mesophilicE. cloacae (BS-2), likeC. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion byP. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase.     

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.     

Metabolism of pectin in rumen bacteria Butyrivibrio fibrisolvens and Prevotella ruminicola

Cultures of Butyrivibrio fibrisolvens 787 and Prevotella ruminicola AR29 grown on pectin produced significantly more acetate and less butyrate, lactate, succinate and hydrogen than corresponding cultures grown on l -arabinose and d -glucose. In both bacteria, fermentation of pectin and arabinose yielded less lactate than fermentation of glucose. Pectin-grown cells of these strains possessed activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4·1.2·14), an enzyme catalysing the pyruvate formation in the Entner–Doudoroff pathway. The activity of fructosediphosphate aldolase (EC 4·1.2·13) was found both in cells cultivated on glucose and on pectin. The phosphoketolase activity (EC 4·1.2·9) could not be detected in rumen bacterial strains studied.     

Studies on the methylation of mercuric chloride by pure cultures of bacteria and fungi

Small amounts of methylmercury were produced during 7 days aerobic growth in the presence of sublethal amounts of mercuric chloride by the following bacterial species studied:Pseudomonas fluorescens, Mycobacterium phlei, Escherichia coli, Aerobacter aerogenes, Bacillus megaterium. Under the same conditions methylmercury was also formed by mycelium of the fungi investigated:Aspergillus niger, Scopulariopsis brevicaulis andSaccharomyces cerevisiae. The concentration of the methylmercury produced by the various organisms did not vary much and was of the same order of magnitude as that found in Swedish experiments with lake sediments. In bacteria most of the methylmercury formed was present in the culture liquid, whereas the remainder was in or on the cells. In contrast, methylmercury formed by fungi was for the greater part present in the mycelium. The production of methylmercury byE. coli andA. aerogenes was lower under anaerobic conditions than under aerobic conditions.     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

A note on fermentation reactions of anaerobic bacteria on a solid medium

A new technique is described for fermentation tests of anaerobes in which the fermentation reactions are carried out on cystine trypticase agar plates to which fermentable substances (Taxo discs) are added. This technique is simple, sensitive and reproducible.     

Acidogenic fermentation of pectin by a mixed population of bacteria in continuous culture

Summary Pectin, dissolved in a mineral salts solution, was degraded anaerobically by a mixed population of bacteria in chemostat cultures (pH=6, T=30°C). At a dilution rate of 0.3 h^–1, the specific volume activity was 45.7 (g substrate). (l reactor)^–1.d^–1.