The effect of the carbohydrate growth substrate on the glycosidase activity of hemicellulose-degrading rumen bacterial isolates

Thirteen strains of hemicellulose-degrading bacteria isolated from the rumen were grown on ten different monosaccharide, disaccharide and hemicellulosic poly-saccharide substrates in vitro , and the range and specific activities of the glycosidase enzymes formed were determined. Alkaline phosphatase activities were also measured. The specific activities were affected by the carbohydrate source supplied in the growth medium and were usually reduced in glucose grown cells, and less frequently following growth on cellobiose; the responses to other substrates were both enzyme and organism dependent. In several of the strains examined many of the enzymes were found following growth on all of the substrates tested, although there were wide variations in the specific activities. The activities of several of the glycosidases were increased after growth on hemicellulosic polysaccharides.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture

Bacteroides ovatus was grown in batch culture on 12 different carbon sources (five polysaccharides, seven monosaccharides and disaccharides). Specific growth rates were determined for each substrate together with polysaccharidase and glycosidase activities. Growth rates on polymerized carbohydrates were as fast or faster than on corresponding simple sugars, demonstrating that the rate of polysaccharide depolymerization was not a factor limiting growth. Bacteroides ovatus synthesized a large range of polymer-degrading enzymes. These polysaccharidases and glycosidases were generally repressed during growth on simple sugars, but arabinose was required for optimal production of alpha-arabinofuranosidase. Polysaccharidase and glycosidase activities were measured in continuous cultures grown with either xylan or guar gum under putative carbon limitation. With the exception of beta-xylosidase, activities of the polymer-degrading enzymes were inversely related to growth rate. This correlated with polysaccharide utilization which was greatest at low dilution rates. These results show that Bact. ovatus is highly adapted for growth on polymerized carbohydrate in the human colon and confirm that the utilization of polysaccharides is partly regulated at the level of enzyme synthesis.     

Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture

Bacteroides ovatus was grown in batch culture on 12 different carbon sources (five polysaccharides, seven monosaccharides and disaccharides). Specific growth rates were determined for each substrate together with polysaccharidase and glycosidase activities. Growth rates on polymerized carbohydrates were as fast or faster than on corresponding simple sugars, demonstrating that the rate of polysaccharide depolymerization was not a factor limiting growth. Bacteroides ovatus synthesized a large range of polymer-degrading enzymes. These polysaccharidases and glycosidases were generally repressed during growth on simple sugars, but arabinose was required for optimal production of α-arabinofuranosidase. Polysaccharidase and glycosidase activities were measured in continuous cultures grown with either xylan or guar gum under putative carbon limitation. With the exception of β-xylosidase, activities of the polymer-degrading enzymes were inversely related to growth rate. This correlated with polysaccharide utilization which was greatest at low dilution rates. These results show that Bact. ovatus is highly adapted for growth on polymerized carbohydrate in the human colon and confirm that the utilization of polysaccharides is partly regulated at the level of enzyme synthesis. and accepted 8 June 1989     

Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates

The rumen fungi Neocallimastix patriciarum, Piromonas communis, and a morphologically distinct but unidentified isolate were cultivated on the polysaccharides starch, cellulose, xylan, and their principal component monosaccharides and disaccharides, and the range and specific activities of the glycoside hydrolases formed were monitored using gluco-oligosaccharide and p-nitrophenyl glycoside substrates. A wide range of enzyme activities was detected in preparations from vegetative growth and zoospores of all three isolates. Enzyme activity was also present in the culture medium. The specific activities were affected by the carbohydrate source available in the growth medium, although the more active hydrolases involved in the degradation of plant structural and storage polysaccharides were formed on all seven carbohydrate sources evaluated. Enzyme activities were increased in the zoospore, vegetative, and extracellular preparations after growth on the appropriate structurally related disaccharide or polysaccharide. The hemicellulolytic glycosidases (alpha-L-arabinofuranosidase, beta-D-xylosidase) were most active after growth on xylan, whereas alpha-/beta-glucosidase activity was increased with the corresponding glucan as growth substrate. However, whereas wide-ranging beta-glucosidase activity was detected following growth on maltose or starch, the alpha-glucosidase activities of P. communis were lower or undetectable in vegetative preparations grown on glucose or the beta-glucans cellobiose and cellulose.     

The production of plant cell wall polysaccharide-degrading enzymes by hemicellulolytic rumen bacterial isolates grown on a range of carbohydrate substrates

Several cultures of bacteria, isolated from the rumen, that were able to utilize plant cell wall structural polysaccharides were grown on a range of carbohydrate substrates and the activities of the principal polysaccharide-degrading enzymes determined. The esterase activity was also monitored. The extent of hemicellulose degradation and utilization by the isolates was comparable with that of the hemicellulolytic type strains. Enzyme activities in all of the cultures examined were affected by the carbon source in the growth medium. Many responses were strain specific, although growth on glucose (or cellobiose and maltose to a lesser extent) resulted in reduced activities in most of the organisms examined, whilst polysaccharidic substrates resulted in higher levels of the appropriate polysaccharidase. However, enzyme activity was detectable in some isolates after culture on mono- or disaccharides in the absence of the principal or related polysaccharide substrate.     

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (1%–2% of total acid production) of branched and straight-chain C₃–C₅ volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.     

Dilution rate increases production of plant cell-wall degrading enzymes by anaerobic fungi in continuous-flow culture

The gut anaerobic fungi,Neocallimastix hurleyensis and aOrpinomyces sp., were grown in 100 mL batch and continuous-flow cultures on wheat straw at a concentration of 80 g dry matter/L of culture liquid. In batch cultures,N. hurleyensis and Orpinomyces sp. degraded only ca. 9% and 5% of the wheat straw, respectively. In continuous-flow cultures, however, the two fungi degraded 52-56% of the apparent dry matter of wheat straw. Both fungi were able to produce greater quantities (up to x 30) of cell-wall degrading enzymes (CMCase, xylanase, beta-glucosidase and beta-xylosidase) in continuous-flow cultures than in the corresponding batch cultures. Increasing the dilution rate in continuous-flow culture resulted in the production of increased enzyme activity for all the measured cell-wall degrading enzymes, with proportional relationships between dilution rate and the cumulative activities of beta-glucosidase and beta-xylosidase. Dilution rates, however, had no consistent effect on the cumulative production of the fermentation end-products, acetate, formate, D- and L-lactate from both fungi. In addition to acetate and formate,N. hurleyens is produced D- and L-lactate in both batch and continuous-flow cultures, whereas only trace amounts of L-lactate were detected in the Orpinomyces sp. cultures.     

Polysaccharide-degrading enzymes formed by three species of anaerobic rumen fungi grown on a range of carbohydrate substrates

The range of polysaccharide-degrading enzymes formed by three anaerobic rumen fungi (Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate (F] was monitored following growth on seven mono-, di-, and poly-saccharide carbohydrate substrates. Enzymes capable of degrading a variety of alpha- and beta-glucans, beta-galactans, galactomannan, and hemicellulosic arabinoxylans were present in all three isolates. Although reducing saccharides were released from pectin, polygalacturonic acid was not degraded by the preparations. Enzyme activity was present in both the zoospore and vegetative stages of the life cycle and was also detected extracellularly in culture supernatants after vegetative growth. The specific activities of the polysaccharidases were affected by the growth substrate, being lowest in preparations grown on mono- and di-saccharides, whereas polysaccharidic growth substrates resulted in increased activity of the corresponding polysaccharidases. The enzymes were, however, formed after growth on all substrates. Oligomers and monosaccharides were produced as a result of polysaccharide breakdown by the unfractionated enzyme preparations. Studies on hemicellulose (arabinoxylan) breakdown by unfractionated vegetative preparations of the three isolates indicated that their modes of action, pH optima, substrate affinities, and response to potential inhibitors were similar.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

The production of hemicellulose-degrading enzymes byBacillus macerans in anaerobic culture

Summary The cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h^-1) although the activity was reduced at higher dilution rates (0.125–0.15 h^-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism.     

Utilization of Chondroitin Sulfate by Bacteroides thetaiotaomicron Growing in Carbohydrate-Limited Continuous Culture

When Bacteroides thetaiotaomicron, an obligate anaerobe from the human colonic flora, was grown in continuous culture with the mucopolysaccharide chondroitin sulfate as the limiting source of carbohydrate, growth yields ranged from 48 g of cell dry weight per mol of equivalent monosaccharide at a growth rate of 3.5 h per generation to 32 g per mol at a growth rate of 24 h per generation. The theoretical maximum growth yield (61 g of cell dry weight per mol of equivalent monosaccharide) was comparable to that of 54 g per mol, which was obtained previously when glucuronic acid, a component of chondroitin sulfate, was the limiting carbohydrate (S. F. Kotarski and A. A. Salyers, J. Bacteriol. 146:853-860, 1981). However, the maintenance coefficient was three times higher when chondroitin sulfate was the substrate than when glucuronic acid was the substrate. The specific activity of chondroitin lyase (EC 4.2.2.4), an enzyme which cleaves chondroitin sulfate into disaccharides, declined by nearly 50% as growth rates decreased from 3.5 to 24 h per generation. By contrast, the specific activities of several glycolytic enzymes and disaccharidases remained constant over this range of growth rates. Although chondroitin sulfate was growth limiting, some carbohydrate was detectable in the extracellular fluid at all growth rates. At rapid growth rates (1 to 2 h per generation), this residual carbohydrate included fragments of chondroitin sulfate having a wide range of molecular weights. At slower growth rates (2 to 24 h per generation), the residual carbohydrate consisted mainly of a small fragment which migrated on paper chromatograms more slowly than the disaccharides produced by chondroitin lyase but faster than a tetrasaccharide. This small fragment may represent the reducing end of the chondroitin sulfate molecule.     

Nitrogenase activity and growth of Frankia in batch and continuous culture.

Frankia grown in batch culture was unable to maintain a high rate of nitrogenase activity and, once a peak level was reached, activity rapidly declined. Addition of 5 mM carbon source of cultures or transfer to fresh medium was followed by brief recovery of nitrogenase activity. The extent of recovery decreased as additions or transfers were made to progressively older cultures. Daily addition of fresh medium (dilution rate = 0.125 day-1) allowed Frankia to be maintained in continuous, derepressed culture with stable rates of growth and nitrogenase activity for more than 30 days. The proportion of active, mature vesicles also remained constant in continuous culture but decreased with time in batch culture.     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.