Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.     

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.     

Cellodextrin efflux by the cellulolytic ruminal bacterium Fibrobacter succinogenes and its potential role in the growth of nonadherent bacteria.

When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was grown in a cellobiose-limited chemostat, cellobiose and cellotriose could both be detected, and the ratio of cellotriose to cellobiose was approximately 1 to 4. On the basis of these results, cellodextrin production is an equilibrium (mass action) function and not just an artifact of energy-rich cultural conditions. Cellodextrins could not be detected in low-dilution-rate, cellulose-limited continuous cultures, but these cultures had a large number of nonadherent cells. Because the nonadherent cells had a large reserve of polysaccharide and were observed at all stages of cell division, it appeared that they were utilizing cellodextrins as an energy source for growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial activity of 2-aminophenoxazin-3-one under anaerobic conditions.

Potential toxicity of 2-aminophenoxazin-3-one to 20 bacterial species and two fungi and its inactivation under anaerobic conditions were investigated. Minimum inhibitory concentration for cellulolytic bacteria was in the range of 50-100 micrograms.mL-1, but at 100 micrograms.mL-1 of 2-aminophenoxazin-3-one, there was no effect on the growth of any of the noncellulolytic bacteria. Four noncellulolytic bacterial strains showed no inhibition of growth, even at 200 micrograms.mL-1 of this compound. Under anaerobic conditions and in the presence of cysteine, the long wavelength absorption band of this compound slowly shifted from about 434 to 320 nm, and its inhibitory effect on RNA synthesis was relieved after one-half of a generation time in cultures of cellulolytic ruminal bacteria. A similar shift of absorption band was observed in rumen fluid filtered through a 0.22-microns Millipore filter. It was concluded that protonation of 2-aminophenoxazin-3-one under anaerobic conditions present in the rumen would considerably reduce its potential toxicity to cellulolytic bacteria.     

Oligosaccharide synthesis in Fibrobacter succinogenes S85 and its modulation by the substrate

In this article we compared the metabolism of phosphorylated and unphosphorylated oligosaccharides (cellodextrins and maltodextrins) in Fibrobacter succinogenes S85 resting cells incubated with the following substrates: glucose; cellobiose; a mixture of glucose and cellobiose; and cellulose. Intracellular and extracellular media were analysed by (1)H-NMR and by TLC. The first important finding is that no cellodextrins were found to accumulate in the extracellular media of cells, regardless of the substrate; this contrasts to what is generally reported in the literature. The second finding of this work is that maltodextrins of degree of polymerization > 2 are synthesized regardless of the substrate, and can be used by the bacteria. Maltotriose plays a key role in this metabolism of maltodextrin. Maltodextrin-1-phosphate was detected in all the incubations, and a new metabolite, corresponding to a phosphorylated glucose derivative, was produced in the extracellular medium when cells were incubated with cellulose. The accumulation of these phosphorylated sugars increased with the degree of polymerization of the substrate.     

Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose

To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic Treponema bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the production of succinic acid, lactic acid, and ethanol. Morphological examination, by electron microscopy, showed that cells of T. bryantii associate with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.     

Sorbose mediated enhancement of cellulase biosynthesis inTrichoderma reesei

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.     

Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.     

Carbohydrate Transport by the Anaerobic Thermophile Clostridium thermocellum LQRI

Clostridium thermocellum is an anaerobic thermophilic bacterium which degrades cellulose and ferments the resulting glucose, cellobiose, and cellodextrins predominantly to ethanol. However, relatively little information was available on carbohydrate uptake by this bacterium. Washed cells internalized intact oligomers as large as cellopentaose. Since cellobiose and cellodextrin phosphorylase activities were detected in the cytosol and were not associated with cell membranes, phosphorylation of carbohydrates occurred intracellularly. Kinetic studies indicated that cellobiose and larger cellodextrins were taken up by a common uptake system while glucose entered via a separate mechanism. When cells were treated with metabolic inhibitors including iodoacetate and arsenate, the uptake of radiolabeled glucose or cellobiose was reduced by as much as 90%, and this reduction was associated with a 95% decline in intracellular ATP content. A combination of the ionophores nigericin and valinomycin abolished the proton-motive force but only slightly decreased transport and ATP. These results suggested that the two modes of carbohydrate transport in C. thermocellum were ATP dependent. This work is the first demonstration of cellodextrin transport by a cellulolytic bacterium.     

Fermentation and aerobic metabolism of cellodextrins by yeasts.

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Establishment of Bacteroides succinogenes and measurement of the main digestive parameters in the rumen of gnotoxenic lambs

We attempted to determine the degree of diversification of the microflora that allow the establishment of Bacteroides succinogenes S85 in the rumen of gnotoxenic lambs. Four lambs (group I) received an inoculum orally, composed of 182 noncellulolytic bacterial strains (inoculum 1) previously isolated from the rumen of conventional young lambs. Two lambs (group II) were inoculated with 32 strains (inoculum 2) selected among the 182 strains of inoculum 1. Two lambs (group III) received an inoculum (inoculum 3) composed of 106 noncellulolytic bacterial strains previously isolated from the rumen of meroxenic lambs. Two lambs (group IV) were inoculated with 16 strains (inoculum 4) chosen among the 106 strains of inoculum 3. All lambs were inoculated from birth except two lambs of group I, which were inoculated from 1 month of age. Each lamb then received orally a pure culture of B. succinogenes. This strain became established more easily in the rumen of lambs that had received complex inocula (group I). Its population reached a level close to that generally observed in conventional lambs (10(7)-10(8) bacteria.mL-1). In contrast, B. succinogenes became established in only one lamb of group II, but bacterial numbers varied considerably. In group III, repeated inoculations were necessary to obtain its definitive establishment (10(7)-10(8) bacteria.mL-1 after weaning). In spite of several inoculations, this cellulolytic species failed to establish in the rumen of lambs of group IV, which had received the less complex inoculum. The volatile fatty acid levels were very different from one lamb group to another. The more complex the inoculum administered to the animals, the higher the concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fermentation and aerobic metabolism of cellodextrins by yeasts

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Cellobiose transport by mixed ruminal bacteria from a Cow.

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 microM for the Km and 10 nmol/min/mg of protein for the Vmax. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher Vmax values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters

Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6 kJ mol⁻¹). A thermodynamic “push” from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.     

In vivo 13C NMR study of glucose and cellobiose metabolism by four cellulolytic strains of the genus Fibrobacter

The metabolism of glucose and cellobiose, products of cellulose hydrolysis, was investigated in four cellulolytic strains of the genus Fibrobacter: Fibrobacter succinogenes S85, 095, HM2 and Fibrobacter intestinalis NR9. In vivo 13C nuclear magnetic resonance was used to quantify the relative contribution of glucose and cellobiose to metabolite production, glycogen storage and cellodextrins synthesis in these four strains. The same features were found in all four strains of the genus Fibrobacter metabolizing simultaneously glucose and cellobiose: i) differential metabolism of glucose and cellobiose; glucose seems preferentially used for glycogen storage and energy production, while part of cellobiose seems to be diverted from glycolysis, ii) synthesis of cellodextrins, mainly from cellobiose not entering into glycolysis, iii) accumulation of glucose 6-phosphate, iv) simultaneous presence of cellobiose phosphorylase and cellobiase activities.Although genetically diverse, the Fibrobacter genus appears to possess a marked homogeneity in its carbon metabolism.     

Fermentation of Cellodextrins by Different Yeast Strains

The fermentation of cellodextrins by eight yeast species capable of fermenting cellobiose was monitored. Only two of these species, Torulopsis molischiana and T. wickerhamii, were able to ferment β-glucosides with a degree of polymerization between one and six. These two species showed exocellular β-glucosidase activity. Four other species were able to ferment cellotriose, and the last two species only fermented cellobiose. These latter six species produced a β-glucosidase capable of attacking cellodextrins, but this enzyme was endocellular.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Cellobiose Transport by Mixed Ruminal Bacteria from a Cow

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the K_m and 10 nmol/min/mg of protein for the V_max. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher V_max values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.