Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana

Summary The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^–1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p-nitrophenyllactoside (p-NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p-NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone. Correspondence to: G. Canevascini     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Influence of carbon source on production of a heat stable protease from Thermomonospora fusca YX

Summary Thermomonospora fusca YX produced a very active heat stable protease when incubated in media containing cellulose as the substrate. Cultures grown on Solka-floc generated the highest amount of protease whereas the protease was produced at significantly lower levels when T. fusca YX was grown on cellobiose or glucose. Negligible growth or protease production was observed when protein was used as a carbon source. The production of the protease did not appear to be constitutive. While rapid growth was observed on either cellobiose or glucose, protease levels were at least two to fourfold lower than for the T. fusca YX cultures grown on Solka-floc wich generated 33% less cell mass. Protease production was four times lower in cultures which employed casein hydrolysate (tryptone) or xylan as carbon sources than for cellulose.     

Kinetic models for growth and product formation on multiple substrates

Hydrolyzates from lignocellulosic biomass contain a mixture of simple sugars; the predominant ones being glucose, cellobiose and xylose. The fermentation of such mixtures to ethanol or other chemicals requires an understanding of how each of these substrates is utilized.Candida lusitaniae can efficiently produce ethanol from both glucose and cellobiose and is an attractive organism for ethanol production. Experiments were performed to obtain kinetic data for ethanol production from glucose, cellobiose and xylose. Various combinations were tested in order to determine kinetic behavior with multiple carbon sources. Glucose was shown to repress the utilization of cellobiose and xylose. However, cellobiose and xylose were simultaneously utilized after glucose depletion. Maximum volumetric ethanol production rates were 0.56, 0.33, and 0.003 g/L-h from glucose, cellobiose and xylose, respectively. A kinetic model based on cAMP mediated catabolite repression was developed. This model adequately described the growth and ethanol production from a mixture of sugars in a batch culture.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Regulation of xylanase activity in cellulomonas sp. IIbc

The regulation mechanism governing the xylanolytic activity in the strain Cellulomonas sp. IIbc was studied. High levels of activity were detected during the cultivation on cellulose as the only carbon source. No activity was produced with glucose, xylose or cellobiose cultures, but in the last one, the synthesis was de-repressed when the sugar concentration dropped to 0.2%. The activity was not inhibited by glucose, cellobiose and xylose up to 1% concentration. A basal constitutive synthesis was detected in nutrient broth cultures. At the same time, xylose and cellobiose acted as inducers of the xylanase activity.     

Metabolism of cellobiose and cellulose byBacteroides cellulosolvens

Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y _H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY _H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY _H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY _H.Issued as NRCC No. 27409.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Utilization of glucose and cellobiose by the microflora of soil enriched with cellulose

The ability of soil microflora to utilize glucose or celloboise was found to depend on previous incubation of the soil with glucose, celloboise or cellulose. Glucose was utilized more rapidly than cellobiose in soil preincubated with glucose or cellobiose. The opposite situation was observed in soil preincubated with cellulose. In the presence of a mixture of both sugars the rate of utilization of one of them was decreased by the second and this decrease could be characterized as competitive inhibition. Glucose accumulated in the medium during utilization of cellobiose alone in soil preincubated with cellulose. This phenomenon was not observed during the utilization of cellobiose in soil preincubated with glucose or cellobiose.     

Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled batch culture

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T.thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.Correspondence to: G. M. Cook     

Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family.

A mesophilic anaerobe, a member of the Bacteroidaceae family (NRC2248), isolated from a cellulose-enrichment culture, digested untreated Whatman cellulose powder and HCl-treated cotton battings while producing hydrogen, carbon dioxide, cellobiose, glucose, and acetic acid as the sole volatile acid. This organism also utilized cellobiose as carbon and energy source but did not utilize glucose. It grew well in synthetic medium containing ammonium salts as nitrogen source and having a pH value of 7.0-7.1 and an Eh value of -160mV or lower. The nutrient requirements of this organism were found to be similar to those of other anaerobes except for Na2S which inhibited cellulose degradation in concentrations above 0.75 mM. Best cellulose degradation occurred under an atmosphere of 80% N2-20% CO2. Use of H2 or 80% H2-20% CO2 as headspace gas inhibited growth. Although accumulation of acetic acid in about 15-16 mM concentrations inhibited the further formation of H2, CO2, and acetic acid in the broth, it did not stop the degradation of cellulose. The results indicate that this organism has the ability to grow in media containing up to 20 g/L of cellulose and to produce industrially important and easily separable end products from cellulose.     

Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter xylinum

The effect of various carbon and nitrogen sources on cellulose membrane production by Acetobacter xylinum was evaluated. Among the carbon sources, sucrose, glucose and mannitol were found to be suitable for optimum levels of cellulose production. The strain was able to utilize a wide range of protein and nitrogen sources such as peptone, soybean meal, glycine, casein hydrolysate, and glutamic acid for cellulose synthesis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pellicle proteins (PP) revealed electrophoretic bands of molecular masses in the range of 116–20 kDa. Furthermore, the strain can be useful for the removal of various nitrogenous and carbon substrates present in waste waters.     

Utilization of carbohydrates by Thermomonospora sp. Grown on glucose,cellobiose, and cellulose

Thermomonospora sp. was grown on glucose, cellobiose, and in order to study its growth characteristics with different carbohydrate substrates and to assess the validity of some of the assumptions made in a previously proposed model for the cellulose fermentation with this microorganism. It was observed that the nitrogen and protein contents of the cells are essentially constant during the fermentation and independent of the carbon source when glucose or cellobiose are utilized. Under oxygen starvation conditions it was shown that unidentification organic compound(s) accumulate(s) in the culture broth. Culture fluorescence was shown to be an excellent variable for monitoring and control of the fermentation process. This microorganism showed a preference for crystalline cellulose (Avicel) as substrate although it grows readily on a more amorphous cellulose (Solka Floc). The production of extra cellular protein is shown to be growth related. Data were obtained confirming the decrease in the number of active adsorption sites as the cause for the decrease in the cellulose digestion rate. It is suggested that a future model should account for the time change of surface characteristics of the cellulose particles.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Characterization of inducible cold-active β-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-Antarctic ecosystem

The psychrotolerant bacterium Shewanella sp. G5 was used to study differential protein expression on glucose and cellobiose as carbon sources in cold-adapted conditions. This strain was able to growth at 4 °C, but reached the maximal specific growth rate at 37 °C, exhibiting similar growing rates values with glucose (μ: 0.4 h⁻¹) and cellobiose (μ: 0.48 h⁻¹). However, it grew at 15 °C approximately in 30 h, with specific growing rates of 0.25 and 0.19 h⁻¹ for cellobiose and glucose, respectively. Thus, this temperature was used to provide conditions related to the environment where the organism was originally isolated, the intestinal content of Munida subrrugosa in the Beagle Channel, Fire Land, Argentina. Cellobiose was reported as a carbon source more frequently available in marine environments close to shore, and its degradation requires the enzyme β-glucosidase. Therefore, this enzymatic activity was used as a marker of cellobiose catabolism. Zymogram analysis showed the presence of cold-adapted β-glucosidase activity bands in the cell wall as well as in the cytoplasm cell fractions. Two-dimensional gel electrophoresis of the whole protein pattern of Shewanella sp. G5 revealed 59 and 55 different spots induced by cellobiose and glucose, respectively. Identification of the quantitatively more relevant proteins suggested that different master regulation schemes are involved in response to glucose and cellobiose carbon sources. Both, physiological and proteomic analyses could show that Shewanella sp. G5 re-organizes its metabolism in response to low temperature (15 °C) with significant differences in the presence of these two carbon sources.     

Cellulolytic and physiological properties of Clostridium thermocellum

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, -cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 moles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 mole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.