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     

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with ¹³C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of ¹³C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.     

Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting

Fibrobacter succinogenes S85 grew rapidly on cellobiose (0.31 h⁻¹) and the absolute rate of increase in fermentation acids was 0.68 h⁻¹. Cultures that were provided with ball-milled cellulose initially produced fermentation acids and microbial protein as fast as those provided with cellobiose, but the absolute cellulose digestion rate eventually declined. If the inoculum size was increased, the kinetics decayed from first to zero order (with respect to cells) even sooner, but in each case the absolute rate declined after only 20 to 30% of the cellulose had been fermented. Congo red binding indicated that the cellulose surface area of individual cellulose particles was not decreasing, and the transition of ball-milled cellulose digestion corresponded with the appearance of unbound cells in the culture supernatant. When bound cells from partially digested cellulose were removed and the cellulose was re-incubated with a fresh inoculum, the initial absolute fermentation rate was as high as the one observed for undigested cellulose and cellobiose. Based on these results, cellulose digestion by F. succinogenes S85 appears to be constrained by cellulose surface area rather than cellulase activity per se. Received: 19 January 2000 / Received revision: 18 April 2000 / Accepted: 1 May 2000     

Conversion of cellulose to sugars and cellobionic acid by the extracellular enzyme system of Chaetomium cellulolyticum

Summary Hydrolyses of cellulosics by crude enzyme yielded glucose, cellobiose, and cellobionic acid. A cellobiose oxidizing enzyme was detected in culture broth of Chaetomium cellulolyticum and identified as a cellobiose dehydrogenase.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014–0.076 h^-1) and extracellular pH (6.11–6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentrationto response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07–0.11 h^-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04–0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11–0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determined that maintenance coefficient (0.04–0.06 g cellulose/g cells · h) and true growth yield (0.23–0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower _max on microcrystalline cellulose.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

The Effect of Cellobiose,Glucose, and Cellulose on the Survival of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> A3C Cultures Grown Under Ammonia Limitation

The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation. If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster. The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide. Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein^–1, but ATP could not be detected in cultures that had an excess of soluble carbohydrates. However, if F. succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added. From these results, it appears that excess soluble carbohydrates can promote the death of F. succinogenes, but cellulose does not.     

The degradation and utilization of wheat-straw cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Cell walls (CW) of untreated wheat straw and sulphur-dioxide (SO₂)-treated wheat straw were used as model substrates for the hydrolysis and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Fibrobacter succinogenes S85 and BL2 strains and their co-cultures with D1 were the best degraders of CW among ruminal cultures, solubilizing 37.2–39.6% of CW carbohydrates of untreated straw and 62.2–74.5% of SO₂-treated straw. Complementary action between Butyrivibrio fibrisolvens D1 and the F. succinogenes strains was identified with respect to co-culture growth and carbohydrate utilization. However, the extent of CW solubilization was determined mainly by the F. succinogenes strains. In both substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of xylan and hemicellulose: 96.5–98.3%, 34.4–40.5% and 33.5–36.2%, respectively. Under scanning electron microscopy visualization, S85 and BL2 cells of the co-cultures comprised the most dense layer of bacterial cell mass attached to and colonized on straw stems and leaves, whereas D1 cells were always nearby. Stems and leaves of the untreated straw were less crowded by attached bacteria than that of the SO₂-treated straw. In both materials, the cell surface topography of S85 and BL2 bacteria attached to CW particles was specified by a coat of characteristic protuberant structures, polycellulosome complexes.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate‐active enzymes with versatile polysaccharide‐degrading capacity

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate‐active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non‐cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV‐pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose‐degrading enzyme cocktail by 2.4‐fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro‐slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.     

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose 1^–1 and 4.4 g ethanol 1^–1 were converted to 2.6 g butyrate 1^–1 and 4.6 g caproate 1^–1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.Mention of specific products is intended only to provide information and does not contitute an endorsement by the U.S. Department of Agriculture over other products not mentioned.     

Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85

Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains d-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove d-limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.     

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium

Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85.

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Kinetics of the utilization of different C sources and the cellulose formation by Acetobacter xylinum

Different methylated glucose derivatives and cellobiose were examined as the carbon sources for growth and cellulose formation by Acetobacter xylinum. HPLC studies were carried out to gain information about the kinetics of the utilization of the C sources used. The type and yields of the synthesized cellulose were described. Besides glucose, cellobiose was a substrate for the synthesis of this polysaccharide by the bacteria. Other methylated derivatives of glucose were not accepted for a comparable synthesis of this polymer. An estimation of citrate in an unmodified culture liquid (SH medium) showed utilization in a late phase of cultivation. The influence of this organic acid on the pH value, cellulose synthesis and growth is described. By the application of citric acid as a sole carbon source “gel-like” forms of cellulose were formed generally.     

Yersinia kristensenii: A new species of enterobacteriaceae composed of sucrose-negative strains (formerly called atypicalYersinia enterocolitica orYersinia enterocolitica-Like)

Induction of cellulase was observed inFusarium sp. with reduction in lag period by lactose-pregrown cells as compared with glucose-pregrown cells. Insoluble cellulose (Sigmacell) induced maximum cellulase production in the induction medium. Supplementation of the culture growing on cellulose by cellobiose or glucose resulted in increased cellular growth and decreased cellulase production. Stepfeeding of cellobiose to the culture growting on carboxymethyl cellulose resulted in decreased cellulase production. Significant cellulase activity was detected in the culture filtrate of cells growing on Sigmacell supplemented with glucose, only when the glucose disappeared from the medium. This suggests that cellulase production may in part be regulated by catabolite repression.     

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