The effect of Aspergillus oryzae fermentation extract on the anaerobic fungus Neocallimastix frontalis EB 188: effects on physiology

 Aspergillus oryzae fermentation extract (Amaferm) was used to stimulate the in vitro growth of the cellulolytic fungus Neocallimastix frontalis EB 188. Soluble and filter-sterilized extract was added either at the start or throughout culture growth. Culture mass, protein secretion and cellulase secretion were measured in stationary test-tubes or round-bottom flasks and a stirred (desktop) fermenter. The soluble extract increased culture mass and protein and cellulase secretion in a dose-dependent manner. Maximum stimulation caused the supernatant cellulase to nearly double (i.e., 87% over controls; P<0.05), cell mass increased by 27% (P<0.05) over controls and secreted protein increased 37% (P<0.05) over controls. The timing of extract addition did not alter the culture response and suggested a recycling of components. The robustness of fungal zoospores used as inoculum, however, greatly influenced the effectiveness of the extract to stimulate secretions. Extracts did not directly influence the pH of the culture medium or the endogenous levels of enzymes. The rate of carbon source utilization and morphology of the fungus were unchanged by soluble-extract additions at any level tested. The extract was inhibitory when added to concentrations exceeding an amount equivalent to 20 g animal^-1 day^-1. Received: 6 December 1995/Received revision: 7 February 1996/Accepted: 4 March 1996     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Summary Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel R _f values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.     

Optimization of protein and cellulase secretion in Neocallimastix frontalis EB188

Variations in culture feeding protocols were used to optimize the secretion of protein and cellulase in Neocallimastix frontalis EB188. High numbers (2000/ml) of zoospores, culture feeding at 55 h using a 1:3 dilution and cotton cellulose [0.25% (w/v) final] as the carbon source increased secretion. Endoglucanase reached 1.6±0.06 IU/ml, exoglucanase reached 0.032±0.006 IU/ml and -glucosidase reached 0.874±0.090 IU/ml. Medium containing twice the concentration of non-carbon-source components failed to increase secretions. Gel electrophoresis demonstrated that eleven cellulases were present. Two cellulases were secreted only in stationary cultures. rotein and cellulase secretion in N. frontalis EB188 may be dependent on the dilution of fermentation products. Correspondence to: R. E. Calza     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima,protease and ion sensitivities

Summary The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono--lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to -endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Carbon source,cyclic nucleotide,and protein inhibitor effects on protein and cellulase secretions inNeocallimastix frontalis EB188

Protein and cellulase secretion inNeocallimastix frontalis EB188 was studied. Induction of secretion in this ruminal fungus was dependent on the amount of medium cellulose and on de novo protein synthesis. Medium glucose repressed secretions and diminished the utilization of medium cellulose. Exogenously added cyclic nucleotides failed to derepress glucose repression. Medium glycerol had no effect on protein and cellulase secretion, would not support cell growth, and was nontoxic.     

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis,EB 188. Part 2. Carbon source utilization and effects on zoospore production

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.     

Effects of live Saccharomyces cerevisiae cells on zoospore germination,growth, and cellulolytic activity of the rumen anaerobic fungus,Neocallimastix frontalis MCH3

The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements.     

Regulation of protein and cellulase excretion in the ruminal fungusNeocallimastix frontalis EB188

Extracellular protein and cellulase excretion secretions were studied in the ruminal fungusNeocallimastix frontalis EB188. Cellulase assay and polyacrylamide gel electrophoresis was used to characterize protein excretion patterns caused by media switches of established cultures. Glucose switch medium caused the excretion of low levels of cellulase and increased amounts of low-molecular-weight proteins. Cellulose switch medium caused the excretion of high levels of cellulase and increased amounts of high-molecular-weight proteins. Several proteins were excreted uniquely or in greater amounts in cellulose cultures than in glucose cultures. Distinct protein excretion patterns suggested that regulation of cellulase was a closely controlled process in ruminal fungi.     

Characterization of<Emphasis Type="Italic"> Aspergillus oryzae</Emphasis> fermentation extract effects on the rumen fungus<Emphasis Type="Italic"> Neocallimastix frontalis</Emphasis>, EB 188. Part 1. Zoospore development and physiology

Experiments were performed to determine the effect of Aspergillus oryzae (AO) fermentation extract on zoospore development in the rumen fungus Neocallimastix frontalis EB 188. Powdered product, or liquid extract prepared from such powder, was added at the recommended value for supplementation in dairy cattle. Stationary and stirred cultures were periodically sampled and assayed for extracellular and intracellular protein and enzymes, gas production, zoospore production and maturation, and carbon source utilization. Soluble extract increased fungal physiology when grown in stirred vessels or stationary cultures. Treated cultures produced higher levels of enzymes (nearly double). Mobile zoospores matured into germination entities more rapidly in treated cultures, and when powdered product was used, nearly 3 times more motile zoospores were produced at 56 h of fungal growth. Levels of the intracellular enzyme malate dehydrogenase increased by 6-fold in the presence of powdered product. Product wheat bran carrier used as soluble extract or powder had very little effect on fungal cultures. Medium cellulose was completely hydrolyzed in all cultures but this occurred earlier in those containing AO treatment.     

Kinetics of Thermoascus aurantiacus solid-state fermentation on sugar-beet pulp – polysaccharide alteration and production of related enzymatic activities

 The fungal solubilization of cell wall components of sugar-beet pulp, during solid-state fermentation of Thermoascus aurantiacus, is reported here. The extracellular fungal enzyme activities related to the substrate degradation were also studied. In 120 h, more than 60% of the main sugar-beet pulp polysaccharides, i.e. pectins, arabinose- and glucose-containing polysaccharides, were rapidly brought into solution by the fungus. The slow accumulation of monosaccharides compared to the fast degradation of the polysaccharides suggested that most of the released sugars were consumed by the fungus. The analysis of the enzymes present in the water extracts of the solid-state cultures proved that the fungus was able to synthesize a complete enzymatic system required for the hydrolysis of the main sugar-beet pulp polysaccharides. The highest enzyme activities measured were β-glucosidase and α-L-arabinofuranosidase. Received: 22 September 1995/Received revision: 15 January 1996/Accepted: 22 January 1996     

Responses of Aquatic Fungal Communities on Leaf Litter to Temperature‐Change Events

Increases of extreme weather events are predicted to occur with ongoing climate change, but impacts to freshwaters have rarely been examined. We assessed the effects of temperature on leaf‐litter associated fungi by exposing leaves colonized in a stream to 18 °C (control), 25 °C, or 18 °C after freezing. Treatments altered fungal dominance on leaves; Lunulospora curvula sporulation was stimulated by increased temperature and stopped by the freeze‐thaw treatment. Fungal biomass and diversity decreased at 18 °C after freezing, but not at 25 °C. Leaf decomposition was retarded by the freeze‐thaw treatment (k = –0.024 day^–1) and stimulated at 25 °C (k = –0.069 day^–1). Results suggest that occasional freezing may constrain fungal diversity and their ecological functions, while warming appears to accelerate plant‐litter decomposition in streams. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

Two-step degradation of pyrene by white-rot fungi and soil microorganisms

  The effect of soil microorganisms on mineralization of ¹⁴C-labelled pyrene by white-rot fungi in solid-state fermentation was investigated. Two strains of white-rot fungi, Dichomitus squalens and a Pleurotus sp., were tested. The fungi were incubated on milled wheat straw contaminated with [¹⁴C]pyrene for 15 weeks. CO₂ and ¹⁴CO₂ liberated from the cultures were determined weekly. To study the effect of soil microorganisms on respiration and [¹⁴C]pyrene mineralization in different periods of fungal development, the fungal substrate was covered with soil at different times of incubation (after 0, 1, 3, 5, 7, 9 or 11 weeks). The two fungi showed contrasting ecological behaviour in competition with the soil microflora. Pleurotus sp. was highly resistant to microbial attack and had the ability to penetrate the soil. D. squalens was less competitive and did not colonize the soil. The resistance of the fungus was dependent on the duration of fungal preincubation. Mineralization of [¹⁴C]pyrene by mixed cultures of D. squalens and soil microorganisms was higher than by the fungus or the soil microflora alone when soil was added after 3 weeks of incubation or later. With Pleurotus sp., the mineralization of [¹⁴C]pyrene was enhanced by the soil microflora irrespective of the time of soil application. With D. squalens, which in pure culture mineralized less [¹⁴C]pyrene than did Pleurotus sp., the increase of [¹⁴C]pyrene mineralization caused by soil application was higher than with Pleurotus sp. Received: 8 March 1996 / Received revision: 1 July 1996 / Accepted: 8 July 1996     

Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains

Tempe fermentations using mixed cultures of Rhizopus oligosporus MS5, R. oryzae EN, Citrobacter freundii, and Brevibacterium epidermidis were investigated. Consumption of 150 g tempe, produced with a pure fungal mixed culture out of strains MS5 and EN, is sufficient to cover the daily requirements of niacin, vitamin K, ergosterol, and tocopherol as well as half of the daily requirement of pyridoxine, riboflavin, and biotin. Moreover, one-fourth of the recommended amount of folate is supplied. Supplementation of the fungal inoculum with C. freundii results in tempe enriched with vitamin B₁₂. Menachinone was produced as a typical bacterial vitamin K derivative. Metabolic activity of C.␣freundii led to an additional decrease of the α-galactosides stachyose and raffinose compared to pure fungal fermentations. No bacterial formation of factor 2 could be observed. Received: 31 July 1996 / Received revision: 4 October 1996 / Accepted: 14 October 1996     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

Respiratory stimulation and generation of superoxide radicals in Pseudomonas aeruginosa by fungal naphthoquinones

The mechanism of action of antimicrobial naphthoquinones from the fungus Fusarium was studied by using Pseudomonas aeruginosa. Bostricoidin, methyl ether fusarubin, and fusarubin stimulated the oxygen consumption of bacterial cells and induced cyanide-insensitive oxygen consumption. These activities of the tested compounds were also observed in bacterial membrane preparations in a dose-dependent manner. Naphthoquinones stimulated the generation of superoxide anion and hydrogen peroxide. The naphthoquinone effectively acted as the electron acceptors for bacterial diaphorase, which could explain the antibacterial activity of Fusarium naphthoquinones since electron acceptors lead to the stimulation of respiratory activity and the generation of oxygen radical species. Received: 12 July 1996 / Accepted: 31 October 1996     

The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes

Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4-dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAME in peptone medium, respectively 15–60 μM and 4–40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus, Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAME production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus. Received: 17 September 1996 / Received revision: 7 February 1997 / Accepted: 15 February 1997     

Fermentation extract effects on the morphology and metabolism of the rumen fungus Neocallimastix frontalis EB188

The effects of Aspergillus oryzae fermentation extract, Amaferm, on the rumen fungus Neocallimastix frontalis EB188 were studied. The secretion of cellulase was increased by 67% and rhyzoid development was increased 3.8-fold in the presence of extract. Strength of fungal response increased in a dose-dependent manner and demonstrated a positive correlation between cell surface area and enzyme secretion. Above certain concentrations of extract, however, the development of the fungus and enzyme secretions remained at control values or slightly diminished. Supernatant fluid appearance of the intracellular enzyme, malate dehydrogenase, paralleled the secretion of cellulase both in the presence and absence of extract. Ether solubilization of extract demonstrated that the active component(s) possessed a moderately polar value between 2.7 and 2.8. Thin layer chromatography separated extract into inert, inhibitory and intensely stimulating fractions. These results support the idea that by accelerating fungal growth and metabolism, Amaferm increases the rate (or extent) of fibre degradation caused by rumen fungi and that this, in turn, may contribute to enhanced animal performance.