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     

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.     

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.     

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.     

The effect of Aspergillus oryzae fermentation extract on the anaerobic fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206: generalized effects and component analysis

Three fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206, representing the predominant cultures isolated from cattle, were shown to respond to the addition of Aspergillus oryzae fermentation extract (i.e., Amaferm; BioZyme Inc., St. Joseph, Mo.) stimulation. Growth rates, protein and cellulase secretion and fungal mass production were all accelerated in the presence of the extract. Analysis of volatile fatty acids produced by these three species suggested that extract addition increased and altered gas production. Fractionation and preliminary analysis of the components present in the soluble extract, which stimulated the growth of the cellulolytic fungus N. frontalis EB 188, were also attempted. Soluble and filtered, sterilized extract was treated prior to use as a stimulant. Pretreatments included dialysis, ultraviolet irradiation, freeze thaw cycling, boiling, autoclaving, digestion with protease, autodigestion, organic extraction, decolorizing-carbon binding and polyethylene glycol concentration. Boiling, protease treatment, organic extraction, freeze thaw cycling and decolorizing-carbon binding reduced the ability of the extract to stimulate fungal cultures. Gel electrophoresis methods demonstrated that protein- and cellulasesecretion profiles were not identical in control and stimulated cultures. High-performance liquid chromatography methods allowed the separation of the extract into a limited number of ultraviolet-absorbing peaks, of which several stimulated the physiology of the fungus. Received: 6 December 1995/Received revision: 7 February 1996/Accepted: 4 March 1996     

Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188.

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.     

Inhibition of the rumen anaerobic fungus Neocallimastix frontalis by fermentation products

The effects of bacterial fermentation products on cellulose degradation by the rumen fungus Neocallimastix frontalis have been investigated. H₂, formate, lactate and ethanol were strong inhibitors, particularly at high concentrations. Acetate and malate also inhibited, whereas succinate had a variable effect. Butyrate and propionate had no inhibitory effects.     

Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

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.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.     

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis.

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Xylanolysis by cocultures of the rumen fungus Neocallimastix frontalis and ruminal bacteria

The effect of fibrolytic and saccharolytic rumen bacteria on xylanolysis by the rumen fungus Neocallimastix frontalis has been investigated. In cocultivations N. frontalis interacted synergistically with Bacteroides ruminicola, Succinivibrio dextrin-osolvens and Selenomonas ruminantium during xylan utilization. Xylan utilization decreased in cocultures containing Lachnospira multiparus or Streptococcus bovis. Ruminococcus flavefaciens appeared to inhibit fungal growth.     

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.     

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.     

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Purification and characterization of two 1,4-beta-xylan endohydrolases from the rumen fungus Neocallimastix frontalis.

Two beta-endoxylanases produced by Neocallimastix frontalis have been purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Xylanase I is a nonglycosylated protein with an apparent molecular mass of 45 kDa. Xylanase II is a glycoprotein with an apparent molecular mass of 70 kDa. The pH optima of these enzymes were 5.5 and 6, respectively, and the temperature optimum was 55 degrees C for each enzyme. The endo mode of action of the enzymes was revealed by thin-layer chromatography of xylan hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, confirming the biochemical specificities of the enzymes. Both enzymes exhibited carboxymethyl cellulase activity, and xylanase I was absorbed on crystalline cellulose, indicating that these enzymes might belong to the F family of beta-1,4-glycanases.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis.

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lasalocid on glucose uptake, hydrogen production and the solubilization of straw by the anaerobic rumen fungus Neocallimastix frontalis

Three isolates of Neocallimastix frontalis grown in pure culture with glucose as substrate differed in their response to the presence of lasalocid in the growth medium. For two strains (RE1 and RK21) hydrogen production and glucose uptake were reduced to 50% or less of the control values in the presence of lasalocid at 0–25 μg/ ml. A third strain (PNK2) retained significant fermentative ability in the presence of at least four times this amount of lasalocid. Lasalocid was rather less inhibitory to the solubilization of straw by these fungi, PNK2 again providing the most resistant strain. It is concluded that the use of lasalocid in ruminant feeds is likely to have a strain selective effect on the rumen population of Neocallimastix.