The nature of anaerobic fungi and their polysaccharide degrading enzymes

Conclusion The discovery of anaerobic fungi has added a new member to the indigenous microorganisms that inhabit the rumen ecosystem. Anaerobic fungi do not appear essential for the survival of ruminants due to their presence in very low numbers, and sometimes absence, in ruminants fed low fiber diets, but their presence may likely be very important in the digestion of fibrous diets. The anaerobic fungi have adapted well to the rumen environment. They are able to ferment a large array of soluble carbohydrates and can synthesize cellular components in an anaerobic environment. The fungi posses hydrogenosomes for the removal of reducing equivalents in the form of molecular hydrogen and the removal of trace oxygen is a accomplished via removal by NADH oxidase. Their positive synergistic interaction with methanogenic bacteria eludes to their highly evolved role in the rumen environment. The fungi also produce resistant sporangia that allows for transfer of species to a new host in an oxygen environment. The anaerobic fungi posses a highly active array of polysaccharide degrading enzymes that may provide an advantage in the highly competitive rumen ecosystem. The production of specific enzymes that hydrolyze the lignocellulosic fraction of plant walls is unique in rumen microorganisms and allows for their attachment and growth on fibrous plant particles that are not available to the rumen bacteria.     

Cereal straw and pure cellulose as carbon sources for growth and production of plant cell-wall degrading enzymes by Sporotrichum thermophile

Sporotrichum thermophile grew well and produced plant cell-wall degrading enzymes on straw (barley and wheat) of different particle sizes and Avicel as carbon sources. Comparable activities of endoglucanase, Avicelase and cellobiase were produced on each substrate. In contrast, activities of xylanase, aryl--glucosidase, -xylosidase, esterase and -l-arabinofuranosidase were higher on straw (either wheat or barley) than on Avicel. The enzyme systems produced on barley straw of different particle sizes degraded finely milled barley straw in vitro more rapidly and to a greater extent than those produced on Avicel. In contrast, the enzyme systems produced on Avicel and very coarse barley straw hydrolysed Avicel to about the same extent while that produced on fine barley straw was slightly less effective. The main hydrolysis product in all cases was glucose. Isoelectric focusing revealed that the plant cell-wall degrading enzyme system produced by S. thermophile on barley straw was qualitatively and quantitatively superior to that produced on Avicel.C. Sugden was and M.K. Bhat is with the Department of Protein Engineering, Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Reading RG6 2EF, UK; C. Sugden is now with the Department of Biochemistry, University of York, Heslington, York YO1 5DD, UK.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Effect of calcium on cell-wall degrading enzymes of Botrytis cinerea

Effective anti-Botrytis strategies leading to reduce pesticides on strawberries are examined to provide the protection that is harmless to humans, higher animals and plants. Calcium treatments significantly inhibited the spore germination and mycelial growth of B. cinerea. The intracellular polygalacturonase and CMCase showed low activities in B. cinerea cultivated by medium containing calcium. On the other hand, calcium-stimulated β-glucosidases production occurred. Our findings suggest that the calcium treatments keep CMCase activity low and cause low activities of cell-wall degrading enzymes of B. cinerea in the late stage of growth.     

The production of hemicellulose-degrading enzymes byBacillus macerans in anaerobic culture

Summary The cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h^-1) although the activity was reduced at higher dilution rates (0.125–0.15 h^-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism.     

Pectinolytic enzymes of anaerobic fungi

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii , A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100–900 and 10–450 μg galacturonic acid h^-1 mg protein^-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).     

Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi

Five anaerobic fungal isolates from the bovine rumen were grown on Coastal Bermuda grass (CBG) leaf blades and monitored over a 9-day period for substrate utilization, fermentation products, cellulase, and xylanase activities. Two of the fungal isolates showed monocentric growth patterns; one (isolate MC-1) had monoflagellated zoospores and morphologically resembled members of the genus Piromyces; the other (isolate MC-2) had multiflagellated zoospores and resembled members of the genus Neocallimastix. Three other isolates (PC-1, PC-2, and PC-3) exhibited polycentric growth and have not yet been described in the literature; these isolates were characterized by differences in morphology. All of the isolates degraded CBG to approximately the same extent (70% [dry weight]) in 9 days. Fermentation product accumulation was concurrent with substrate utilization. The major fermentation products for all isolates were formate, acetate, D-(-)-lactate, L-(+)-lactate, ethanol, carbon dioxide, and hydrogen. Succinate was produced by all cultures, with the exception of MC-1. Fermentation balances revealed different profiles for each isolate. As a group, monocentric isolates produced a greater ratio of oxidized to reduced products when grown on glucose or CBG than did the polycentric isolates, which produced a nearly equal ratio of these products. All isolates exhibited cellulolytic and xylanolytic activities, including endoglucanase, exoglucanase, beta-glucosidase, xylanase, and beta-xylosidase activities. Increasing enzyme activity correlated with the accumulation of fermentation products and substrate utilization. The optimum pH for the enzymatic activity of polycentric isolates was within a more narrow range (pH 6.4 to 7.0) than that of the monocentric isolates (pH 5.5 to 7.5). Activity toward cellulosic substrates was not detected until after the disappearance of reducing sugars. Xylanase activity was found to be five to seven times that of carboxymethyl cellulase activity for all cultures grown on CBG.     

Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi.

Five anaerobic fungal isolates from the bovine rumen were grown on Coastal Bermuda grass (CBG) leaf blades and monitored over a 9-day period for substrate utilization, fermentation products, cellulase, and xylanase activities. Two of the fungal isolates showed monocentric growth patterns; one (isolate MC-1) had monoflagellated zoospores and morphologically resembled members of the genus Piromyces; the other (isolate MC-2) had multiflagellated zoospores and resembled members of the genus Neocallimastix. Three other isolates (PC-1, PC-2, and PC-3) exhibited polycentric growth and have not yet been described in the literature; these isolates were characterized by differences in morphology. All of the isolates degraded CBG to approximately the same extent (70% [dry weight]) in 9 days. Fermentation product accumulation was concurrent with substrate utilization. The major fermentation products for all isolates were formate, acetate, D-(-)-lactate, L-(+)-lactate, ethanol, carbon dioxide, and hydrogen. Succinate was produced by all cultures, with the exception of MC-1. Fermentation balances revealed different profiles for each isolate. As a group, monocentric isolates produced a greater ratio of oxidized to reduced products when grown on glucose or CBG than did the polycentric isolates, which produced a nearly equal ratio of these products. All isolates exhibited cellulolytic and xylanolytic activities, including endoglucanase, exoglucanase, beta-glucosidase, xylanase, and beta-xylosidase activities. Increasing enzyme activity correlated with the accumulation of fermentation products and substrate utilization. The optimum pH for the enzymatic activity of polycentric isolates was within a more narrow range (pH 6.4 to 7.0) than that of the monocentric isolates (pH 5.5 to 7.5). Activity toward cellulosic substrates was not detected until after the disappearance of reducing sugars. Xylanase activity was found to be five to seven times that of carboxymethyl cellulase activity for all cultures grown on CBG.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

Production of plant protoplasts by enzymes from selected wood-destroying fungi

The goal of this work was to select higher fungi for the production of enzymes able to produce plant protoplasts. The hydrolytic capacity of various species of wood-detsroying fungi was examined to select those with the maximal hydrolytic capacity. The selection criteria included attack on cellulose, and oligo-and polysaccharides by each fungus species. Lenzites elegans, gonoderma applanatum and Pycnoporus sanguineus produced thr richest enzymatic mixtures, which were evaluated for their protoplasting ability. L. elegans has the best enzymatic mixture able to [roduced plant protoplasts with the shortest time for maceration of plant tissues, followed by G. applanatum and P. sanguineus. The potential of these fungi and the simplicity of enzyme proudtcion suggest the possibility of commercial applications.Correepondence to: A. R. Sampietro     

AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production

Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.     

Adaptive control of specific growth rate based on proton production in anaerobic fed-batch culture

Summary This paper develops a practical and useful computer control scheme to control the specific growth rate as accurately as possible by measuring the released protons in anaerobic alcohol fermentation. In the case of current study, most of the released protons are due to the uptake of cationic ammonium ion via the conversion: NH₄ ⁺ NH₃(cell) + H⁺. Correlating the Proton Production (PP) and the Proton Production Rate (PPR) with specific growth rate () proved PP as a better measured variable. Using a simple adaptive control algorithm, was successfully controlled in a Zymomonas mobilis fed-batch culture.     

In vitro degradation of cell-wall and digestibility of cereal straws treated with anaerobic ruminal fungi

Ruminal fungal isolates (Orpinomyces sp.; C-14, Piromyces sp.; C-15, Orpinomyces sp.; B-13 and Anaeromyces sp.; B-6), were evaluated under anoxic conditions for their effect on in vitro dry matter digestibility, neutral detergent fibre, acid detergent fibre and acid detergent lignin using rice and wheat straw as substrate. There was no significant effect of the fungal isolates on the disappearance of the substrates along with rumen liquor when compared to control. The doses of 10(6) cfu/ml of the isolate were found to have maximum degradation of straws in comparison to the doses of 10(3) cfu/ml.     

Callus culture as substrate for the production of specific cell wall degrading enzymes for derived protoplast isolation

Callus culture of spruce (Picea excelsa LINK) appears to be a suitable substrate for the fungusTrichoderma reesei to produce an efficient extracellular lytic system for protoplast isolation. In comparison with Onozuka R-10 cellulase, a yield of protoplasts from the spruce callus 2·5 higher was obtained. Another testea commercial cellulase DK was less efficient. The addition of Macerozyme R–10 significantly enhanced release of protoplasts within all tested enzyme preparations. No difference in the viability of protoplasts has been observed.     

An anaerobic continuous-flow culture model of interactions between intestinal microflora and Candida albicans

Summary The finding by earlier workers that Escherichia coli suppressed the growth of Candida albicans in vitro or in gnotobiotic mice has led to numerous, erroneous conclusions regarding the identity of the organisms and mechanisms responsible for the suppression of Candida in the gut. This is due, in part, to the fact that nearly all studies to date have not reflected interactions as they occur in the intestinal tract. This paper describes a series of experiments that establish that an anaerobic continuous-flow (CF) culture model, of the ecology of the large intestinal flora reproduces interactions between bacteria and Candida as they occur in the large intestine. This was determined in the following ways. (i) Bacterial counts in CF cultures of conventional mouse cecal flora or human fecal flora closely resembled that found in the mouse intestine and human feces. (ii) Dense layers of bacterial growth that formed on the glass walls of the CF culture vessels resembled bacterial populations that colonize intestinal mucosa. (iii) Total and individual levels of certain metabolic end-products of the predominant anaerobic bacterial flora present in CF cultures coincided with those found in the large intestine of conventional mice or human feces used to establish the CF cultures. (iv) C. albicans was eliminated from CF cultures of mouse cecal flora at a rate similar to that of untreated experimental animals. (v) Contents of CF cultures fed to antibiotic-treated mice redressed several cecal abnormalities, and suppressed Candida populations to levels found in conventional animals. Thus, a number of complex ecological mechanisms were maintained in CF cultures which normally control Candida populations in the large intestine. It is suggested, therefore, that the CF culture model should help to further define the mechanisms which control C. albicans and other fungi in the intestinal tract, as well as define which components of the indigenous microflora are responsible for suppression of Candida in the gut.     

Dilution rate as a determinant of mycelial morphology in continuous culture

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched "colonial" variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h(-1). The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.     

Cultivation of anaerobic fungi in a 10-l fermenter system for the production of (hemi-)cellulolytic enzymes

 Two species of anaerobic fungi, i.e. Piromyces strain E2 and Neocallimastix patriciarum strain N2, were cultivated in a 10-l batch fermenter with filter- paper cellulose as the carbon source. The accumulation of fermentation products, production of extracellular protein and (hemi-)cellulolytic enzymes were monitored during growth. Growth of Piromyces E2 in the fermenter resulted in a shift in the fermentation pattern to more acetate and formate and less ethanol, lactate, succinate and malate, possibly because of removal of hydrogen. The specific activities of Avicelase, endoglucanase, β-glucosidase and xylanase were up to threefold higher compared to small batch cultures. Enzyme activities produced per gram of cellulose were up to four times the values reported for Piromyces E2 grown in a semi-continuous coculture with the methanogen Methanobacterium formicicum. The performance of fermenter enzyme preparations from the anaerobic fungi with respect to hydrolysis of Avicel compared well to that of preparations of Trichoderma reesei. However, addition of exogenous β-glucosidase was indispensible with the latter preparation for the complete conversion to glucose. Received: 14 December 1995/Received revision: 19 March 1996/Accepted: 25 March 1996