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.     

Fermentation of woods by rumen anaerobic fungi

Abstract The potential of rumen anaerobic fungi for fermenting untreated woods has been assessed using two Neocallimastix species isolated from sheep. When a strain of N. frontalis was incubated for 11 days with wood from 12 hardwood (angiosperm) species, many woods were measurably fermented, with wood from Populus tremuloides (32%) and Fagus sylvatica (21%) being the most highly degraded. This N. frontalis solubilised celulose, hemicellulose and lignin in P. tremuloides wood. Lower degradation (17%) of P. tremuloides wood by a different species of Neocallimastix showed that the choice of fungus as well as the structure and chemistry of the wood influenced the amount of wood cell wall degraded by anaerobic fungi. The amount of degradation was not related to the length of fungal rhizoids.     

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen

Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.     

The fermentative characteristics of anaerobic rumen fungi

Substrate utilization and fermentation characteristics of rumen fungi of the genus Neocallimastix are described. Preliminary observations on the removal of monosaccharides from plant cell walls and the effect of fermentation products on growth of Neocallimastix sp. (isolate R1) are presented. The properties of rumen fungi are discussed in relation to their role in the rumen.     

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).     

Study of the nuclear cycle of four species of strictly anaerobic rumen fungi by fluorescence microscopy

The nuclear cycles of four species of strictly anaerobic rumen fungi (Neocallimastix frontalis, Piromonas communis, Neocallimastix joyonii, andSphaeromonas communis) were compared by fluorescence microscopy. The existence of two types of sporangium development and thalli was confirmed in this study. The first type involved monocentric species fitted with an endogenously developing single sporangium and with rhizoids, such asN. frontalis, P. communis, andS. communis, characterized by the presence of rhizoid-like vesicles. The second type concerned polycentric species with an exogenous sporangium development such asN. joyonii. This species is characterized by a rhizomycelium and gamma particle-like bodies within the zoospore.     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen.

Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.     

Characterization of chitinases of polycentric anaerobic rumen fungi

Chitinolytic systems of anaerobic polycentric rumen fungi of genera Orpinomyces and Anaeromyces were investigated in three crude enzyme fractions - extracellular, cytosolic and cell-wall. Endochitinase was found as a dominant enzyme with highest activity in the cytosolic fraction. Endochitinases of both genera were stable at pH 4.5-7.0 with optimum at 6.5. The Orpinomyces endochitinase was stable up to 50 degrees C with an optimum for enzyme activity at 50 degrees C; similarly, Anaeromyces endochitinase was stable up to 40 degrees C with optimum at 40 degrees C. The most suitable substrate for both endochitinases was fungal cell-wall chitin. Enzyme activities were inhibited by Hg(2+) and Mn(2+), and activated by Mg(2+) and Fe(3+). Both endochitinases were inhibited by 10 mmol/L SDS and activated by iodoacetamide.     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi

J. KOPEČNÝ, B. HODROVÁ AND C. S. STEWART. 1996. The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium . The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.     

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.     

Morphology of fibroblasts grown on substrates formed by dielectrophoretically aligned carbon nanotubes

Multiwall carbon nanotube templates formed on the surfaces of planar interdigitated microelectrode arrays by means of AC electric field-guided assembly are being explored as potential substrates for tissue engineering. The objective of the present study is to examine whether surface patterns of aligned multiwall carbon nanotubes can have an effect on cell growth, morphology, and alignment. Bovine fibroblasts grown on aligned carbon nanotubes for a period of 2 weeks were found to have raised bodies and pronounced cell extensions for anchoring themselves to the substrate similar to that of the cells found in native tissues. On the other hand, cells grown on various control surfaces had a flat, circular morphology. The cell cultures were visualized by means of SEM imaging and the resulting morphologies were statistically analyzed and compared.     

Effect of anaerobic fungi on in vitro feed digestion by mixed rumen microflora of buffalo

Five strains of anaerobic fungi isolated from the faeces of wild (hog deer, Cervus porcinus; blackbuck, Antelope cervicapra; spotted deer, Axis axis; nilgai, Baselophus tragocamelus) and rumen liquor of domestic (sheep, Ovies aries) ruminants showing high fibrolytic enzyme producing ability were added to mixed rumen microflora of buffalo to study their effect on the digestibility of lignocellulosic feed (wheat straw and wheat bran in the ratio of 80:20), enzyme production and fermentation end products in in vitro conditions. Among the 5 isolates studied, FNG5 (isolated from nilgai) showed the highest stimulating effect on apparent digestibility (35.31 +/- 1.61% vs. 28.61 +/- 1.55%; P < 0.05), true digestibility (43.64 +/- 1.73% vs. 35.37 +/- 1.65%; P < 0.01), neutral detergent fiber digestibility (29.30 +/- 2.58% vs. 18.47 +/- 2.12; P < 0.01) of feed 24 h after inoculation compared to the control group. The production of carboxymethyl cellulase, xylanase, acetyl esterase and beta-glucosidase was significantly (P < 0.05) higher in the FNG5 inoculated incubation medium. There was no improvement in the digestibility and enzyme production on the addition of the other 4 isolates. Total volatile fatty acid levels as well as the concentration of acetate, propionate, isobutyrate and valerate were significantly higher in the FNG5 added group as compared to the control group. The fungal isolate FNG5 from nilgai, a wild ruminant, was found to be superior to the other isolates tested and appears to have a potential to be used as a feed additive for improving fiber degradation in domestic ruminants.     

In vitro methane formation and carbohydrate fermentation by rumen microbes as influenced by selected rumen ciliate species

Ciliate protozoa contribute to ruminal digestion and emission of the greenhouse gas methane. Individual species of ciliates co-cultured with mixed prokaryote populations were hypothesized to utilize carbohydrate types differently. In an in vitro batch culture experiment, 0.6 g of pure cellulose or xylan was incubated for 24 h in 40-mL cultures of Entodinium caudatum, Epidinium ecaudatum, and Eudiplodinium maggii with accompanying prokaryotes. Irrespective of ciliate species, gas formation (mL) and short-chain fatty acids (SCFA) concentrations (mmol L^?1) were higher with xylan (71; 156) than with cellulose (52; 105). Methane did not differ (7.9% of total gas). The SCFA profiles resulting from fermentation of the carbohydrates were similar before and after removing the ciliates from the mixed microbial population. However, absolute methane production (mL 24 h^?1) was lower by 50% on average after removing E. caudatum and E. maggii. Methanogen copies were less without E. maggii, but not without E. ecaudatum. Within 3 weeks part of this difference was compensated. Butyrate proportion was higher in cultures with E. maggii and E. ecaudatum than with E. caudatum and only when fermenting xylan. In conclusion, the three ciliate species partly differed in their response to carbohydrate type and in supporting methane formation.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Susceptibility and resistance of anaerobic rumen fungi to antimicrobial feed additives

Listeria monocytogenes survived in meat, cheese and egg ravioli stored at 5°C for 14 d. Ravioli were considered edible for the first 9 d of storage. Initial L. monocytogenes populations of 3 × 10⁵ cfu/g of ravioli were reduced to non-detectable levels after heat treatment simulating that which would be used by the consumer.     

Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates

The rumen fungi Neocallimastix patriciarum, Piromonas communis, and a morphologically distinct but unidentified isolate were cultivated on the polysaccharides starch, cellulose, xylan, and their principal component monosaccharides and disaccharides, and the range and specific activities of the glycoside hydrolases formed were monitored using gluco-oligosaccharide and p-nitrophenyl glycoside substrates. A wide range of enzyme activities was detected in preparations from vegetative growth and zoospores of all three isolates. Enzyme activity was also present in the culture medium. The specific activities were affected by the carbohydrate source available in the growth medium, although the more active hydrolases involved in the degradation of plant structural and storage polysaccharides were formed on all seven carbohydrate sources evaluated. Enzyme activities were increased in the zoospore, vegetative, and extracellular preparations after growth on the appropriate structurally related disaccharide or polysaccharide. The hemicellulolytic glycosidases (alpha-L-arabinofuranosidase, beta-D-xylosidase) were most active after growth on xylan, whereas alpha-/beta-glucosidase activity was increased with the corresponding glucan as growth substrate. However, whereas wide-ranging beta-glucosidase activity was detected following growth on maltose or starch, the alpha-glucosidase activities of P. communis were lower or undetectable in vegetative preparations grown on glucose or the beta-glucans cellobiose and cellulose.     

Influence of three species of arbuscular mycorrhizal fungi on the persistence of aromatic hydrocarbons in contaminated substrates

Aromatic hydrocarbons are pollutants which have mutagenic and carcinogenic properties as well as relatively high hydrosolubility. Their presence in soils makes techniques such as bioremediation an important topic for research. In this work, the effect of arbuscular mycorrhiza (AM) on the persistence of benzene, toluene, ethylbenzene and xylene (BTEX) in artificially contaminated substrates was evaluated. Leek plants were grown with three AM fungal species using a specially designed mesocosm system, in which internal air and substrate samples were analyzed by gas chromatography for BTEX content. Strong reductions in the BTEX concentration in substrates were generally observed in the presence of mycorrhizal plants. Residual BTEX content ranged between nearly total disappearance (<2%) and 40% of the original concentration, whereas there was a high persistence of hydrocarbons in the samples of substrate alone or with non-mycorrhizal plants. These results provide first evidence for an influence of AM activity in reducing pollution of substrates by aromatic hydrocarbons.Andrea Volante and Guido Lingua contributed equally to this paper