Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture.

AIMS: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. METHODS AND RESULTS: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. CONCLUSIONS: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan-lignin complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.

Polysaccharidase and glycosidase production of avicel grown rumen fungus Orpinomyces sp. GMLF5.

Extracellular and cell-associated enzyme preparations were obtained from ruminal anaerobic fungi Orpinomyces sp. GMLF5 grown in culture containing microcrystalline cellulose (avicel) as sole energy source and degradation capacities of the preparations towards several polysaccharides and glycosides were studied. Fungus showed substantial increases in xylanase, carboxymethyl cellulase (CMCase), lichenase, amylase, beta-xylosidase, beta-glucosidase and alpha-L-arabinofuranosidase activities between 72 and 168 hours. High amounts of cell associated beta-xylosidase were noted in 4 and 5 days old cultures. Optimum temperature and pH of the polysaccharidases were found at 50 degrees C and 6.0-6.5, respectively. Xylanase was found to be virtually stable at 50 degrees C, CMCase and lichenase were stable at 40 degrees C for 200 min, however amylase was found more sensitive to heat treatment. The fibrolytic enzymes of the isolate GMLF5 were observed to be capable of hydrolyze the avicel.

Effect of administration of rumen fungi on production performance of lactating buffaloes.

Anaerobic fungi were orally dosed to lactating buffaloes to study their effect on the digestibility of a diet (composed of 50% wheat straw and 50% concentrate along with six kg maize green/animal/day), rumen fermentation patterns and milk production. Group I (control) was administered with fungus-free anaerobic broth, while group II and III were administered with Orpinomyces sp. C-14 or Piromyces sp. WNG-12 (250 ml; 3-5 days of growth/animal/ week), respectively. Milk production was higher in group II and III (8.42 and 8.48 kg/d) than in the control (8.03 kg/d) with virtually the same feed intake (i.e. 11.50 and 10.62 and 11.79 kg, respectively). There was an increase of 6% fat-corrected milk yield/animal/day in group II and III, respectively compared to the control. The milk fat was higher in the fungal culture administered groups than in the control group. The digestibility of dry matter, crude protein, neutral detergent fibre, acid detergent fibre, cellulose and digestible energy also increased significantly in group II and III. The pH and ammonia nitrogen were lower, whereas total volatile fatty acids, total nitrogen, trichloroacid precipitable nitrogen and number of zoospores/ml of rumen liquor were higher in group II and III when compared to the control. Hence, it can be stated that rumen fungi can be used as a direct-fed microbial in lactating buffaloes, to enhance the digestibility of wheat straw based diets leading to higher production.

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen.

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.

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.

In vitro degradation of wheat straw by anaerobic fungi from small ruminants.

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome.

The anaerobic gut fungi occupy a unique niche in the intestinal tract of large herbivorous animals and are thought to act as primary colonizers of plant material during digestion. They are the only known obligately anaerobic fungi but molecular analysis of this group has been hampered by difficulties in their culture and manipulation, and by their extremely high A+T nucleotide content. This study begins to answer some of the fundamental questions about the structure and organization of the anaerobic gut fungal genome. Directed plasmid libraries using genomic DNA digested with highly or moderately rich AT-specific restriction enzymes (VspI and EcoRI) were prepared from a polycentric Orpinomyces isolate. Clones were sequenced from these libraries and the breadth of genomic inserts, both genic and intergenic, was characterized. Genes encoding numerous functions not previously characterized for these fungi were identified, including cytoskeletal, secretory pathway and transporter genes. A peptidase gene with no introns and having sequence similarity to a gene encoding a bacterial peptidase was also identified, extending the range of metabolic enzymes resulting from apparent trans-kingdom transfer from bacteria to fungi, as previously characterized largely for genes encoding plant-degrading enzymes. This paper presents the first thorough analysis of the genic, intergenic and rDNA regions of a variety of genomic segments from an anaerobic gut fungus and provides observations on rules governing intron boundaries, the codon biases observed with different types of genes, and the sequence of only the second anaerobic gut fungal promoter reported. Large numbers of retrotransposon sequences of different types were found and the authors speculate on the possible consequences of any such transposon activity in the genome. The coding sequences identified included several orphan gene sequences, including one with regions strongly suggestive of structural proteins such as collagens and lampirin. This gene was present as a single copy in Orpinomyces, was expressed during vegetative growth and was also detected in genomes from another gut fungal genus, Neocallimastix.

The effects of sequential inoculation of mixed rumen protozoa on the degradation of orchard grass cell walls by anaerobic fungus Anaeromyces mucronatus 543.

The effects of protozoa on the degradation of plant cell walls (CW) during different growth stages of the fungus Anaeromyces mucronatus have been investigated. Since fungi show a marked lag in their in vitro cultures and many protozoa rapidly die during a prolonged incubation time, the effects of protozoa may vary according to the growth phase of the fungi. Therefore, the approach adopted was (i) to inoculate CW with fungus monoculture, (ii) to inoculate CW with fungus-protozoa coculture, or (iii) to sequentially inoculate fungal cultures that had been grown in CW for 24 (initial stage of growth), 48, and 72 h (late stage of growth) with mixed protozoa. When a fungus was associated with protozoa, a growth phase dependent effect was observed. Ruminal protozoa adversely affected the growth and activity when introduced in the initial growth stage of A. mucronatus, but a synergetic interaction was detected when added to late growth stage cultures. Although there is no immediate explanation for these results, the data suggested that protozoa can engulf the fungal zoospores, which are in ruminal fluids and (or) attached to small feed particles, but cannot engulf the fungal thallus that is tightly attached to feed particles by a rhizoidal system. Our data indicated that the protozoa did not influence cellulolysis by the fungi in exponential and (or) stationary phase, but they had a marked inhibitory effect on fungi that were in lag phase. Inhibition during lag phase could result from the protozoal predation of fungal zoospores that had failed to attach to substrates.

Glycoside hydrolase production by an anaerobic rumen fungus Caecomyces communis.

The ruminal fungus Caecomyces communis was grown anaerobically either in a discontinuous cultivation system or in a fermentor with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The best culture conditions for glycoside hydrolase production were obtained in Lowe medium with daily fresh medium addition, whereas the Orpin medium with ruminal fluid was favourable to fungal growth and to the enzyme export process. Among glycoside hydrolases assessed in both culture fluid and cellular homogenate, beta-D-fucosidase activity was preponderant. Most studied enzymes were mainly associated with cells (from 50% to 99%). Glycoside hydrolase activities were constitutive, but their level was regulated by a carbon source. beta-D-fucosidase and beta-D-xylosidase activity production was activated by the association of glucose plus cellobiose, whereas beta-D-glucosidase activity production was stimulated by cellobiose alone. Enzyme release could be favoured by glucose alone or by Ray grass hay added to glucose plus cellobiose.