Cryopreservation of the anaerobic rumen fungus Neocallimastix patriciarum

A rapid extraction and purification procedure is described for the preparation of toxic peptides from freshwater blooms and laboratory isolates of Microcystis aeruginosa . Extraction with methanol/butanol, followed by C₁₈ cartridge concentration; gel filtration and high performance liquid chromatography yields discrete toxin peaks. Elution profiles for the laboratory isolates and bloom extracts are compared and the applicability of the method for detecting cyanobacterial toxins in natural waters is discussed.     

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.     

Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.     

Solubilization of lignin by the ruminal anaerobic fungus Neocallimastix patriciarum.

The ability of the ruminal anaerobic phycomycete Neocallimastix patriciarum to digest model lignin compounds and lignified structures in plant material was studied in batch culture. The fungus did not degrade or transform model lignin compounds that were representative of the predominant intermonomer linkages in lignin, nor did it solubilize acid detergent lignin that had been isolated from spear grass. In a stem fraction of sorghum, 33.6% of lignin was apparently solubilized by the fungus. Solubilization of ester- and either-linked phenolics accounted for 9.2% of the lignin released. The amounts of free phenolic acids detected in culture fluid were equivalent to the apparent loss of ester-linked phenolics from the sorghum substrate. However, the fungus was unable to cleave the ether bond in hydroxycinnamic acid bridges that cross-link lignin and polysaccharide. It is suggested that the majority of the solubilized lignin fraction was a lignin carbohydrate complex containing ether-linked hydroxycinnamic acids. The lignin carbohydrate complex was probably solubilized through dissolution of xylan in the lignin-xylan matrix rather than by lignin depolymerization.     

Proteolytic activity of a rumen anaerobic fungus

Abstract A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn²⁺, Ca²⁺ or Co²⁺, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p -Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl- l -lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p -nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high- M _r form.     

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii

A new strain of strictly anaerobic fungi was isolated from the rumen of sheep. This strain is characterized by a polycentric thallus, an extensive and polynuclear rhizomycelium, polyflagellated zoospores with gamma particle-like bodies. We propose to assign this strain in a new species: Neocallimastix joyonii.     

Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus Neocallimastix patriciarum

Canola seed oil-bodies were investigated as a production vehicle and immobilization matrix for xylanases. A recombinant xynC gene from Neocallimastix patriciarum encoding a xylanase (XynC) was fused to an oleosin coding sequence suitable for targeting the xylanase to the oil-body membrane. This fusion gene was introduced into Brassica napus using Agrobacterium-mediated transformation. Transgenic Canola plants were obtained expressing xylanase which was targeted to the oil-bodies of seeds as shown by analysis with XynC-specific antibodies. Oil-bodies extracted from transgenic seeds exhibited xylanase activity, indicating the immobilization of XynC on the surface of oil bodies and the functioning of the xylanase as a fusion protein. The immobilized XynC retained its optimal temperature, K_m value and specificity. However, it exhibited reduced sensitivity to pH. Furthermore, it was shown that the enzyme immobilized on oil-bodies could be recycled by flotation several times without loss of activity.     

Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus

A new species of strictly anaerobic fungus was isolated from the cow rumen. It is characterized by a polycentric thallus, a polynuclear rhizomycelium, mucronate zoosporangia and uniflagellated zoospores. It is also singular in that the sporocysts do not react to the specific lectins of L-fucose, N-acetyl-D-galactosamine and diacetyl chitobiose. These characteristics justify the creation of a new genus.     

A highly active extracellular cellulase from the anaerobic rumen fungus Neocallimastix frontalis

Abstract An extracellular cellulase which was highly active in solubilizing the highly hydrogen bond-ordered cellulose in cotton fibre was found in a culture filtrate of the anaerobic fungus, Neocallimastix frontalis , isolated from the rumen of a sheep. The cellulase was several-fold more active in solubilizing cotton fibre per unit of endo-1,4-β-glucanase than the cellulase of the aerobic fungus Trichoderma reesei mutant strain C-30, which is one of the most active cellulases isolated so far.     

Feasibility of installing and maintaining anaerobiosis using Escherichia coli HD701 as a facultative anaerobe for hydrogen production by Clostridium acetobutylicum ATCC 824 from various carbohydrates

Using Escherichia coli for installing and maintaining anaerobiosis for hydrogen production by Clostridium acetobutylicum ATCC 824 is a cost-effective approach for industrial hydrogen production, as it does not require reducing agents or sparging with inert gases. This study was devoted for investigating the feasibility for installing and maintaining anaerobiosis of hydrogen production by C. acetobutylicum ATCC 824 when using E. coli HD701 utilizable versus non utilizable sugars as a-carbon source. Using E. coli HD701 for installing anaerobiosis showed a comparable hydrogen production yield and efficiency to the use of reducing agents and nitrogen sparging in case of hydrogen production from the E. coli HD701 non utilizable sugars. In contrast, using E. coli HD701 for installing anaerobiosis showed a lower hydrogen production yield and efficiency than the use of reducing agents and nitrogen sparging in case of using glucose as a substrate. This is possibly because E. coli HD701 when using glucose compensate for the substrate, and produce hydrogen with lower efficiency than C. acetobutylicum ATCC 824. These results indicated that the use of E. coli HD701 for installing anaerobiosis would not be economically feasible when using E. coli HD701 utilizable sugars as a carbon source. In contrast, the use of this approach for installing anaerobiosis for hydrogen production from sucrose and starch would have a high potency for industrial applications.     

Expression of a cellulase gene, celA, from the rumen fungus Neocallimastix patriciarum in Streptococcus bovis by means of promoter fusions

The aerotolerant rumen bacterium, Streptococcus bovis, has been used as a host for expression of genes of eukaryotic origin. The coding regions of the celA cellulase gene from the rumen fungus, Neocallimastix patriciarum, were fused with bacterial promoter/signal peptide regions from the Ruminococcus flavefaciens xynD and S. bovis -(1,3-1,4)-glucanase genes. Fusion cassettes were built into shuttle vector constructs based on pIL253 or pTRW10 and constructs carrying celA were transformed into S. bovis JB1. Active N. patriciarum cellulase was produced in S. bovis with either promoter, although better expression levels were obtained with the native S. bovis -glucanase promoter fragment.     

An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans

A Neocallimastix patriciarum acetylxylan esterase (BnaA) was expressed from the cloned gene in Escherichia coli. Purified recombinant BnaA efficiently released acetate from soluble acetylated birchwood xylan (ABX), with a specific activity of 76 U mg⁻¹. In contrast, release of acetate was very inefficient from the insoluble substrates, spear grass and delignified spear grass. Addition of a recombinant xylanase, XynA, also expressed from a cloned N. patriciarum gene, had no effect on the release of acetate from ABX. However, the combination of recombinant BnaA and XynA released more acetate from spear grass and delignified spear grass than did BnaA alone. Significantly more reducing sugar was also released from all three substrates by the combination of recombinant XynA and BnaA than by XynA alone. Thus the extent of digestion of acetylated xylans by XynA appears to be limited by the acetylation. In this system BnaA does not appear to increase the rate of cleavage of insoluble substrates by XynA, but probably allows the release of shorter xylose oligomers from already solubilised acetylated xylan polymers. Received: 11 January 1999 / Accepted: 28 February 1999     

Isozyme and morphological characteristics of the anaerobic fungus Piromyces mae isolated from the duodenum, rumen and faeces of sheep

Abstract Isolates of anaerobic fungi obtained from the rumen, duodenum and faeces of sheep were identified as Piromyces mae based on their morphological characteristics observed using light microscopy. There was no significant morphological variation among the isolates of P. mae from the rumen, duodenum and faeces. Isozymes of 12 isolates of P. mae (one each from the rumen, duodenum and faeces from 4 different sheep) were analysed by PAGE. A total of 12 isozymes were studied and 5 isozyme loci were successfully typed. They were malic enzyme, malate dehydrogenase, shikimate dehydrogenase, α-esterase and β-esterase. All the isolates of P. mae regardless of whether they were from the rumen, duodenum or faeces or from different animals produced very similar isozyme banding patterns for each of the enzyme systems. The similar isozyme profiles of the isolates indicate that they are of the same species although they exist in different regions of the alimentary tract.     

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2

Abstract The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H₂ and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.     

Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads

Bacteria were isolated from the mycelial surface of Pleurotus ostreatus and their role in fruiting body induction (fructification) of the edible mushroom P. ostreatus was investigated. Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P. ostreatus. In particular, the population size of fluorescent pseudomonads increased during fruiting body induction. An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp. isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus. Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

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.