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.     

Production of cellulases and saccharification of lignocellulosics by A. Micromonospora sp.

A locally isolated strain of Micromonospora sp. when grown on different natural cellulosic substrates gave the highest activity of carboxymethylcellulase (34 U/ml) and Avicelase (0.9 U/ml) on rice straw. Sugar cane bagasse was also a good substrate for growth and cellulase production. With commercial cellulosic substrates, highest carboxymethylcellulase (90 U/ml) and Avicelase (2.8 U/ml) activities were when the organism grew on xylan. Saccharification of sugar cane bagasse and rice straw by enzyme preparations of the organism grown on the respective substrates released 5.6 and 5.8 mg reducing sugar/ml. With all enzyme preparations, bagasse was more easily saccharified than rice straw.The authors are with the Atomic Energy Research Establishment, GPO Box 3787, Dhaka 1000, Bangladesh; N.A. Chowdhury, M. Moniruzzaman, and N. Choudhury in the Institute of Food and Radiation Biology, and N. Nahar in the Institute of Nuclear Science and Technology.     

Ultrastructural study of two rumen fungi:Piromonas communis andSphaeromonas communis

Two species of strictly anaerobic rumen fungi,Sphaeromonas communis andPiromonas communis, were examined by light and electron microscopy (scanning and transmission). Although morphologically different (vegetative body, number of flagella per zoospore), the ultrastructure of these two microorganisms was rather similar to that ofNeocallimastix frontalis andN. patriciarum. Two types of organelles were regularly found, i.e., isolated or associated ribosomes in the form of aggregates and hydrogenosome-like organelles with an amorphous content that may be involved in energy generation for these mitochondria-free strictly anaerobic fungi. UnlikeN. frontalis, the distribution of organelles was homogenous.     

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.     

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and<Emphasis Type="Italic"> m</Emphasis>-xylene metabolism

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.     

内生真菌与苍术粉对连作花生根际微生物区系和微量元素的影响

通过盆栽试验,研究了内生真菌拟茎点霉B3(Phomopsis liquidambari)及苍术(Atractylodes lancea)粉联合施用对连作花生根际土壤微生物区系、酶活性及有效态微量元素(Mo、B、DTPA-Fe、Zn、Cu、Mn)含量的影响。结果表明:内生真菌B3和苍术粉复合处理比内生真菌B3处理的荚果和秸秆产量分别增加10.28%和14.11%,内生真菌B3处理与正常施肥相比显著提高了根瘤数量、荚果和秸秆产重,各处理组与正常施肥对照相比分枝数和根长无显著差异。B3处理与对照相比显著提高了种子期、结荚期和成熟期根际土壤可培养细菌和放线菌数量,B3和苍术粉复合处理与对照相比显著提高种子期、花期和成熟期可培养真菌和放线菌数量;细菌DGGE指纹图谱聚类分析表明,B3和苍术粉复合处理相对于正常施肥处理,显著改变种子期、苗期、花期和成熟期花生根际土壤细菌群落结构,同时苗期、花期和结荚期的细菌条带数和香农指数也有所提高,真菌DGGE指纹图谱聚类分析表明,B3和苍术粉复合处理对真菌群落影响较大,除种子期以外的生育期真菌条带数和香农指数都有明显提高,花期真菌群落结构变化最大,相似度仅为49.6%。花生关键生育期(花期和结荚期)根际土壤脲酶和蔗糖酶活性B3处理和复合处理都显著高于正常施肥对照,促进了连作花生生态系统的物质循环和能量流动。B3和苍术粉复合处理促进了花生生长发育必需微量元素Mo、B、Fe、Zn、Mn的活化,花生叶片和籽粒中微量元素Mo、B、Fe的积累显著增加。研究结果表明,内生真菌和苍术粉联合施用能有效改善连作花生根际微生物区系,提高土壤酶活性,促进微量元素的活化和吸收,对缓解花生连作障碍具有重要意义。     

Effect of vanillin on the production of wood-decomposing enzymes from a wood-rotting fungus, <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

To examine the effect of vanillin on the production of the wood-decomposing enzymes of a wood-rotting fungus, vanillin was added as a model of lignin-related phenols to Coriolus versicolor cultures containing cellulosic and xylan substrates. Among five conditions tested, cellobiose alone was the most effective inducer of cellulolytic and xylanolytic enzymes. Addition of vanillin enhanced the effect of cellobiose on enzyme production. However, vanillin did not act as greatly in other cultures, except for cellobiose. Analytical isoelectric focusing and active staining of endo--1,4-glucanase demonstrated that isozyme patterns in the presence of vanillin were the same as those in absence of vanillin, indicating that vanillin does not induce novel isozymes but rather enhances enzyme production. On the other hand, vanillin, which enhanced production of phenol-oxidizing enzymes, was not always determined in all cultures, suggesting that the action of vanillin depends on the kinds of carbohydrates. Therefore, the effect of a monolignol vanillin on enzyme production was associated with coexistent carbohydrates.     

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.     

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.     

Nitrogen regulation in fungi

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems inAspergillus nidulans andNeurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation ofareA, the principle regulatory gene fromA. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities inA. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by theareA product, in particular localisation of the domain within theareA product which mediates the regulatory response within the protein.     

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing α-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and β-glucoside hydrolyzing activities. All strains synthesized β-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on α-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10–13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. β-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and β-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi. These studies were supported byPakistan Atomic Energy Commission. Some chemicals were purchased from funds allocated byUnited States Agency for International Development, Washington (DC, USA), under PSTC proposal 6.163.     

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa. Xyn10A exhibited extremely high (92%) amino acid sequence identity with Xyn10B (CAP0116) of this strain and had 42% and 32% identity with the catalytic domains of Rhodothermus marinus xylanase I and Thermoascus aurantiacus xylanase I, respectively. Xyn10A enzyme was purified from recombinant Escherichia coli and was highly active toward oat-spelt and Birchwood xylan and slightly active toward carboxymethyl cellulose, arabinogalactouronic acid, and various p-nitrophenyl monosaccharides. Xyn10A hydrolyzed xylan and xylooligosaccharides larger than xylobiose to produce xylose. This enzyme was optimally active at 60°C and had an optimum pH of 5.0. This is one of a number of related activities encoded on the large plasmid in this strain.     

Differential responses of three fungal species to environmental factors and their role in the mycorrhization of<Emphasis Type="Italic"> Pinus radiata</Emphasis> D. Don

Three ectomycorrhizal (ECM) isolates of Rhizopogon luteolus, R. roseolus and Scleroderma citrinum were found to differ markedly in their in vitro tolerance to adverse conditions limiting fungal growth, i.e. water availability, pH and heavy metal pollution. S. citrinum was the most sensitive, R. luteolus intermediate and R. roseolus the most tolerant species. Pinus radiata D. Don seedlings were inoculated in the laboratory and in a containerised seedling nursery with spore suspensions of the three ECM species. Colonisation percentage was considerably lower under nursery conditions, probably due to competition by native fungi. The effects of nursery ECM inoculation on seedling growth depended on the fungal species. Only R. roseolus-colonised plants showed a significantly higher shoot growth than non-mycorrhizal plants. All three fungi induced significantly higher root dry weights relative to control plants. Despite the low mycorrhizal colonisation, mycorrhization with all three species improved the physiological status of nursery-grown seedlings, e.g. enhanced root enzyme activity, shoot nutrient and pigment content, net photosynthesis rate and water use efficiency. Of the three fungal species, R. roseolus was the most effective; this species was also the most adaptable and showed the greatest range of tolerance to adverse environmental conditions in pure culture. It is, therefore, proposed as a promising fungal species for ECM inoculation of P. radiata in the nursery.     

Utilisation of carbon substrates by multiple genotypes of ericoid mycorrhizal fungal endophytes from eastern Australian Ericaceae

The abilities of six genotypes of two putative Helotiales ascomycete ericoid mycorrhizal fungal taxa from Woollsia pungens and Leucopogon parviflorus (Ericaceae) to utilise glucose, galactose, mannose, cellobiose, carboxymethylcellulose, crystalline cellulose, starch and xylan as sole carbon sources were tested in axenic liquid culture. With the exception of all taxon II isolates on carboxymethylcellulose, all genotypes of both taxa produced measurable biomass on all substrates. Significant intraspecific variation was observed in biomass production on all substrates. While pooled data for all genotypes of each taxon revealed significant interspecific differences in biomass production on carboxymethylcellulose, glucose, cellobiose, and starch, mean biomass production for each taxon on the latter three substrates differed less than threefold, suggesting that the saprotrophic abilities of the two taxa are broadly similar.     

Multisubstrate specifics amylase from mushroomTermitomyces clypeatus

An amylase was purified from the culture filtrate ofTermitomyces clypeatus by ammonium sulphate precipitation, DEAE-Sephadex chromatography and gel filtration on Bio-Gel P-200 column. The electrophoretically homogeneous preparation also exhibited hydrolytic activity (in a decreasing order) on amylose, xylan, amylopectin, glycogen, arabinogalactan and arabinoxylan. The enzyme had characteristically endo-hydrolytic activity on all the substrates tested and no xylose, glucose, arabinose or glucuronic acid could be detected even after prolonged enzymatic digestion of the polysaccharides. Interestingly the enzyme had similar pH optima (5.5), temperature optima (55°C), pH stability (pH 3–10) and thermal denaturation kinetics when acted on both starch and xylan (larch wood) .K _m values were found to be 2.63 mg/ml for amylase and 6.25 mg/ml for xylanase activity. Hill’s plot also indicated that the enzyme contained a single active site for both activities. Hg²⁺ was found to be most potent inhibitor. Ca²⁺, a common activator for amylase activity, appeared to be an inhibitor for this enzyme. Thus it appeared that the enzyme had multisubstrate specificity acting as α-amylase on starch and also acting as xylanase on side chain oligosaccharides of xylan containing α-linked sugars.     

Activities and properties of cellulase and xylanase associated with Phragmites leaf litter in a seawater lake

The activities and properties of cellulases and xylanases associated with Phragmites communis leaves were followed during the course of their decomposition from autumn to summer in a seawater lake. Cellulases and xylanases of low optimum pH (4 or less) were detected on aerial dead leaves before submergence. These enzymes remained on the leaves immediately after submergence, but were replaced by enzymes with a higher optimum pH (about 5.5 to 6.5), the activity of which increased rapidly during the initial two weeks. The enzyme activities then declined with water temperature until about day 100, but gradually increased again thereafter. This successive change in enzyme activities closely correlated with that of the decomposition rates of cellulose and xylan. The molecular weight distribution of the cellulases and xylanases changed markedly in the latter half of the experimental period, which suggested a change in the flora of active decomposer microorganisms.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Production of xylanases byAspergillus fumigatus andAspergillus oryzae on xylan-based media

Aspergillus fumigatus andA. oryzae were cultivated in laboratory fermenters on media containing xylan as the main carbon source.A. fumigatus produced xylanase on unsubstituted, insoluble beech xylan but growth and enzyme production on soluble xylo-oligosaccharides from the steaming of hardwood were poor due to the presence of inhibitors. An essential prerequisite for good xylanase production byA. fumigatus was decrease in the pH of the cultivation below 3.0 At higher pH values, the production of proteolytic enzymes caused degradation of the xylanase activity already produced.A. oryzae produced rather less xylanase activity thanA. fumigatus on the beech xylan medium but, after adaptation, was capable of efficient enzyme production on the steamed substrate.M.J. Bailey and L. Viikari are with the VTT, Biotechnical Laboratory, PO Box 202, SF-02151 Espoo, Finland