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)     

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)     

Continuous degradation of trichloroethylene by Xanthobacter sp. strain Py2 during growth on propene.

Propene-grown Xanthobacter sp. strain Py2 cells can degrade trichloroethylene (TCE), but the transformation capacity of such cells was limited and depended on both the TCE concentration and the biomass concentration. Toxic metabolites presumably accumulated extracellularly, because the fermentation of glucose by yeast cells was inhibited by TCE degradation products formed by strain Py2. The affinity of the propene monooxygenase for TCE was low, and this allowed strain Py2 to grow on propene in the presence of TCE. During batch growth with propene and TCE, the TCE was not degraded before most of the propene had been consumed. Continuous degradation of TCE in a chemostat culture of strain Py2 growing with propene was observed with TCE concentrations up to 206 microns in the growth medium without washout of the fermentor occurring. At this TCE concentration the specific degradation rate was 1.5 nmol/min/mg of biomass. The total amount of TCE that could be degraded during simultaneous growth on propene depended on the TCE concentration and ranged from 0.03 to 0.34g of TCE per g of biomass. The biomass yield on propene was not affected by the cometabolic degradation of TCE.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Effect of phenolic monomers on biomass and hydrolytic enzyme activities of an anaerobic fungus isolated from wild nil gai (Baselophus tragocamelus)

AIMS: To test the anaerobic fungus, Piromyces sp. FNG5, for its tolerance to phenolic monomers released in the rumen by degradation of lignocellulosic poor-quality feeds. METHODS AND RESULTS: Effects of phenolic monomers on biomass and fibrolytic enzyme activities of a pure culture of lignocellulolytic anaerobic fungus (Piromyces sp. FNG5) isolated from faeces of wild nil gai (blue bull, Baselophus tragocamelus) were evaluated. There was a reduction in fungal biomass at 1 mm concentration of catechol with complete inhibition at 10 mm. p-Coumaric acid caused a reduction in biomass at 10 mm and no growth was observed above 20 mm concentration. The fungal isolate could tolerate up to 5 mm of ferulic acid without any reduction in biomass level, and was able to grow to some extent up to the highest level of ferulic acid tested (20 mm). Vanillic acid had no effect on biomass of the fungus even up to 50 mm level. The phenolic monomers varied in their potential to inhibit the secretion of carboxymethyl cellulase, xylanase, beta-glucosidase and acetyl esterase activities with catechol being the most inhibitory and vanillic acid being the least inhibitory. After 14 days of incubation, 38.49-65.14%p-Coumaric acid, 65.22-74.10% ferulic acid and 34.13-66.78% vanillic acid disappeared from the medium under anaerobic conditions. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the anaerobic fungus Piromyces sp. FNG5 is tolerant to phenolic monomers and has ability to degrade them. Therefore, such anaerobic fungi may play an important role in fibre degradation in the rumen.     

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.     

Hydrolytic activities of anaerobic fungi from wild blue bull (Boselaphus tragocamelus)

The anaerobic fungi play an active role in the plant fibre degradation by producing a wide array of potential hydrolytic enzymes in the rumen. In present study, 12 anaerobic fungal strains were isolated from the faecal samples of wild blue bull, and identified as species of Piromyces, Anaeromyces, Orpinomyces and Neocallimastix based on their morphological characteristics. Isolate WNG-12 (Piromyces sp.), showed maximum filter paper cellulase (23 mIU ml(-1)) and xylanase (127 mIU ml(-1)) activity, while WNG-5 (Piromyces sp.) showed maximum carboxymethyl cellulase activity (231 mIU ml(-1)). Based on the results obtained, it can be stated that Piromyces sp. WNG-12 may be a promising isolate in utilizing fibre rich diets in the rumen as evidenced by the production of hydrolytic enzymes in vitro.     

Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium

Anaerobic fungi were isolated from rumen fluid of a domestic sheep (Ovis aries; a ruminant) and from faeces of five non-ruminants: African elephant (Loxodonta africana), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Indian elephant (Elephas maximus) and mara (Dolichotis patagonum). The anaerobic fungus isolated from the sheep was a Neocallimastix species and the isolates from non-ruminants were all species similar to Piromyces spp. A defined medium is described which supported growth of all the isolates, and was used to examine growth characteristics of the different strains. For each fungus the lipid phosphate content was determined after growth on cellobiose and the resulting values were used to estimate fungal biomass after growth on solid substrates. The ability of isolates from ruminants and non-ruminants to digest both wheat straw and cellulose was comparable. More than 90% and 60%, respectively, of filter paper cellulose and wheat straw were digested by most strains within 60-78 h. Growth of two fungi, isolated from rumen fluid of a sheep (Neocallimastix strain N1) and from faeces of an Indian rhinoceros (Piromyces strain R1), on cellobiose was studied in detail. Fungal growth yields on cellobiose were 64.1 g (mol substrate)-1 for N1 and 34.2 g mol-1 for R1. The major fermentation products of both strains were formate, lactate, acetate, ethanol and hydrogen.     

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene.

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic activity of rumen fungi belonging to the genera Neocallimastix and Piromyces

Proteolytic activity of two rumen fungal isolates Neocallimastix sp. strain N1 and Piromyces sp. strain P1 was examined. Proteases are active between pH 6.5 and 9.0 with maximum at 7.9 for isolate N1 and between 6.5 and 10.5 with maximum at 8.8 for isolate P1. Proteolytic activity increased as temperature increased until 50°C and a sudden decrease at 60°C was observed in both isolates. EDTA, 1,10-phenanthroline, p -chloromercuribenzoate (PCMB), merthiolate and phenylmethylsulphonyl fluoride (PMSF) were effective proteolytic inhibitors against both isolates.     

Noncatalytic docking domains of cellulosomes of anaerobic fungi

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.     

Biodegradation of dibutyl phosphite by Sphingobium sp. AMGD5 isolated from aerobic granular biomass

In the present study, cultivation of aerobic granular biomass capable of biodegradation of dibutyl phosphite, an organophosphite, and isolation of dibutyl phosphite degrading bacterial strains, are reported for the first time. The strain AMGD5, identified as Sphingobium sp., based on 16S rRNA sequencing, degraded dibutyl phosphite efficiently and utilised it as the sole source of carbon and phosphorus. Microbial degradation of dibutyl phosphite caused a significant decrease in medium pH, leading to cessation of growth and further degradation of dibutyl phosphite. Under buffered conditions, complete degradation of up to 3 mM of dibutyl phosphite was achieved within 60 h. The strain showed almost similar growth pattern when either phosphite or dibutyl phosphite was used as the phosphorous source. A 4-fold enhancement in phosphatase activity was evident in dibutyl phosphite fed cells, implying their role in dibutyl phosphite degradation. Sphingobium sp. AMGD5 can be a potential candidate for bioremediation of dibutyl phosphite contaminated waters or sites.     

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.     

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.     

Microcalorimetric study of aerobic growth of Cellulomonas sp. 21399 on various carbohydrates

Summary Microcalorimetry was used to study the energetic aerobic growth of Cellulomonas sp. 21399 on glucose, cellobiose and amorphous and crystalline cellulose. The thermochemical aspect of growth on glucose was established with regard to the anabolic contribution. The results obtained allowed the use of glucose as a reference substrate for cellulose degradation. The experimental enthalpy change and the maximum catabolic activity, calculated from the maximum power evolved by the culture, were, respectively,-1079 kJ/mol and 0.85 mmol glucose per hour per dry weight of cells. The growth response on amorphous cellulose was equivalent to that demonstrated on glucose. However, on crystalline cellulose media, Cellulomonas sp. 21399 exhibited eight times less power and the quantity of heat evolved during growth showed that 50% of the cellulose was degraded. Quantitative results and the shape of power-time curves achieved indicate that the structural features of cellulose strongly influence its microbial degradability.     

The physical action of cellulases revealed by a quartz crystal microbalance study using ultrathin cellulose films and pure cellulases

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.     

Degradation and transformation of anthracene by white-rot fungus Armillaria sp. F022

Characterization of anthracene metabolites produced by Armillaria sp. F022 was performed in the enzymatic system. The fungal culture was conducted in 100-mL Erlenmeyer flask containing mineral salt broth medium (20 mL) and incubated at 120 rpm for 5–30 days. The culture broth was then centrifuged at 10,000 rpm for 45 min to obtain the extract. Additionally, the effect of glucose consumption, laccase activity, and biomass production in degradation of anthracene were also investigated. Approximately, 92 % of the initial concentration of anthracene was degraded within 30 days of incubation. Dynamic pattern of the biomass production was affected the laccase activity during the experiment. The biomass of the fungus increased with the increasing of laccase activity. The isolation and characterization of four metabolites indicated that the structure of anthracene was transformed by Armillaria sp. F022 in two routes. First, anthracene was oxidized to form anthraquinone, benzoic acid, and second, converted into other products, 2-hydroxy-3-naphthoic acid and coumarin. Gas chromatography–mass spectrometry analysis also revealed that the molecular structure of anthracene was transformed by the action of the enzyme, generating a series of intermediate compounds such as anthraquinone by ring-cleavage reactions. The ligninolytic enzymes expecially free extracellular laccase played an important role in the transformation of anthracene during degradation period.     

The effect of bovicin HC5, a bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro

A Pseudomonas monteilli strain (designated C11) that uses the phosphotriester coroxon as its sole phosphorus source has been isolated. Native PAGE and activity staining identified a single isozyme with significant phosphotriesterase activity in the soluble fraction of the cell. This phosphotriesterase could hydrolyse both coumaphos and coroxon. The hydrolysis product of coroxon, diethylphosphate, and the thion analogue, coumaphos, could not serve as phosphorus sources when added to the growth medium. The majority of the phosphotriesterase and phosphatase activity was contained in the soluble fraction of the cell. Phosphatase activity was inhibited by vanadate as well as by dialysis against the metal chelator, EDTA. Phosphotriesterase activity was not affected by either vanadate or dialysis with EDTA or 1,10-phenanthroline. Phosphotriesterase activity was regulated by the amounts of both phosphate and coroxon in the medium, whereas total phosphatase activity was regulated by phosphate but not coroxon. A lack of hybridisation using a probe against the opd (organophosphate degradation) gene encoding a phosphotriesterase from Flavobacterium sp. ATCC27551 against bulk DNA from P. monteilli C11 suggested that this strain does not contain opd. The work presented here indicates the presence of a novel phosphotriesterase in P. monteilli C11.