Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.

Crystal Structure and Biochemical Characterization of Xylose Isomerase from Piromyces sp. E2.

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a D-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize D-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino acid sequence comparison with other reported XIs and the relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XI. Moreover kinetic analysis of PsXI was also performed using Mn²âº, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance D-xylose utilization for biofuel production.

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography.

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity.

Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae.

The conversion of abundant levels of xylose in lignocellulosic materials into viable products would generate economic benefits. The heterologous expression of the xylose isomerase (XI) gene is considered a direct and effective strategy for establishing the xylose metabolic pathway in Saccharomyces cerevisiae. However, only limited sources of xylA are functionally expressed in S. cerevisiae and are capable of driving effective xylose consumption. In this study, Ru-xylA (where Ru represents the rumen), which was screened from the contents of the bovine rumen metagenomic library, was functionally expressed in S. cerevisiae, and the enzyme activity was 1.31 U mg(-1) protein. This is a new source of XI that can exhibit high activity levels in S. cerevisiae. The activity of this enzyme is comparable to those of the Piromyces sp. XI. Then, the Ru-XI activity was further improved through mutagenesis and growth-based screening in a centromeric plasmid. A variant containing two mutations (K11T/D220V) that exhibited a 68% increase in enzyme activity was isolated. Our work identified a new xylose isomerase that can be functionally expressed in S. cerevisiae and results in a higher XI enzyme activity through mutagenesis.

Immobilization of Piromyces rhizinflata ß-glucanase on poly(dimethylsiloxane) and Si wafer and prediction of optimum reaction for enzyme activity.

EglA, a ß-1,4-glucanase isolated from the ruminal fungus Piromyces rhizinflata, shows promise in a wide range of industrial applications because of its broad substrate specificity. In this study, EglA was immobilized on different supporting materials including poly(dimethylsiloxane) (PDMS), Si wafer, textured Si wafer, and indium tin oxide-coated (ITO-coated) glass. The binding abilities of PDMS and Si wafer toward EglA were significantly higher than those of the other supporting materials. The optimized temperature and pH conditions for EglA immobilized on PDMS and on Si wafer were further determined by a response surface methodology (RSM) combined with a central composite design (CCD). The results indicated that the optimum pH and temperature values as well as the specific ß-glucanase activity of EglA on PDMS were higher than those of free-form EglA. In addition, EglA immobilized on PDMS could be reused up to six times with detectable enzyme activity, while the enzyme activity of Eg1A on Si wafer was undetectable after three cycles of enzyme reaction. The results demonstrate that PDMS is an attractive supporting material for EglA immobilization and could be developed into an enzyme chip or enzyme tube for potential industrial applications.

Substrate binding of a GH5 endoglucanase from the ruminal fungus Piromyces rhizinflata.

The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (ß/α)(8)-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the -3, -2 and -1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the -3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (ß/α)(8)-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem.

Molecular cloning and characterization of a ß-glucanase from Piromyces rhizinflatus.

Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a ß-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. To examine the enzyme activities, CbhYW23-2 was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis was then employed for the planned statistical optimization of the ß-glucanase activities of CbhYW23-2. The optimal conditions for the highest ß-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the high activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications.

Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications.

AIMS: The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. METHODS AND RESULTS: The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). CONCLUSION: The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain.

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose-xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L(-1) acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L(-1)) in all cultures. Xylose fermentation in acetic -acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.

Isolation and identification of cellulose-binding proteins from sheep rumen contents.

To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

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.

Expression of rumen microbial fibrolytic enzyme genes in probiotic Lactobacillus reuteri.

This study was aimed at evaluating the cloning and expression of three rumen microbial fibrolytic enzyme genes in a strain of Lactobacillus reuteri and investigating the probiotic characteristics of these genetically modified lactobacilli. The Neocallimastix patriciarum xylanase gene xynCDBFV, the Fibrobacter succinogenes beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene, and the Piromyces rhizinflata cellulase gene eglA were cloned in a strain of L. reuteri isolated from the gastrointestinal tract of broilers. The enzymes were expressed and secreted under the control of the Lactococcus lactis lacA promoter and its secretion signal. The L. reuteri transformed strains not only acquired the capacity to break down soluble carboxymethyl cellulose, beta-glucan, or xylan but also showed high adhesion efficiency to mucin and mucus and resistance to bile salt and acid.

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

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.

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.

Genomic DNA analysis of genes encoding (hemi-)cellulolytic enzymes of the anaerobic fungus Piromyces sp. E2.

Anaerobic fungi contain more than one copy of genes encoding (hemi-)cellulases in their genome. The arrangement of these genes on the chromosomes was not known. A genomic DNA (gDNA) library of Piromyces sp. E2 was screened with different probes specific for (hemi-)cellulolytic enzymes. This screening resulted in three gDNA clones with genes encoding glycoside hydrolase enzymes of families 1 (beta-glucosidase), 6 (exoglucanase) and 26 (mannanase). Each clone contained two or more genes of the same family. Comparison of the gene copies on a clone revealed that they were highly homologous, and in addition, 54-75% of the substitutions was synonymous. One of the mannanase genes contained an intron. PCR with selected primers resulted in a gDNA clone with a new representative (cel9B) of glycoside hydrolase family 9 (endoglucanase). Comparison with cel9A revealed that cel9B had 67% homology on the nucleotide level. Furthermore, three introns were present. All results of this paper taken together provided evidence for duplications of (hemi-)cellulolytic genes, which resulted in clusters of almost identical genes arranged head-to-tail on the genome. In contrast to other eukaryotes, this phenomenon appears frequently in anaerobic fungi.

Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway.

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25+/-13 nmol min(-1)mg protein(-1)). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.

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.