Screening of white-rot fungi for bioprocessing of wheat straw into ruminant feed.

AIM: In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal-treated wheat straw. METHODS AND RESULTS: Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0·05) IVGP compared to the control after 7 weeks (227·7 ml g-1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g-1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g-1 OM, respectively. CONCLUSION: Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.

Differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value of wheat straw for ruminants.

AIM: This study evaluated differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value and in vitro degradability of wheat straw. METHODS AND RESULTS: Wheat straw was treated with the fungi for 7 weeks. Weekly samples were analysed for ergosterol content, in vitro gas production (IVGP), chemical composition and lignin-degrading enzyme activity. Ergosterol data showed CS1 to have a faster initial growth than CS2 and reaching a stationary phase after 3 weeks. The IVGP of CS1-treated wheat straw exceeded the control earlier than CS2 (4 vs 5 weeks). CS1 showed a significantly higher (P < 0·001) selectivity in lignin degradation compared to CS2. Both strains showed peak activity of laccase and manganese peroxidase (MnP) at week 1. CS1 showed a significantly higher (P < 0·001) laccase activity, but lower (P = 0·008) MnP activity compared to CS2. CONCLUSION: Both CS strains improved the nutritive value of wheat straw. Variation between strains was clearly demonstrated by their growth pattern and enzyme activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences among the two strains provide an opportunity for future selection and breeding programs in improving the extent and selectivity of lignin degradation in agricultural biomass.

Fungal treated lignocellulosic biomass as ruminant feed ingredient: a review.

In ruminant nutrition, there is an increasing interest for ingredients that do not compete with human nutrition. Ruminants are specialists in digesting carbohydrates in plant cell walls; therefore lignocellulosic biomass has potential in ruminant nutrition. The presence of lignin in biomass, however, limits the effective utilization of cellulose and hemicellulose. Currently, most often chemical and/or physical treatments are used to degrade lignin. White rot fungi are selective lignin degraders and can be a potential alternative to current methods which involve potentially toxic chemicals and expensive equipment. This review provides an overview of research conducted to date on fungal pretreatment of lignocellulosic biomass for ruminant feeds. White rot fungi colonize lignocellulosic biomass, and during colonization produce enzymes, radicals and other small compounds to breakdown lignin. The mechanisms on how these fungi degrade lignin are not fully understood, but fungal strain, the origin of lignocellulose and culture conditions have a major effect on the process. Ceriporiopsis subvermispora and Pleurotus eryngii are the most effective fungi to improve the nutritional value of biomass for ruminant nutrition. However, conclusions on the effectiveness of fungal delignification are difficult to draw due to a lack of standardized culture conditions and information on fungal strains used. Methods of analysis between studies are not uniform for both chemical analysis and in vitro degradation measurements. In vivo studies are limited in number and mostly describing digestibility after mushroom production, when the fungus has degraded cellulose to derive energy for fruit body development. Optimization of fungal pretreatment is required to shorten the process of delignification and make it more selective for lignin. In this respect, future research should focus on optimization of culture conditions and gene expression to obtain a better understanding of the mechanisms involved and allow the development of superior fungal strains to degrade lignin in biomass.

Effect of fungal treatments of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and methane production.

Maize stover, rice straw, oil palm fronds and sugarcane bagasse were treated with the white-rot fungi Ceriporiopsis subvermispora, Lentinula edodes, Pleurotus eryngii, or Pleurotus ostreatus at 24 °C for 0-6 weeks. The fungi increased total gas production from oil palm fronds by 68-132%, but none of the fungi improved the in vitro rumen fermentability of maize stover. C. subvermispora and L. edodes increased total gas production of sugarcane bagasse by 65-71%, but P. eryngii and P. ostreatus decreased it by 22-50%. There was a linear relationship (P<0.05) between the proportion of lignin in the original substrate and the increase in in vitro gas production observed for C. subvermispora and L. edodes treatments (R2=0.92 and 0.96, respectively). It is concluded that C. subvermispora and L. edodes have a particularly high potential to improve the nutritive value of highly lignified ruminant feeds.

Fungal strain and incubation period affect chemical composition and nutrient availability of wheat straw for rumen fermentation.

Eleven white-rot fungi were examined for their potency to degrade lignin and to improve the rumen fermentability of wheat straw. The straw was inoculated with the fungi and incubated under solid state conditions at 24°C for 0-49 days to determine changes in in vitro gas production and chemical composition. Results show that some fungi could degrade lignin by as much as 63%, yet the delignification was highly correlated with the degradation of hemicellulose (r=0.96). Reduction in lignin was poorly (r=0.47), but the ratio between lignin and cellulose loss was strongly (r=0.87) correlated with the increase in gas production. Treatment with Ceriporiopsis subvermispora for 49 days increased total gas production of the straw from 200 to 309 ml/g organic matter (OM). It was concluded that some fungi highly selective for lignin and not for cellulose are able to improve the nutritive value of wheat straw as a ruminant feed.

Isolation, characterization, and expression patterns of a DMC1 homolog from the basidiomycete Pleurotus ostreatus.

Here we describe the isolation of a Pleurotus ostreatus gene PoDMC1. The predicted amino acid sequence of the oyster mushroom gene is 62% identical to the yeast DMC1 and 60% identical to human DMC1. The highest degree of amino acid identity (88%), however, was shown with Coprinus CoLIM15, a DMC1 homolog recently found in Coprinus cinereus. The exact matching of sizes and positions of most introns in both basidiomycete genes underlines the close relationship between these DMC1 orthologs. The RecA homolog DMC1 from yeast and its orthologs from other species have been reported to be meiosis specific and essential for sporulation. Here we show that PoDMC1 is exclusively expressed in the lamellae/basidiospore fraction of fruit bodies and not in somatic cells of fruiting bodies or in vegetative mycelium. Furthermore, the gene is not expressed in the lamellae/basidiospore fraction of a nonsporulating mutant of P. ostreatus. Since one of the major problems in cultivating the oyster mushroom is the abundant sporulation that causes allergic reactions in man, PoDMC1 could be an important target gene in constructing sporeless Pleurotus strains.

Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens.

Agrobacterium tumefaciens is known to transfer parts of its tumor-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus. Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection marker is stable during mitosis and meiosis. The Agrobacterium system allows the transformation of both homokaryons and heterokaryons of A. bisporus. Also, both karyotypes of an heterokaryon can be transformed simultaneously. Furthermore, this is the first report on the transformation of vegetative mycelium of a commercial strain of A. bisporus.

Molecular cloning of a widely distributed microsatellite core sequence from the cultivated mushroom Agaricus bisporus.

An Agaricus bisporus microsatellite with the tetranucleotide motif TATG tandemly repeated was isolated from an A. bisporus library enriched in repeated sequences. The use of the 16-mer oligonucleotide (TATG)4 indicates that many loci contain nearby copies of the microsatellite in opposite orientations. The wide distribution of the microsatellite in the A. bisporus genome was assessed (i) by polyacrylamide gel electrophoresis of the products generated by directed amplification of microsatellite-region DNA (DAMD) and (ii) by hybridization of these products with A. bisporus chromosomes separated by pulsed-field gel electrophoresis. This is, to our knowledge, the first microsatellite reported in the cultivated edible mushrooms. DAMD-PCR products were generated using DNA of three Pleurotus species (P. pulmonarius, P. sajor-caju, and P. florida), indicating that (TATG)4 repeats are also present in these cultivated species. The variability found within closely related strains indicates that such microsatellites are useful in fingerprinting and studying genetic variability in wild and commercial mushrooms.

Abr1, a transposon-like element in the genome of the cultivated mushroom Agaricus bisporus (Lange) Imbach.

A 300-bp repetitive element was found in the genome of the white button mushroom, Agaricus bisporus, and designated Abr1. It is present in approximately 15 copies per haploid genome in the commercial strain Horst U1. Analysis of seven copies showed 89 to 97% sequence identity. The repeat has features typical of class II transposons (i.e., terminal inverted repeats, subterminal repeats, and a target site duplication of 7 bp). The latter shows a consensus sequence. When used as probe on Southern blots, Abr1 identifies relatively little variation within traditional and present-day commercial strains, indicating that most strains are identical or have a common origin. In contrast to these cultivars, high variation is found among field-collected strains. Furthermore, a remarkable difference in copy numbers of Abr1 was found between A. bisporus isolates with a secondarily homothallic life cycle and those with a heterothallic life cycle. Abr1 is a type II transposon not previously reported in basidiomycetes and appears to be useful for the identification of strains within the species A. bisporus.

The Agaricus bisporus pruA gene encodes a cytosolic delta 1-pyrroline-5-carboxylate dehydrogenase which is expressed in fruit bodies but not in gill tissue.

A fortuitously cloned 3'-truncated cDNA encoding the Agaricus bisporus delta 1-pyrroline-5-carboxylate dehydrogenase was used to characterize the complete gene. The gene would encode a cytosolic polypeptide of 546 amino acids, and the basidiomycetous gene was evenly expressed in various parts of the mushroom except for the gills. No expression was detected in compost-grown mycelium. The steady-state mRNA level of the gene in the vegetative phase was determined on simple synthetic media and was two- to threefold higher with ammonium or proline as the sole nitrogen source compared to glutamate as the sole nitrogen source. Moreover, the steady-state mRNA level was not markedly influenced by addition of ammonium phosphate to proline- or glutamate-utilizing cultures. The results suggest that ammonium and the amino acids proline and glutamate are equally preferred nitrogen sources in this organism and are consistent with previous observations of H. M Kalisz, D.A. Wood, and D. Moore (Trans. Br. Mycol. Soc. 88:221-227, 1987) that A. bisporus continues to degrade protein and secrete ammonium even if ammonium and glucose are present in the culture medium.

Isolation of expressed sequence tags of Agaricus bisporus and their assignment to chromosomes.

The genome of the cultivated basidiomycete Agaricus bisporus Horst U1 and of its homokaryotic parents has been characterized by using an optimized method of pulsed-field gel electrophoresis. Expressed sequence tags obtained as expressed cDNAs from a primordial tissue-derived cDNA library and a number of previously isolated genes were used to identify the individual chromosomes of the parental lines of Horst U1. The genome consists of 13 chromosomes, and its total size is 31 Mb. For those chromosomes that could not be resolved by contour-clamped homogeneous electric field electrophoresis, the segregation of marker genes was studied in a set of 86 homokaryotic offspring of Horst U1. At least two markers were assigned to each individual chromosome. In this way all individual chromosomes were unequivocally identified. The large size difference observed between the homologous chromosomes IX, harboring the rDNA repeat, was shown to be largely due to a higher copy number of rDNA in parental strain H97 than in parental strain H39.

The Agaricus bisporus hypA gene encodes a hydrophobin and specifically accumulates in peel tissue of mushroom caps during fruit body development.

Differential screening of a cDNA library was used to clone genes that are specifically expressed during mushroom development in the basidiomycete Agaricus bisporus. One of the isolated genes encodes a polypeptide of 112 amino acid residues and belongs to the fungal gene family encoding hydrophobins. This gene, hypA, has the characteristic pattern of eight cysteine residues at conserved positions and a hydrophobicity pattern that is very similar to class I hydrophobins. Elucidation of the genomic structure of hypA led to the identification of a second copy, hypC, located downstream of hypA. Although at a much lower level, hypC is like hypA specifically expressed on fruit bodies. The hypA mRNA level is transiently increased ten days after fruit body induction and expression appears to be associated with rapid expansion of the mushroom caps. In mushroom caps, very high concentrations of hypA messengers were found in the (outer) peel tissue, where they accumulate to more than 60% of the total mRNA mass. The corresponding protein with a molecular mass of 8 to 9 kDa was purified from this peel tissue and was identified by N-terminal sequencing. Our results suggest that HYPA forms a protective hydrophobic layer instrumental in cap formation.

Nucleotide sequence and expression of the gene encoding NADP+- dependent glutamate dehydrogenase (gdhA) from Agaricus bisporus.

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from an Agaricus bisporus recombinant phage lambda library. The deduced amino acid sequence would specify a 457-amino acid protein that is highly homologous in sequence to those derived from previously isolated and characterized genes coding for microbial NADP+-GDH. The open reading frame is interrupted by six introns. None of the introns is located at either one of the positions of the two introns conserved in the corresponding open reading frames of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. Northern analysis suggests that the A. bisporus gdhA gene is transcriptionally regulated and that, unlike the case in ascomycetes, transcription of this gene is repressed upon the addition of ammonium to the culture.

Investigation of Mitochondrial Transmission in Selected Matings between Homokaryons from Commercial and Wild-Collected Isolates of Agaricus bisporus (= Agaricus brunnescens).

Ten heterokaryons of Agaricus bisporus (= Agaricus brunnescens) were shown to carry four different mitochondrial (mt) genotypes by analysis of mt restriction fragment length polymorphisms (RFLPs). Fifteen homokaryons derived from these strains were used to investigate mt inheritance in A. bisporus. One hundred eighty-nine pairings were performed in 25 different combinations. Pairings in 15 different combinations produced heterokaryons on the basis of nuclear RFLP analyses and/or fruiting trials. The mt genotype of each new intraspecies hybrid was examined by using mt RFLPs as genetic markers. Our results suggest the following. (i) Recombination between the mt genomes was not a common event. (ii) From most individual pairings, all heterokaryons carried the same mt genotype. (iii) Heterokaryons carrying either of the two possible mt genotypes were observed in certain crosses after modification of the pairing procedure. A biparental transmission pattern was demonstrated for some crosses, but there appears to be a preference for one of the mt genotypes to predominate in any specific pairing.

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts.

Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.

Generation of an electrochemical proton gradient in Streptococcus cremoris by lactate efflux.

Recently an energy-recycling model was proposed that postulates the generation of an electrochemical gradient in fermentative bacteria by carrier-mediated excretion of metabolic end products in symport with protons. In this paper experimental support for this model is given. In batch cultures of Streptococcus cremoris with glucose as the sole energy source the maximal specific growth rate decreased by 30% when the external lactate concentration was decreased from 50 to 90 mM. In the same range of external lactate concentrations the molar growth yield Y for glucose as measured in energy-limited chemostat cultures also showed a 30% drop. From Y max lactose values of S. cremoris grown in the presence and absence of added lactate it was calculated that the net energy gain from the lactate efflux system was at least 12%. Lactate efflux from de-energized cells loaded with lactate could drive the uptake of leucine. This uptake was sensitive to carbonylcyanide p-trifluoromethoxyphenylhydrazone and was only partly inhibited by dicyclohexylcarbodiimide (DCCD). The limited inhibition by DCCD of lactate-induced leucine uptake indicates that ATP hydrolysis was not the driving force for transport of leucine. Uptake studies with the lipophilic cation tetraphenylphosphonium demonstrated that lactate efflux increased the electrical potential across the membrane by 51 mV. The generation of an electrical potential by lactate efflux and the demonstration of a potassium efflux-induced uptake of lactate indicates that lactate is translocated across the membrane by a symport system with more than one proton.