C. Adinarayana Reddy

The potential for white-rot fungi in the treatment of pollutants

Abstract Lignin-degrading white-rot fungi have the unique ability to degrade/mineralize a broad spectrum of structurally diverse toxic environmental pollutants. Extracellular peroxidases are important in degrading some, but not all, xenobiotic compounds. More research is needed to realize the potential of white-rot fungi in field-scale applications. Recent progress in our knowledge of the biochemistry and molecular biology of the key enzymes involved in xenobiotic degradation should pave the way for the eventual development of rational and enhanced bioremediation strategies.

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.

Effects of Turning Frequency, Leaves to Grass Mix Ratio and Windrow vs. Pile Configuration on the Composting of Yard Trimmings

Because of proposed bans on the landfilling and incineration of leaves, grass and brush, large-scale composting is fast becoming the primary disposal option for yard trimmings in many states. Few systematic studies have been done to compare the effects of turning regime, feedstock mix ratio, or windrow vs. pile configuration on composting and the characteristics of finished compost. In this study, various ratios of leaves, grass and brush were mixed and composted in two series of windrows; and one set of static piles. One windrow series (#1) was turned seven times every four weeks, while the other windrow series (#2), and the piles, were turned once every four weeks. The effects of the different treatments were examined by measuring compost temperature, oxygen concentration, pH, organic matter and moisture content, volatile fatty acid content, bulk density, stability, humification and seed germination indices, total and available nutrient levels, and particle size distribution. Results showed that turning...

Identification of glucose oxidase activity as the primary source of hydrogen peroxide production in ligninolytic cultures of Phanerochaete chrysosporium

The primary enzymatic activity involved in hydrogen peroxide (H2O2) production by extracts of ligninolytic cultures of Phanerochaete chrysosporium was investigated. Glucose supported the highest level of oxygen-dependent H2O2 production by cell extracts compared to a number of other substrates tested. No H2O2 production was observed anaerobically under N2. Polyacrylamide gel electrophoresis of extracts from ligninolytic cultures followed by diaminobenzidine/horseradish peroxidase staining procedure showed that only one protein band exhibited glucose-dependent H2O2 production. This protein band was not seen in extracts of non-ligninolytic cultures or in the extracellular fluid of ligninolytic cultures. Extracts of cells grown with either xylose, succinate or cellobiose showed the presence of only one glucose-dependent H2O2-producing band, with electrophoretic mobility similar to that observed in extracts of glucose-grown cells. Both glucose oxidase activity and lignin degradation were triggered in response to nitrogen (N) or carbohydrate starvation, and were repressed in media containing high levels of N (24 mM) or carbohydrate (56 mM), or on addition of exogenous N sources such as glutamate to the low N medium (2.4 mM N). The results indicate that glucose oxidase activity is the primary source of H2O2 in ligninolytic cultures of P. chrysosporium and that nutritional parameters which affect lignin degradation have a parallel effect on glucose oxidase activity.

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.

Yard Waste Composting: Studies Using Different Mixes of Leaves and Grass in a Laboratory Scale System

Composting has become a widely used method of recycling yard wastes such as leaves and grass. However, very little information is available on the chemical changes that occur during the composting of different mixtures of leaves and grass. In this study, three different mixes of leaves and grass were composted at approximately 60% moisture in a temperature controlled laboratory scale system. The mixes, which consisted of all leaves (Mix 1); 2/3 leaves + 1/3 grass (Mix 2); and 1/3 leaves + 2/3 grass (Mix 3), had initial C:N ratios of 48, 30 and 22, respectively. The compost process was monitored by measuring the rate of CO2 evolution, pH, stability, the degree of humification and changes in polysaccharide, carbon, nitrogen and organic matter content. Results showed that the greater the grass content of the mix, the higher the initial pH and the faster the rate of CO2 evolution, organic matter loss and nitrogen loss. After 43 days of composting, Mixes 1, 2 and 3, lost, respectively 61%, 74% and 78% of the c...

Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain.

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO2, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations≤4 mg l-1, but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of14C-ring-LAS to 14CO2 by P. chrysosporium was <1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain-length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition toβ-oxidation in the chain-shortening process.

In vitro degradation of natural insoluble lignin in aqueous media by the extracellular peroxidases of Phanerochaete chrysosporium.

The lignin peroxidases (LIP) and manganese peroxidases (MNP) of Phanerochaete chrysosporium catalyze a wide range of lignin depolymerization reactions with lignin models and synthetic lignins in solution. However, their ability to degrade insoluble natural lignin in aqueous media has not been demonstrated. Insoluble isolated poplar lignin similar to natural lignin was treated in vitro in aqueous media for 12 h with LIP, MNP, and both. Treatment with MNP alone slightly increased the solid mass and produced measurable amounts of lignin-derived 2,6-dimethoxyhydroquinone and 2-methoxyhydroquinone but did not appreciably decrease the total lignin content. Treatment with LIP alone did not decrease the mass but produced measurable amounts of lignin-derived p-hydroxybenzoic acid and slightly decreased the lignin content. Finally, treatment with LIP and MNP together decreased the solid mass by 11%, decreased the lignin content by 5%, and released low-concentration compounds with mass spectra containing the typical lignin-derived electron-impact fragments of mass 107, 137, 151, 167, and 181. These results suggest that MNP increases the effectiveness of LIP-mediated lignin degradation.

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

SummaryLignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.

Use of a shuttle vector for the transformation of the white rot basidiomycete, Phanerochaete chrysosporium

A novel shuttle vector based spheroplast transformation system for the lignin degrading filamentous fungus P. chrysosporium is described. The transformation vector, designated pRR12, consists of the yeast integration plasmid YIp5, a putative autonomous replication sequence (ars) of P. chrysosporium, and a 2.2 kb PvuII fragment carrying kanr determinant from plasmid pNG35, which confers resistance against both kanamycin and the related antibiotic G418. Two different strains of P. chrysosporium (ME446 and BKM-F) were transformed to G418 resistance using vector pRR12. Approximately 20 transformants per micrograms of vector DNA were obtained. The transforming vector pRR12 could be recovered from the total DNA of transformants by E. coli transformation, albeit at a low frequency.