Ronald L. Crawford

Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium

Ligninase-I (Mr 42,000-43,000; carbohydrate, 21%) and peroxidase-M2 (Mr 45,000-47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzymes substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.

Bioremediation : principles and applications

Preface D. Crawford 1. Introduction R. Crawford 2. Engineering of bioremediation processes W. Admassu and R. A. Korus 3. Bioremediation in soil: influence of soil properties on organic contaminants and bacteria M. J. Morra 4. Biodegradation of BTEX hydrocarbons under anaerobic conditions L. Crumholtz, M. E. Caldwell and J. M. Suflita 5. Bioremediation of petroleum contamination E. Rosenberg and E. Zon 6. Bioremediation of environments contaminated by polycyclic aromatic hydrocarbons J. Mueller, C. Cerniglia and P. Pritchard 7. Bioremediation of nitroaromatic compounds S. B. Funk, D. Crawford and R. Crawford 8. A history of PCB biodegradation R. Unterman 9. Bioremediation of chlorinated phenols J. Puhakka and E. Melin 10. Biodegradation of chlorinated aliphatic compounds L. Wackett 11. Microbial remediation of metals T. Roane, I. Pepper and R. Miller 12. Molecular techniques in bioremediation M. Shields and S. Francesconi Index.

Microbial degradation of lignin

Abstract The transformations of lignin that occur during its biodegradation are complex and incompletely understood. Certain fungi of the white-rot group, and possibly other fungi and bacteria, completely decompose lignin to carbon dioxide and water. Other fungi and bacteria apparently degrade lignin incompletely. Differences in lignin-degrading abilities observed for different organisms may result from differences in the completeness of their ligninolytic enzyme systems. Not all lignin components may be attacked by a particular organism. Alternatively, different organisms may differ in their basic mechanisms of attack on lignin. The basic pathways of lignin degradation have been elucidated only for certain representatives of the white-and brown-rot fungi. Although it is known that each of the principal structural components of lignin is attacked by other fungi and bacteria, the biochemistry of that attack has not been elucidated. Work with low molecular weight lignin models has provided only limited information on possible pathways of lignin degradation by microorganisms. There is little evidence to suggest a correlation between abilities to degrade single-ring aromatic or lignin model compounds and the ability to degrade polymeric lignin. More evidence has come from analysis of spent culture media for lignin breakdown products and from comparative chemical analyses of sound lignins versus decayed lignin residues. Accumulated evidence with the most thoroughly studied white-rot fungi suggests that with these fungi lignin degradation proceeds by way of extracellular mixed-function oxygenases and dioxygenases, which catalyse demethylations, hydroxylations and ring-fission reactions within a largely intact polymer, concomitant with some release of low molecular weight lignin fragments. There are also apparent relationships between lignin, carbohydrate and nitrogen metabolism for some organisms, but the relationships may vary from one organism to another. Although research is now mostly at a basic level, industrial applications may result from lignin degradation research. Considerable potential exists for the development of bioconversions which might produce low molecular weight chemicals from waste lignins, and thereby reduce our dependence on petroleum as a source of these chemicals. Alternatively, such bioconversions might produce chemically altered forms of polymeric lignin that may be valuable industrially.

Microbiological removal of pentachlorophenol from soil using a Flavobacterium

Abstract Experiments reported here show that it is possible to remove pentachlorophenol (PCP) from a variety of contaminated soils, including actual waste-dump soils, by inoculating such soils with cells of a PCP-degrading Flavobacterium. However, soil conditions such as temperature, water content, PCP concentration and density of bacterial cells must be maintained within optimum ranges. Manipulation of the natural microflora so that it degrades PCP may be the preferred decontamination method in many instances. Very highly contaminated soils are so toxic that methods such as soil leaching followed by decontamination of the leachate may be required, as direct inoculation or self-purification are ineffective.

Recent advances in studies of the mechanisms of microbial degradation of lignins

Abstract Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ∪c-|_.), hydroxyl radical (·OH) or singlet oxygen ( 1 O 2 ) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H 2 O 2 is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus , scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C α −C β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H 2 O 2 -requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignins aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.

Microbial degradation of chlorinated phenols

Abstract The chlorinated phenols comprise a large group of toxic, man-made chemicals that are serious environmental pollutants. Microorganisms can degrade many, but not all, of the chlorinated phenols, often using chlorophenol-specific catabolic enzymes. Novel technologies are evolving for using specific microorganisms to clean contaminated soils and waters of chlorophenols.

Chemistry of softwood lignin degradation byStreptomyces viridosporus

Polymeric lignin isolated from ground spruce phloem/bark tissue following decay by the actinomyceteStreptomyces viridosporus (T7A) was characterized chemically and compared to undergraded lignin from the same source. The chemical transformations resulting from degradation were compared to those that result from fungal degradation of softwood lignins by brown- and white-rot fungi. Degradative chemical analyses showed thatS. viridosporus-degraded lignin was significantly altered in structure. Much of the integrity of the basic 4-hydroxy-3-methoxyphenylpropane subunit structure was lost. Actinomycete-decayed lignin was decreased in carbon and enriched in oxygen and hydrogen contents. It also had been extensively demethylated. Chemical analysed indicated that phenylpropanoid side-chains had been oxidized by introduction of α-carbonyls and by side-chain shortening reactions. Although the degraded lignin remained polymeric, it was significantly dearomatized. These changes are similar to those previously reported for white-rotted lignins, except for the increased hydrogen content. The evidence indicated that lignin degradation byS. viridosporus is oxidative and involves demethylations, ring cleavage reactions, and oxidative attack on phenylpropanoid side-chains. Also, some reduced structures accumulate in the polymer and some low molecular weight intermediates are released into the growth medium.

Biodegradation of ‘BTEX’ hydrocarbons under anaerobic conditions

Introduction Fossil energy reserves are valuable natural resources that underpin most major world economies. The extraction, transport and utilization of these resources inevitably leads to the release of these substances to environmental compartments where they are deemed undesirable. For instance, petroleum or petroleum distillation products often occur as contaminants in soils, aquifers and surface waters via a myriad of mechanisms including leaking underground storage tanks, aboveground spills and release by marine transport vessels. Since environmental matrices (air, water and soil) are completely integrated, all are susceptible to a reduction in quality and often quantity due to the release of pollutant materials. When this occurs, a great deal of concern is associated with the impact of the hydrocarbons on humans and recipient environments. However, this impact cannot be correctly gauged without information on the transport and fate characteristics of the individual contaminants. A major factor governing the transport and fate of contaminant hydrocarbons is their susceptibility to metabolism by aerobic and anaerobic microorganisms. While a plethora of information is available on the prospects for aerobic biodegradation, comparatively little is understood about anaerobic hydrocarbon biotransformation. However, it is well recognized that anaerobic microbial activities directly or indirectly impact all major environmental compartments. In many environments, most notably the terrestrial subsurface, oxygen concentrations are often initially low. With rapid utilization by hydrocarbonoclastic microorganisms and limited rates of reoxygenation, oxygen becomes depleted. Without the reactive power of molecular oxygen, the biodegradation rate of hydrocarbons slows down and contamination problems are exacerbated. Nevertheless, some of the most toxicologically important components of petroleum can be metabolized, even in the absence of oxygen.

Catabolism of the lignin substructure model compound dehydrodivanillin by a lignin-degrading Streptomyces

The lignin-degrading actinomycete Streptomyces viridosporus T7A readily degrades the lignin model compound dehydrodivanillin. Four mutants of this organism (produced by irradiation of spores with ultraviolet light) were shown to have lost the ability to catabolize dehydrodivanillin. These mutant strains retained an undiminished ability to degrade Douglas-fir lignin (14C-lignin → 14CO2) as compared to the wild-type strain. None of the strains accumulated detectable quantities of dehydrodivanillin when grown on lignocellulose. Thus it appears that the enzymes involved in dehydrodivanillin catabolism are not a part of the streptomycetes system for degrading polymeric lignin. It is concluded that dehydrodivanillin is probably not a relevant model compound for study of lignin polymer degradation by Streptomyces viridosporus. Since many stable mutants completely lacking DHDV-degrading ability were readily obtained, it is suggested that the relevant catabolic enzymes may be encoded on a plasmid.

Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces

Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources. These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer. The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization. APPLs were shown to be mostly free of nonlignin components. As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups. The yield of APPL from different lignocelluloses correlated with their biodegradability. Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces. In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin. A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate. APPL produced by S. viridosporus was examined for its properties and possible use as an antioxidant. The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL. Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions. As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered. Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation.