David J. Dickinson

Methanotrophs, Methylosinus trichosporium OB3b, sMMO, and Their Application to Bioremediation

One of the most problematic groups of the USEPA and EU priority pollutants are the halogenated organic compounds. These substances have a wide range of industrial applications, such as solvents and cleaners. Inadequate disposal techniques and accidental spillages have led to their detection in soil, groundwater, and river sediments. Persistence of these compounds in the environment has resulted from low levels of biodegradation due to chemical structural features that preclude or retard biological attack. Research has indicated the idea that treatment systems based on methanotrophic co-metabolic transformation may be a cost-effective and efficient alternative to physical methods because of the potential for high transformation rates, the possibility of complete compound degradation without the formation of toxic metabolites, applicability to a broad spectrum of compounds, and the use of a widely available and inexpensive growth substrate. A substantial amount of work concerning methanotrophic cometabolic transformations has been carried out using the soluble form of methane monooxygenase (sMMO) from the obligate methanotroph Methylosinus trichosporium OB3b. This NADH-dependent monooxygenase is derepressed when cells are grown under copper stress. sMMO has a wider specificity than the particulate form. sMMO has been shown to degrade trichloroethylene (TCE) at a rate of at least one order of magnitude faster than obtained with other mixed and pure cultures, suggesting it has a wider application to bioremediation. Furthermore, sMMO catalyzes an unusually wide range of oxidation reactions, including the hydroxylation of alkanes, epoxidation of alkenes, ethers, halogenated methanes, cyclic and aromatic compounds including compounds, that are resistant to degradation in the environment. However, the practical application of methantrophs and Methylosinus trichosporium OB3b to the treatment of chlorinated organics has met with mixed success. Although oxidation rates are rapid, compound oxidation with M. trichosporium OB3b is difficult. This fastidious organism grows relatively slowly, which limits the speed with which sMMO expressing biomass can be generated. Furthermore, product toxicity toward the cell, affecting the stability of the enzyme when transforming certain compounds has been observed, for example, by the products of 1,2,3 trichlorobenzene hydroxylation (2,3,4- and 3,4,5-trichlorophenol) and of TCE degradation (chloral hydrate). Because of this toxicity and the inability of sMMO to further oxidize its own hydroxylation products, the ability of methane monoxygenase to carry out the monooxygenation of a wide variety of substituted aromatics and polyaromatics cannot be fully exploited in M. trichosporium OB3b. Many of these problems could be overcome by the use of either a mixed downstream heterotrophic population of organisms that could accommodate the products of hydroxylation or to express sMMO in an organism that could metabolize the products of hydroxylation. The latter of these two approaches would have several advantages. The main benefit would be the removal of the need for methane, which is required to induce sMMO in M. trichosporium OB3b, and supply carbon and energy to the cells that continuously oxidise the target compound, but also acts as a competitive inhibitor of sMMO. Instead, the recombinant could utilize the products of sMMO-mediated hydroxylation as a carbon source.

The effect of diffusible metabolites of Trichoderma harzianum on in vitro interactions between basidiomycete isolates at two different temperature regimes

As part of a study aimed at developing a biological control of decay in freshly felled pine the action of Trichoderma harzianum on the interactions between target decay basidiomycetes was investigated. An in vitro bilayer system allowed the action of the moulds diffusible metabolites, as distinct from the other modes of antagonism, to be studied. Seven basidiomycete isolates, representing commonly found decay fungi in pine, were challenged against each other in the presence of Trichoderma metabolites and also on control (uninoculated) bilayers. Challenges were carried out at two temperatures, 10° and 22 °C. Both temperature and the presence of Trichoderma influenced the outcome of the interactions seen on the bilayers. Temperature had a marked effect on some of the challenges for instance switching a deadlock interaction to overgrowth of one individual by the other. Similarly, the same challenge could have contrasting outcomes depending on whether the interaction occurred on Trichoderma or control bilayers. The relevance of the results to biological control of wood decay is discussed.

The development of ideas in biological control applied to forest products

After giving an overview of biological control agents and their application to forest products, the review considers the main biocontrol mechanisms that are thought to operate in wood. Competition for nutrients, antibiosis, mycoparasitism and production of extracellular enzymes have all been implicated in the biological control of wood decay fungi, although the relative importance of these potential mechanisms in vivo remains obscure. Attempts at biologically controlling basidiomycetes in forest products on a commercial scale have met with limited success and the environmental factors influencing efficacy are reviewed. It is concluded that the chances of achieving successful biocontrol of decay rely as much on appreciating the limitations of biological treatments as upon a comprehensive understanding of the mechanisms involved.

Investigation of the extracellular mucilaginous materials produced by some wood decay fungi

The morphology of the extracellular mucilaginous material (ECM) produced by Coniophora puteana and Coriolus versicolor during colonization of Scots pine and beech was studied using SEM. Wood specimens were examined in the frozen hydrated (FH) condition using low-temperature SEM, and in the freeze-dried (FD) and critical point dried (CPD) state, using conventional SEM. All techniques produced artifacts but the ECM was best preserved when examined in the FH state. Very little difference was observed between FH and FD preparations, but critical point drying damaged the ECM extensively. Copious amounts of ECM were produced by both fungi. It was found to line much of the lumen surface, establishing contact between the mycetium and the wood substratum. Most aerial hyphae were coated with ECM, appearing glued together in a bundle-like fashion. The ECM thickness varied within the same wood cell and from one cell to another. A peculiar granular pattern, in which the ECM was definitely involved, was seen on occasion to encircle the infecting hyphae where they contacted the wood surface. Other morphological patterns of ECM distribution were also observed. Calcium oxalate crystals of varying shapes and sizes were often seen associated with the mycelia and mucilage of the two fungi in beech but not in Scots pine. The probable roles played by the ECM in wood decay mechanisms are discussed.

The production of extracellular mucilaginous material (ECMM) in two wood-rotting basidiomycetes is affected by growth conditions

The ability of two wood-decay basidiomycetes to produce extracellular mucilaginous material (ECMM) and its relationship with total biomass production has been investigated. Growth and ECMM production by the white-rot fungus Coriolus versicolor and the brown-rot fungus Gloeophyllum trabeum were assessed in liquid culture under different nutritional and environmental conditions. Nutritional, pH and temperature factors all influenced significantly the proportion of ECMM in the total biomass produced. When total biomass production was reduced due to unfavorable growth conditions (stress), the proportion of ECMM in the biomass was elevated. The results are discussed with regard to the possible role(s) of ECMM in the responses of these fungi to stress.

Analysis of the hyphal load during early stages of wood decay by basidiomycetes in the presence of the wood preservative fungicides CuSO4 and cyproconazole

Abstract This study considers variations in hyphal load in decaying wood in the presence of the fungicides CuSO4 and cyproconazole. Variations in the chitin content of hyphae following exposure to both fungicides have been detected. Increasing concentrations of CuSO4 and cyproconazole in wood caused an increase in the amount of N-acetyl glucosamine in the mycelia of Coriolus versicolor and Gloeophyllum trabeum, which may be associated with increased deposition of chitin. This may in turn be an expression of the formation of a thicker cell wall at increased fungicide concentrations. Low concentrations of both fungicides also caused an increase in the amount of mycelium produced by G. trabeum. However, the same concentrations were effective at preventing mass loss, indicating that the fungal mycelium was less effective at decaying wood, despite being present in relatively large amounts. In the case of C. versicolor, this effect was not observed, as increasing concentrations of both fungicides caused an initial decrease in the amount of mycelium, followed by a recovery phase at intermediate chemical concentrations. Again, mass loss was greatly inhibited by the presence of low concentrations of both fungicides.