Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate
Introduction
Environmental pollution with hazardous wastes containing recalcitrant xenobiotic chemicals has become one of the major ecological problems. Unlike the naturally occurring organic compounds that are readily degraded upon introduction into the environment, some of these synthetic chemicals are extremely resistant to biodegradation by native microorganisms (Fernando and Aust, 1994). Ligninolytic fungi causing white rot of the wood have been shown to degrade and mineralize a large variety of recalcitrant compounds due to the nonspecificity of their enzyme machinery. Many of those compounds are major environmental pollutants such as munitions waste, pesticides, organochlorines, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), synthetic dyes, wood preservatives and synthetic polymers (Pointing, 2001).
White rot fungi (such as Phanerochaete chrysosporium, Trametes versicolor, etc.) typically secrete one or more of the three principal ligninolytic enzymes (Hatakka, 1994), i.e. lignin peroxidase (LiP, E.C. 1.11.1.14), Mn-dependent peroxidase (MnP, E.C. 1.11.1.13) and phenol oxidase (laccase) (LAC, E.C. 1.10.3.2) (Thurston, 1994, Orth and Tien, 1995). A number of other enzymes are produced in parallel including other peroxidases, enzymes producing H2O2 required by peroxidases (e.g. glyoxal oxidase and superoxide dismutase), and enzymes linked to lignocellulose degradation pathways (e.g. glucose oxidase and aryl alcohol oxidase), whose role in lignin degradation is poorly understood (Pointing, 2001).
The physiology of LiP, MnP and LAC has been studied extensively (Hatakka, 1994, Thurston, 1994). These enzymes have been shown to take part in vitro transformation of nonpolymeric, recalcitrant pollutants such as nitrotoluenes (Van Acken et al., 1999), PAHs (Hammel et al., 1991, Johannes et al., 1996), organic and synthetic dyes (Ollikka et al., 1993, Heinfling et al., 1998), and pentachlorophenol (Lin et al., 1990). The importance of high extracellular levels of these enzymes to enable the efficient degradation of recalcitrant compounds under in vivo conditions relates to the sorption and complexing of enzymes in soil and the probable loss of much of their activity once externalized (Stotzky and Burns, 1982). The reason for our poor understanding is the high complexity of the biodegradation mechanisms involved, where in addition to the above ligninolytic enzymes, other biochemical systems and interactions may influence the rate of the bioremediation process, namely cytochrome P450 monooxygenase system (Bezalel et al., 1997), hydroxyl radicals and the level of H2O2 produced by the fungal organism (Kotterman et al., 1996, Tanaka, 1999). The limited bioavailability of pollutants results from their sorption to soil particles or a covalent coupling with soil organic matter (Rüttmann-Johnson and Lamar, 1997, Kästner et al., 1999) as well as from the sorption to the fungal mycelium due to their hydrophobic properties (Providenti et al.,1993, Wang and Yu, 1998, Gramss et al., 1999).
The research reported here was aimed at evaluating the importance of elevated levels of extracellular, ligninolytic enzyme activities for rapid and efficient degradation of selected recalcitrant compounds under a variety of conditions. Different strains of ligninolytic fungi and several major organopollutants, such as synthetic dyes, PAHs and PCBs were used and their degradation studied in both water and soil.
Section snippets
Microorganisms
Phanerochaete chrysosporium strain ME 446 (ATCC 34541), Trametes versicolor strain CCBAS 614, Coriolopsis polyzona strain CCBAS 740, Pleurotus ostreatus strain 3004 and Irpex lacteus strain 617/93 were obtained from the Culture Collection of Basidiomycetes of the Academy of Sciences, Prague (CCBAS). Fungal cultures were maintained on malt extract/glucose medium agar slants (Novotný et al., 2000).
Growth in liquid media and in soil
In all of the experiments the cultures were run in triplicate. The data shown represent the mean
Results
The biodegradation of recalcitrant pollutants by ligninolytic fungal enzymes in vitro has been documented (Pointing, 2001). However, under in vivo conditions, other biochemical systems, processes and interactions can either contribute to degradation of the pollutant (e.g. fungal cytochrome P450 monooxygenase system, hydroxyl radical formation by the fungus) or limit the degradation rate (e.g. low bioavailability of the pollutant due to sorption to soil particles, hydrophobicity of the pollutant
Discussion
Although a number of studies exist that deal with the involvement of peroxidases and laccases in biodegradation of recalcitrant compounds, not many of them focus on the relationship between the extracellular enzyme levels and degradation rates under in vivo conditions. This relationship can be obscured by the complexity of the biodegradation process, where various interactions and limitations may determine which step will become rate-limiting.
Our study showed that in a majority of experiments
Conclusions
In spite of the existence of other factors known to limit the degradation of xenobiotics by white rot fungi in liquid media and soil (e.g. H2O2 production, limited bioavailability, hydrophobic properties of compounds), evidence was gathered that significant levels of extracellular ligninolytic enzymes was an important factor in ensuring high biodegradation rates. This was especially so in liquid media. MnP and to a lesser extent, LAC were shown to be the most important enzymes in our study.
Acknowledgements
We thank to Jitex Pisek a.s. for kindly providing us the samples of textile coloring bath liquids. The work was supported by the projects GA ČR 526/00/1303, KONTAKT 2003-14 and 011-2004-05, and Institutional Research Concept AV0Z5020903.
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