White rot fungi are the major organisms that can decompose the entire wood structure of cellulose, hemi-cellulose and lignin. They secrete a range of enzymes including several cellulases, hemicellulases and ligninases to attack these major components. The ligninases are an interesting group of enzymes including peroxidases (Lignin-peroxidase and Mn -dependent peroxidases) and polyphenol oxidases known as laccases (Evans et al. 1994). Laccases are produced in substantial quantities by the white rot fungus Trametes versicolor (Evans, 1985). In addition to attacking lignin, laccases can use other phenolic and non-phenolic compounds as substrates including the toxic chlorophenols. 

Chlorophenols have abundant uses as wood preservatives and as products of the pharmaceutical industry. They are also produced as waste by products of pulp and paper manufacture. Effluent waters can contain toxic levels of chlorophenols that are difficult to remove by conventional processes. T. versicolor can tolerate high concentrations of chlorophenols, if 'adapted' gradually to increasing levels (Leontievsky et al. 2002). In liquid cultures, by increasing amounts of pentachlorophenol (PCP), and 2,4-dichlorophenol (DCP) gradually from 200 ppm to 2000 ppm, mixtures of chlorophenols up to 3500 ppm can be transformed by T. versicolor reducing the toxicity of the cultures. This process can be hastened by growing the fungus in an immobilized form that results in higher cell density, higher and extended enzyme activities, greater stability and frequently a reduction in treatment cost (Ruggiero et al. 1989).

Removal of chlorophenols from aqueous effluents

To evaluate the ability of T. versicolor to transform large quantities of PCP and DCP, free-cell cultures were grown in 2-litre bioreactors in which varying concentrations of PCP and DCP were added during a 42-day fermentation. Two additions of 200 ppm PCP were made at 4 and 8 days after inoculation, 1000 ppm PCP added at 19 days and a mixture of 2000 ppm PCP and 2000 ppm DCP added at 34 days. The rate of removal of the chlorophenols from the liquid culture was determined by analyzing for chlorophenols in samples from the culture fluid using HPLC (Leontievsky et al. 2002). At the end of the fermentation the amount of chlorophenols attached to components of the fungal biomass was also measured.

Secretion of laccase into the liquid medium by the fungus increased ten-fold after the first addition of PCP, but rapidly fell to basal levels once all PCP had been removed from the medium. Laccase then remained more or less constant for the remainder of the fermentation. Data from these free cell cultures were compared with that from parallel cultures in which T. versicolor was immobilized on nylon mesh that was mounted on metal baffles around the internal perimeter of the bioreactor. The rate of removal of the chlorophenols in the immobilized cultures was higher at all stages of addition of chlorophenols to the fermentations, than in the free-cell cultures (Table 1).

After 42 days of fermentation the fungal biomass was removed from the bioreactor and analysed for any adsorption of chlorophenols to the external polysaccharide layer around the cell walls. In free-cell cultures from a total addition of 2000 ppm DCP and 3400 ppm of PCP, up to 60% of DCP and 80% of PCP were adsorbed on to the biomass, whereas in immobilized cultures less than 5% of DCP and 28% of PCP were adsorbed. In the free-cell culture 7% of DCP and 22% of PCP remained in the medium, whereas in the immobilized culture only 8% of DCP and 1% of PCP remained in the medium after 42 days. Laccase activity accounted for transformation of only 20% of DCP and 12% of PCP in the free-cell cultures, but in the immobilized cultures laccase activity accounted for transformation of 85% of DCP and 70% of PCP. Clearly the immobilized culture was more efficient in degrading the chlorophenols in comparison with the free-cell culture. Adsorption of the chlorophenols to the polysaccharide layer around the hyphae was an intermediate stage in transformation of the chlorophenols by the fungus, as enzymes pass through the polysaccharide layer on secretion by the fungus and will act on the adsorbed chlorophenols.

The use of nylon mesh as an immobilization matrix for removal of the chlorophenols from liquid medium facilitated more efficient removal and could be scaled-up readily for application to larger volumes of effluent waters containing chlorophenols. Its simplicity, as a nylon mesh fixed to a metal frame around the perimeter of the bioreactor, provides an easy method for scale-up of bioreactor design. It will permit ready removal of spent biomass and replacement with new, young mycelia already immobilized on replacement mesh, without serious interruption of effluent treatment. Commercial development of such a system may have advantages for removal of high toxicity effluents that are a problem for current technologies.

EVANS, Christine S. Laccase activity in lignin degradation by Coriolus versicolor: in vivo and in vitro studies. FEMS Microbiology Letters, 1985, vol. 27, p. 339-343.

EVANS, Christine S.; DUTTON, Martin V.; GUILLEN, Francisco and VENESS, Robert G. Enzymes and small molecular mass agents involved with lignocellulose degradation. FEMS Microbiology Reviews, March 1994, vol. 13, no. 2-3, p. 235-240.

LEONTIEVSKY, Alexey A.; MYASOEDOVA, Nina M.; GOLOVLEVA, Ludmila A.; SEDARATY, Mohammed and EVANS Christine S. Adaptation of the white rot basidiomycete Panus tigrinus for transformation of high concentrations of chlorophenols. Applied Microbiology and Biotechnology, 2002, vol. 59, no. 4, p. 599-604.

RUGGIERO, P.; SARKAR, Jawed M. and BOLLAG, Jean-Marc. Detoxification of 2,4-dichlorophenol by a laccase immobilized on soil or clay. Soil Science, 1989, vol. 147, p. 361-370.