White-rot fungi : formulation for inoculation and biodegradation of pentachlorophenol in contaminated sites

Abstract

The use of technical grade pentachlorophenol (PCP) as a fungicide in the New Zealand timber industry has left an estimated 800 contaminated sites throughout New Zealand. Technical grade pentachlorophenol is reported to be carcinogen, and persists in the environment for a long time. Maximum acceptable levels of this contaminant in soil have been passed into law by the New Zealand Government. Therefore, remediation of these highly contaminated sites is necessary.

The cost of standard physical and chemical remediation technologies, and nature of the physical and chemical properties of PCP that promote its sorption to soil organic matter, has led researchers to investigate white-rot fungi as a bioremediation treatment for site remediation; in particular, the organisms Phanerochaete chrysosporium, Phanerochaete sordida and Trametes versicolor. White-rot fungi require a cosubstrate for growth before inoculation/bioaugmentation or "delivery" into soil for expression of the necessary extracellular enzymes responsible for PCP degradation (lignin peroxidase, manganese peroxidase and laccase) and earlier research in white-rot fungal bioremediation neglected this area. Recent work has started to address this gap. However, there are still gaps in the literature in particular the use of actual aged residues, which are soils that have been contaminated years or decades previously, for use in treatment studies. In addition, the bioremediation of soils with levels of pentachlorophenol contamination above 1000 mg kg⁻¹need to be studied.

The aim of the project was to improve formulation techniques for inoculation of New Zealand white-rot fungi into soil to remove pentachlorophenol from a 10 743 mg kg⁻¹ pentachlorophenol concentration aged residue, with 16 900 mg kg⁻¹ fuel oil as a co contaminant. The aged pentachlorophenol residue was diluted to lower contaminant concentrations with an uncontaminated clay loam soil or a sterile co-substrate amendment to enhance white-rot fungal survival.

Two New Zealand Trametes isolates, T. versicolor isolate HR131 and Trametes sp. isolate HR577 were used for the majority of the bioaugmentation work. The formulation chosen as a white-rot fungal carrier for bioaugmentation in the majority of the experiments was a Monterey pine-kibbled rye-calcium carbonate formulation with a C:N ratio of 50. The sterile co-substrate amendment used for the majority of the experiments was a Douglas fir (Pseudotsuga menziesii) com grits mix. Poplar (Populus deltoides) and Monterey pine (Pinus radiata) formulates with com grits were also used. PCP induced manganese peroxidase activity but not laccase in-vitro from the two New Zealand Trametes isolates. Upon bioaugmentation the aged PCP residue induced both laccase and manganese peroxidase activity from both New Zealand Trametes isolates.

The two New Zealand Trametes isolates inoculated into 150-1100 mg kg⁻¹ PCP concentration aged residue-clay loam mixes demonstrated PCP reductions of 40-80% over 3-7 weeks in preliminary experiments. The percentage of PCP reduction depended on culture age, PCP concentration, substrate amendment addition, isolate, and percentage dry weight of the fungal inoculum.

Two North American P. chrysosporium isolates (ATCC 24725 and ATCC 3541) and a North American P. sordida isolate (ATCC 90628) were compared against the New Zealand Trametes isolates for PCP biodegradation. P. sordida degraded 66% of the PCP in a 1092 mg kg⁻¹ PCP concentration aged residue compared with 40-55% for the New Zealand isolates and the two P. chrysosporium isolates after 7 weeks. The majority of PCP (> 60 %) removed was converted to undesirable pentachloroanisole by all three Phanerochaete isolates. The New Zealand Trametes isolates produced minimal amounts of pentachloroanisole.

The inoculation of Trametes sp. isolate HR577 into a 1920 mg kg⁻¹ PCP concentration aged residue resulted in PCP removal varying between 20-55% after 7 weeks for the different bioaugmentation conditions. The culture age of the fungus had a significant effect. The mixing of organobentonite clay in the aged PCP residue before bioaugmentation aided white-rot fungal colonisation but did not result in increased PCP removal over 7 weeks

The maximum reduction of PCP was 64% over 7 weeks in a 6414 mg kg⁻¹ PCP concentration aged residue-sterile substrate amendment mix, by the Trametes sp. isolate HR577. In contrast, T. versicolor isolate HR131 reduced PCP by 46% in the same aged PCP residue mix under the same conditions.

The New Zealand Trametes isolates reduced significant amounts of PCP in soils exceeding the originally proposed maximum PCP concentration thought possible (1800 mg kg⁻¹) for survival and biodegradation of PCP by New Zealand white-rot fungi in an aged residue, with seven treatment combinations. The PCP reductions in these treatments were highly correlated with laccase activity (R² = 0.97). The use of these techniques to remedy highly contaminated, aged PCP residues above 5000 mg kg⁻¹ PCP concentration may be possible as part of a treatment train or multistage process in the future.