Synergistic rhizosphere degradation of γ-hexachlorocyclohexane (lindane) through the combinatorial plant-fungal action

Abstract

Fungi are usually involved in degradation/deterioration of many anthropogenic wastes due to their verse enzyme secretions and adaptive capabilities. In this study, five dominant fungal strains were isolated from an aged lindane polluted site, they were all mixed (100 mg each) together with pent mushroom compost (SMC) and applied to lindane polluted soil (5 kg) at 10, 20, 30, 40% and control 0% (soil with no treatment), these were used to grow M. maximus Jacq for 3 months. To establish lindane degradation, deductions such as Degradation rate (K1), Half-life (t1/2) and Degradation efficiency (DE) were made based on the analyzed lindane concentrations before and after the experiment. We also tested the presence and expressions of phosphoesterases (mpd and opd-A) and catechol 1,2-dioxygenases (efk2 and efk4) genes in the strains. The stains were identified as Aspergillus niger (KY693970); Talaromyces atroroseus (KY488464), Talaromyces purpurogenus (KY488468), Yarrowia lipolytica (KY488469) and Aspergillus flavus (KY693973) through morphological and molecular methods. Combined rhizospheric action of M. maximus and fungi speed up lindane degradation rate, initially detected lindane concentration of 45 mg/kg was reduced to 11.26, 9.34 and 11.23 mg/kg in 20, 30 and 40% treatments respectively making 79.76, 85.93 and 88.67% degradation efficiencies. K1 of 1.29 was recorded in control while higher K1 of 1.60, 1.96 and 2.18 /day were recorded in 20, 30 and 40% treatments respectively. The best t1/2 of 0.32 and 0.35 /day were recorded in 40 and 30% compared to control (0.54 /day). All the strains were also affirmed to possess the tested genes; opd was overexpressed in all the strains except KY693973 while mpd was overexpressed in KY693970, KY488464 but moderately expressed in KY488468, KY488469 and KY693973. However, efk genes were under-expressed in most of the strains except KY488469 and KY693973 which showed moderate expression of efk4. This work suggests that the synergistic association of the identified rhizospheric fungi and M. maximus roots could be used to remove lindane in soil at a limited time period and this combination could be used at large scale.

Fig 1. Phylogenetic tree of lindane degrading fungal strains, based on a distance matrix analysis of the ITS sequences.

(1a) Aspergillus niger asemoC KY693970; (1b) Talaromyces atroroseus asemoG KY488464 and Talaromyces purpurogenus asemoN KY488468; (1c) Yarrowia lipolytica asemoO KY488469; (1d) Aspergillus flavus asemoP KY693973.

Fig 2. PCR Detection of genes encoding phosphoesterase and catechol 1,2-dioxygenase in in lindane utilizing fungal strains.

mpd and opd-A = phosphoesterases; afk2 and afk4 = catechol 1,2-dioxygenase; asemoC = A. niger KY693970; asemoG = T. atroroseus KY488464; asemoN = T. purpurogenus KY488468; asemoO = Y. lipolytica KY488469, and asemoP = A. flavus KY693973.

Fig 3. RT-PCR expression of genes encoding phosphoesterase and catechol 1,2-dioxygenase in lindane utilizing fungi.

mpd and opd-A = phosphoesterases; afk2 and afk4 = catechol 1,2-dioxygenase; asemoC = A. niger KY693970; asemoG = T. atroroseus KY488464; asemoN = T. purpurogenus KY488468; asemoO = Y. lipolytica KY488469, and asemoP = A. flavus KY693973.

Fig 4. Proposed degradation of γ-Hexachlorocyclohexane (lindane) through synergistic rhizosphere degradation by the rhizospheric fungal strains and M. maximus.

Compounds: 1. (γ-Hexachlorocyclohexane) 2. Pentachlorocyclohexene (γ-PCCH) 3. 1,3,4,6-Tetrachloro-1,4-cyclohexadiene (1,4-TCDN) 4. 2,4,5-Trichloro- 2,5-cyclohexadiene-1-ol (2,4,5-DNOL) 5. 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) or 2,5-Dichlorohydroquinone (2,5-DCHQ), 6. Chlorohydroquinone (CHQ) 7. 2,5-Dichlorophenol (2,5- DCP) 8. 1,2,4-Trichlorobenzene (1,2,4-TCB) 9. 2,5-Dichlorobenzene (2,5-DCB) 10. Chlorobenzene 11. benzene 12. β-Ketoadipate (3-oxoadipate) 13. 3-Oxoadipyl-CoA 14. γ-Hydroxymuconic semialdehyde 15. Glycolic acid 16. Dichloroacethaldehide 17. Dichloroethanol.