Role of Fungal-bacterial Interactions for Improved Hexachlorocyclohexane (Hch) Biodegradation in Soil

Abstract

Organochlorine pesticides (OCPs), such as hexachlorocyclohexane (HCH) were extensively used across the globe for agricultural and public health purposes due to their effectiveness, relatively low cost, and ease of use. Their use was however restricted or banned in most countries across the world owing to their toxicity to non-target organisms, persistence in the environment, and ability to bio-accumulate in the food chain. Despite the restricted use or complete ban, HCH continues to pose serious environmental and health risks. However, the use of naturally existing microorganisms for the bioremediation of hazardous compounds and their detoxification has been proposed as a viable strategy to maintain environmental health. Therefore, the present study was aimed to (i) isolate fungal-bacterial couple from HCHcontaminated soil, (ii) test whether fungal mycelia can act as effective transport networks for HCH isomers, and (iii) test the effect of mycelial-mediated nutrient transfer to HCH degrading bacteria on the biodegradation of HCH isomers. Culture-dependent approaches were applied to isolate and characterize two HCH-degrading bacterial species and a fungal species from HCH-contaminated soil collected from a former obsolete pesticide store in Kitengela, Kenya (GPS: 01.49 S, 37.048E). Using a combination of the 16S gene, ITS gene, and whole genome sequencing the two bacteria were identified as, Sphingobium sp. strain S6 and S8 respectively, while the fungus was identified as F. equiseti strain K3. Both bacterial isolates were shown to effectively degrade all four HCH isomers. The degradation rates of γ-HCH were higher than those of α- and δ-HCH (p < 0.041) while no significant difference (p > 0.12) in the degradation rates of α- and δ-HCH was observed in both Sphingobium strains, while β-HCH had the lowest removal rate. Therefore, the removal rates of HCH isomers in both bacteria were in the order γ > α ≈ δ > β. Subsequently, lin genes responsible for HCH degradation, identical to those found in other HCH degrading sphingomonads were identified by gene sequencing and from their 4.1 Mb draft genomes consisting of 4,015 and 4,039 protein-coding sequences (CDS) for Sphingobium sp. strain S6 and Sphingobium sp. strain S8 respectively. The fungal isolate, on the other hand, poorly degraded HCH isomers in the order β > α > δ > γ. ANOVA revealed statistically significant differences in the degradation of the HCH isomers (p < 0.0039). To test the effectiveness of mycelia as transport vectors for HCH isomers, a laboratory-based microcosm system designed to mimic air-water interfaces in soil was used while the F. equiseti species was used as a model organism. The fungus transported 0.09 – 0.6 μg of different HCH isomers in the order γ > α > δ ≈ β over a 3cm distance and the isomer-specific translocation was likely influenced by their octanol-air partition coefficients (log KOA). To test the effect of

mycelial-mediated nutrient transfer to HCH-degrading bacteria on HCH biotransformation in a nutrient-deprived environment, the poorly HCH-degrading fungus (Fusarium equiseti strain K3) and the HCH-degrading bacterium (Sphingobium sp. strain S8) were used in a spatially structured laboratory-based model ecosystem. Subsequently, a combination of 13C-labelled fungal biomass and protein-based stable isotope probing (protein-SIP) was used to trace the incorporation of 13C fungal metabolites into bacterial proteins while simultaneously determining the biotransformation of the HCH isomers. Relative isotope abundance (RIA, 7.1 – 14.2%), labeling ratio (LR, 0.13 – 0.35), and the shape of isotopic mass distribution profiles of bacterial peptides indicated the transfer of 13C-labeled fungal metabolites into bacterial proteins. The distinct 13C incorporation into the haloalkane dehalogenase (linB) and 2,5- dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (LinC), key enzymes in metabolic HCH degradation, underpinned the role of mycelial nutrient transfer in co-metabolic bacterial HCH degradation in heterogeneous habitats. Bacterial nutrient uptake from mycelia increased HCH removal by twofold as compared to bacterial monocultures. The findings from this study forms an important basis for the development of efficient bioremediation strategies in which either natural or artificial HCH degrading fungal-bacterial couple can be introduced into contaminated sites by bio-augmentation to improve biotransformation of micropollutants such as HCH.