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.
Publisher
University of Nairobi
Rights
Attribution-NonCommercial-NoDerivs 3.0 United StatesUsage Rights
http://creativecommons.org/licenses/by-nc-nd/3.0/us/Collections
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