Plant-fungal mutualism as a strategy for the bioremediation of hydrocarbon polluted soils

Inasmuch as coal remains the linchpin for the generation of electricity and liquid petroleum products in South Africa, hydrocarbon waste and coal discard will continue to pose a threat to the environment. Therefore, the onus is on the associated industries to develop and implement efficient and sustainable strategies to mitigate the negative impacts of energy generating activities on the environment. Most conventional efforts in this regard, although successful for soil repair and the initiation of vegetation, have been deemed unsustainable. In an effort to find a sustainable remediation strategy a novel technology termed “FungCoal” was conceptualized and patented as a strategy for the rehabilitation of open cast coal mines, carbonaceous-rich spoils and coal wastes. This biotechnology, which exploits plant-fungal mutualism to achieve effective biodegradation of coal on discard dumps and the breakdown of the carbonaceous component in spoils, promotes revegetation to facilitate rehabilitation of mining-disturbed land. However, one limiting factor of the FungCoal bioprocess is that it requires oxidized weathered coal, a highly complex and variable resource for use as a co-substrate, for growth and proliferation of the coal degrading microorganisms. To fully exploit the potential of plant-fungal mutualism and its interaction for use in the remediation of coal contaminated soils, this study investigated the proposed relationship between plant roots, root exudate and the coal degrading fungus “Aspergillus sp.” (previously Neosartorya fischeri) strain 84 in more detail, in an effort to gain further insight into the mechanisms underpinning plant-fungal mutualism as a strategy for re-vegetation of coal discard dumps and the rehabilitation of hydrocarbon-contaminated soil using the FungCoal approach. A pot-on-beaker (PoB) method was developed for the easy cultivation and collection of extracellular polymeric substance (EPS)-containing exudates from Zea mays L. (maize) and Abelmuschus esculentus (okra). Characterisation of the EPS material from these exudates was carried out using a combination of physicochemical and biochemical methods. The results from analysis of phenolics and indoles showed that exudates contain some form of indoles and phenolic compounds, although in little proportions, which may fulfil a signalling function, responsible for attracting soil microorganisms into the rhizosphere. Spectroscopic analysis of the exudates using FT-IR revealed vibrations corresponding to functional groups of alkanes, alkenes, alkynes, and carboxylic acids. These compounds likely provide an easily accessible source of carbon to soil microorganisms and are also a better alternative to the poly-aromatics which are an inherent component locked-up in the supposed recalcitrant coal material. The results from biochemical analyses also revealed the presence of carbohydrate, proteins, lipids, and low amounts of α-amino-nitrogen in the EPS of maize and okra. These components of EPS are all essential for the stimulation of enzymatic activities in soil microorganisms and, which may in turn aid biodegradation. The action of the root EPS from maize was further tested on three coal-degrading fungal isolates identified as Aspergillus strain ECCN 84, Aspergillus strain ECCN 225 and Penicillium strain ECCN 243 for manganese peroxidase (MnP) and laccase (LAC) activities. The results revealed that the Aspergillus species, strains ECCN 84 and ECCN 225, showed with or without EPS, observable black halos surrounding each of the colonies after 7d incubation indicative of positive MnP activity, while no activity was observed for the Penicillium sp. strain ECCN 243. Analysis for LAC revealed little or no activity in any of the coal degrading fungi following addition of pulverized coal to the growth medium. Interestingly, the addition of EPS-containing exudate to the coal-containing medium resulted in increased LAC activity for all fungal isolates. This finding affirmed the positive contribution of EPS to extracellular LAC activity, purported as an important enzyme in the coal biodegradation process. Finally, the impact of plant-derived exudate on the colonisation and biodegradation of coal was investigated in situ using rhizoboxes, to simulate a coal environment, and was carried out for 16 weeks. Microscopic examination of coal samples after termination of the experiment showed fungal proliferation and attachment to coal particles. All of the rhizoboxes that contained plants had higher medium pH and EC, and the concentration of phenolics, indoles and humic acids was greater than that of control treatments. These observations indicated better rhizosphere colonisation, substrate biodegradation and humification. Therefore, root exudate appears to play a significant role in coordination of soil microorganisms within the rhizosphere and likely serves both as a scaffold for rhizospheric interactions by providing microorganisms with accessible carbon and as a likely ‘trigger’ for induction of coal-degrading enzymes such as fungal LAC for mobilisation of recalcitrant carbon. This study has shown that EPS exuded from roots of Zea mays together with coal degrading fungus Aspergillus strain ECCN 84 can alkalinise the coal substrate and facilitate introduction of oxygen, possibly as a result of increased laccase activity, and increase availability of nutrients (as indicated by higher EC) in a coal-polluted rhizosphere, to provide plants and their associated mycorrhizae and presumably other beneficial microorganisms a more mesic environment for sustained phytoremediation with enhanced rehabilitation potential. In conclusion, this study confirms the positive role of root exudate in mediating a mutualistic rehabilitation strategy involving plants and fungi such as the FungCoal bioprocess.

Coal-Degrading Bacteria Display Characteristics Typical of Plant Growth Promoting Rhizobacteria

Coal mining produces large quantities of discard that is stockpiled in large dumps. This stockpiled material, termed coal discard, poses an environmental threat emphasising the need for appropriate bioremediation. Here, metagenomic analysis of the 16S rRNA from ten coal-degrading strains previously isolated from coal slurry from discard dumps and from the rhizosphere of diesel-contaminated sites was used to establish genetic relatedness to known plant growth-promoting (PGP) bacteria in the NCBI database. Measurement of indole and ammonium production and solubilisation of P and K were used to screen bacteria for PGP characteristics. BLAST analysis revealed ≥ 99% homology of six isolates with reference PGP strains of Bacillus, Escherichia, Citrobacter, Serratia, Exiguobacterium and Microbacterium, while two strains showed 94% and 91% homology with Proteus. The most competent PGP strains were Proteus strain ECCN 20b, Proteus strain ECCN 23b and Serratia strain ECCN 24b isolated from diesel-contaminated soil. In response to L-trp supplementation, the concentration of indolic compounds (measured as indole-3-acetic acid) increased. Production of ammonium and solubilisation of insoluble P by these strains was also apparent. Only Serratia strain ECCN 24b was capable of solubilising insoluble K. Production of indoles increased following exposure to increasing aliquots of coal discard, suggesting no negative effect of this material on indole production by these coal-degrading bacterial isolates and that these bacteria may indeed possess PGP characteristics.

Geochemical Study of Weathered Coal, a Co-Substrate for Bioremediation of South African Coal Discard Dumps

Coal mining creates large volumes of waste in the form of discard coal that is stockpiled. In South Africa, rehabilitation of coal discard dumps remains a challenge due to reliance on topsoil for establishment of vegetation. Exploitation of fungal bio liquefaction/degradation of coal resulted in the emergence of Fungcoal as a bioprocess for the rehabilitation of coal discard dumps and opencast spoils. In this process, a suite of fungi is used to bio liquefy/degrade recalcitrant waste coal to form a soil-like material which promotes reinvigoration of the microbial component, grass growth, and re-vegetation. Here, the role of outcrop weathered coal as a mineral/carbon source to ensure biologically induced humic acid-like substance enrichment of discard and spoil to increase efficacy of fungi-plant mutualism and stimulate revegetation without the need for topsoil was investigated. Mineralogical, elemental, and pyrolysis gas chromatography-mass spectroscopic analyses show that outcrop weathered coal has decreased volatile material and increased humics, ash, and mineral bound water in comparison to bituminous coal. These changes occur coincidently with decreased C, N, and H contents, and a substantial increase in O concentration. No apparent stoichiometric relationship between sulphur and iron oxide content of weathered coal could be discerned suggesting little residual pyrite in this material and a dominance of oxy-hydroxides of Fe. Organic analysis showed weathered coal to be enriched in C-16 and C-18 fatty acids and the presence of the indicator, 17α(H),21β(H)-homohopane but not the β,β-stereoisomer, was interpreted to indicate that bacteria may only have been active prior to transformation of hard coal into weathered coal.