Free, Andrew

Horsfall, Louise

McDonald, Michael Anthony

2024-08-07T13:39:38Z

2024-08-07T13:39:38Z

2024-08-07

The generation of extremely acidic and metal-contaminated effluent from mining can cause large scale environmental destruction, degradation of surrounding freshwater and soil and poses risks to both ecosystem and human health. Due to the chemistry of this synthetic ‘acid mine drainage’ (AMD), the diversity and function of microbial consortia native to such sites has been largely neglected. Firstly, in order determine the chemical composition of the acidic sediment, elemental analysis was performed, and compared to two non-extreme freshwater sites not impacted by mining- derived pollution. The acid mine drainage sediment (collected from a previous mined site in Central Scotland) demonstrated an extremely low pH, carbon levels reflective of oligotrophic conditions, elevated concentrations of heavy metals, and high sulphate levels. Taken together, these chemical characteristics demonstrated the abiotic conditions and nutritional scarcity associated with microbial proliferation in heavily contaminated systems. Subsequent metabolic profiling of sediment communities involving carbon-source degradation again demonstrated the acidophilic consortia’s more restricted diversity and reduced capacity to catabolise organic compounds compared to control environments and revealed significantly different carbon source utilisation patterns compared to copiotrophic and an additional oligotrophic microbial assemblage. Using a novel cultivation strategy, a member of the genus Thiomonas was successfully isolated, demonstrating the premise that extremophiles can be successfully recovered from their native conditions using non-conventional culture-based approaches. Further amplicon-based molecular analyses allowed elucidation of key taxonomic groups of microorganisms, and quantitation of their functional marker gene abundance. qPCR demonstrated the presence of methane cycling organisms via detection of the mcrA and pmoA genes, responsible for functions previously poorly, or not reported for, in members of acid mine drainage assemblages. Fungal presence was confirmed via quantification of the intergenic ITS2 region, expanding current knowledge on the biogeographical distribution of eukaryotes in low pH, synthetically derived effluents. Thereafter, the routes to community characterisation focused on metagenomic and metataxonomic approaches; allowing the elucidation of members of the acidophilic population and functional attributes which promote microbial survival in hazardous conditions. Long read DNA sequencing using Oxford Nanopore Technology successful allowed the reconstruction of metagenomic assembled genomes (MAGs); assemblies pertaining to organisms from the Pseudomonadota and Actinomycetota phyla were selected for further genomic analysis. Metabolic functions previously unreported for members of acid mine drainage communities were evident within the reassembled bacterial chromosomes. The highest quality assembly pertaining to a genome recovered from Metallibacterium scheffleri demonstrated multiple polyextremophilic adaptations, including resistance mechanisms to counteract acid influx into the cell. The Metallibacterium MAG sequence also demonstrated metabolic strategies employed by this taxon to survive in otherwise deleterious concentrations of pollutants, chiefly exposure to transition metals (via the presence of zitB, copA/B, cutA, merA and ATM-1 genes) and the metalloid arsenic (via arsB/C). Importantly, the Metallibacterium assembled genome also demonstrated more diverse carbon utilisation routes than previously reported, with three complete glycolytic pathways noted. Sulphur cycling capabilities previously contested for Metallibacterium were also evident within the content of the assembled chromosome. These results indicated Metallibacterium’s importance in underpinning biogeochemical function in acidic waste streams and the array of metabolic strategies that could exploited if using Metallibacterium as a future candidate for bioremediation. The Metallibacterium MAG also demonstrated an array of sulphur cycling mechanisms unreported in the previous literature. Additional MAGs relating to Acidiphilum and Mycobacterium are also presented; these MAGs also indicate novel functions, including extreme acid tolerance, autotrophy, and hydrocarbon degradation. Lastly, bioreactor-based experiments were employed to explore whether a specific community function (methanogenesis) could be established from sediment communities. A strong derivation of methanogenic function was achieved using copiotrophic communities. Methane output when using the acidophilic assemblage as a novel inocula was sporadic, however methane was detected in a single bioreactor. In this case, the archaeal community from Benhar Bing within the starting inocula appeared to withstand the bioreactor conditions and produced biogas (with over 65% methane detected within the headspace gas). Based on phylogenetic analysis of both the starting sample and the end point bioreactor samples following dismantling of the reactors (by pre-amplification of the archaeal 16S rRNA gene and subsequent analysis of the V4 region), it is likely that methanogens native to the extremely acidic conditions belong to the Thermoplasmatales which like their sister lineage, the Methanoplasmatales, may utilise a methylotrophic methanogenic pathway to conserve energy. Overall, acidophilic assemblages underpin ecological function in ecosystems degraded by anthropogenic mining activities and can promote pollutant mobility. Despite this, metagenomic results demonstrated that microbial species present can also aid in ecosystem recovery and can promote metal detoxication and the closure of metal redox cycles. Future work could continue to focus on methane cycling occurring in non-standard systems which have been overlooked regarding potential greenhouse gas emissions, which have likely been underestimated. Acidophilic assemblages hold an untapped metabolic repertoire that could be exploited in both the remediation of contaminated systems and potential low pH waste to resource biotechnologies.

https://hdl.handle.net/1842/42067

http://dx.doi.org/10.7488/era/4789

en

The University of Edinburgh

Diversity-function relationships in natural, applied, and engineered microbial ecosystems Free, A., McDonald, M. A. & Pagaling, E., 9 Aug 2018, (E-pub ahead of print) Advances in Applied Microbiology. Vol. 105. p. 131-189 59 p. (Advances in applied microbiology)

polyextremophilic microbial consortia

acid mine drainage

effluent from mining

‘acid mine drainage’ (AMD)

acid mine drainage sedimen

metagenomic assembled genomes (MAGs)

Metallibacterium

Metagenomic, metabolic and functional characterisation of polyextremophilic microbial consortia endogenous to acid mine drainage

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy