Acid Mine Drainage (AMD) sites are found throughout the world, formed through the runoff of acidic water and heavy metals from mining activity. Microorganisms that are able to inhabit these environments are known as acidophiles, growing at an optimal pH of < 3. The Parys Mountain located in Anglesey (UK) is an AMD site subject to studies over the years. These studies have detected the presence of many acidophiles belonging to Archaea such as Cuniculiplasma, Ferroplasma, ‘’Candidatus Scheffleriplasma hospitalis’’ (also known as ‘’B_DKE’’) and an uncultured archaeon known as ‘’E-plasma’’ all from the same phylum Euryarchaeota (or ‘’Ca. Thermoplasmatota’’). One issue in the research of acidophiles is the ability to cultivate them in vitro, as acidophiles (and archaea in general) are difficult to cultivate. Additionally, unique nano sized archaea known as ‘’Ca. Microcaldota’’ (formally known as ‘’Ca. Micrarchaeota’’) has very few successful cultivations in vitro.
The primary goal of this study was to cultivate the microbial diversity of the PM5 site (Parys Mountain). The second goal of this study was to investigate cultivation techniques in an attempt to successfully enrich elusive microorganisms such as ‘’Ca. Micrarchaeota’’. To achieve these goals, aerobic and anaerobic enrichment cultures were set-up and monitored over the course of seven months. Variations included enrichment cultures containing two types of media, as well as acidic water collected from the sample site. Substates included sulfur and yeast extract, beef extract and tryptone added at 0.1% (w/v) or 0.02% (w/v). Finally, some enrichment cultures had the addition of Cuniculiplasma biomass at 10% (w/v). The microbial composition of cultures was monitored by the use of 16S rRNA V4 primers (as well as ITS primers on enrichment cultures that showed fungal growth) at two timepoints. Timepoint 3 and timepoint 7 (2 and 6 months after initial enrichment cultures set-up).
Sequencing of enrichment cultures revealed archaea affiliated (99.22% identity) with C. divulgatum in high abundance in both timepoints 3 and 7, as well as sequences affiliated with Ferroplasma acidiphilum (identity 100%) in near 100% abundance in a number of enrichment cultures. Sequences that could only be affiliated at the family level with Thermoplasmataceae were also detected in a number of enrichment cultures at timepoint 3, while timepoint 7 cultures were able to show an affiliation to ‘’Ca. S. hospitalis’’ (100% identity). Additionally, sequences affiliated (100% identity) with ‘’E-plasma’’ were detected in low abundance in several enrichment cultures. In both timepoints 3 and 7, sequences affiliated (99.61% identity) with ‘’Ca. Micrarchaeota Mia-14’’ were also detected in a number of enrichment cultures. Bacterial sequences were detected in low abundances in comparison to archaeal sequences. The highest abundance sequences were affiliated with the phyla Actinobacteriota and Nitrospirota. Actinobacteria contained sequences that could only be affiliated (100% identity) to the class Acidimicrobiia. Sequencing of fungal cultures also revealed the presence of sequences affiliated with phylum Ascomycota, specifically the genus Aspergillus (99.20% identity).
This study resulted in successful cultivation of ‘’Ca. Micrarchaeota ‘’and has confirmed the tendency for this ‘’Ca. Micrarchaeota’’ phylotype, similar to ‘’Ca. Mancarchaeum acidiphilum, Mia-14’’ to be associated with Cuniculiplasma sp. under certain laboratory conditions and shown evidence of a pH growth range of 1.7-2.2 and at 0.02% (w/v) polypeptide presence. As preferential characteristics, both parameters found to be slightly different than that of another ‘’Ca. Micrarchaeota’’/ C. divulgatum association, isolated from the PM4 site (Parys Mountain, UK). This study has also shown the first growth of ‘’E-plasma’’ in vitro representing an important step in studying this microorganism. Finally, detection of sequences affiliated with ‘’Ca. S. hospitalis’’ also indicate the potential aerobic growth of this microorganism previously only enriched and cultivated under anoxic conditions.