The archaeal Domain is understudied and under-sampled, comprising much of the uncultivated majority of microorganisms known as microbial “dark matter”. Metagenomic studies have revealed a large diversity of archaea remaining uncharacterised through lack of cultivation, and a dominance of Thermoplasmatales related archaea in Acid Mine Drainage (AMD) sites. The AMD site at Parys Mountain (Wales, UK) is dominated by the uncultured archaeon “E-Plasma”, and the present work aims to cultivate these organisms. Archaeal Richmond Mine Acidophilic Nano-organisms (ARMAN) related organisms were also detected in this ecosystem in association with Cuniculiplasma divulgatum. Since no representatives of ARMAN related organisms have been validly described through cultivation, it is the aim of the present work to cultivate these organisms to uncover details regarding their physiology.
Enrichment cultures were established with samples from the Parys Mountain AMD site using five different polypeptide substrates. The microbial content was monitored during cultivation using PCR based techniques and 16S rRNA gene amplicon sequencing. Bacterial and archaeal sequence affiliations were checked with the GreenGenes and National Centre for Biotechnology Information (NCBI) databases respectively. No significant enrichment of “E-Plasma” was detected in the first cultivation attempts, but ARMAN related organisms were detected in later cultures. Subsequent cultivation experiments focussed on their enrichment.
The microbial composition of cultures was affected by pH and the choice of polypeptide substrate used during cultivation. Archaea of the order Thermoplasmatales grew in different quantities in all cultures, with a higher abundance in more acidic conditions. The bacterial content of cultures resembled similar AMD ecosystems, with mostly Actinobacteria, Proteobacteria and Firmicutes represented. After nine months the ARMAN related organism Mia14 was enriched to 35.39 % in a culture containing elemental sulfur at 37.5 °C under microaerophilic conditions with a pH of 1.7. This represents the highest abundance of these organisms to have been cultivated. The removal of sulfur yielded a lower Mia14 content and no additional supplements stimulated its growth further. Here, it is proposed that Mia14 has an association with sulfur and a possible association with Leptospirillum sp. Previous studies are supported by the present work regarding its dependence on C. divulgatum as a host organism. The role of sulfur in the metabolism of Mia14 is unclear: it may be reduced or oxidised as an energy source, or act as an attachment target for cells.
The importance of exploring the diversity of microbial “dark matter” through cultivation is emphasised by the present work. Despite the domination of “E-Plasma” in the environment, cultivation attempts proved unsuccessful. This represents the difficulties associated with Thermoplasmatales cultivation, as highly specific growth conditions are required. The lack of essential nutrients may have prevented the stimulation of growth, possibly due to a symbiosis or other conditions which were not replicated in cultures. With the advent of new cultivation methodology, further cultivation attempts may be made in relation to “E-Plasma”. Further research using the present cultures will continue to investigate the physiology and metabolism of both Mia14 and C. divulgatum.