Metal pollution in freshwater bodies is a long-standing challenge with large expense required to clean-up pollutants such as Cd. There is widespread interest in the potentially low-cost and sustainable use of biological material to perform bioremediation, such as the use of microalgae. Efficient metal bioremediation capacity requires both the ability to tolerate metal stress and metal accumulation. Here, the role of a Chlamydomonas reinhardtii metal tolerance protein (MTP) was examined for enhanced Cd tolerance and uptake. The CrMTP4 gene is a member of the Mn-CDF clade of the cation diffusion facilitator family of metal transporters but is able to provide tolerance and sequestration for Mn and Cd, but not other metals, when expressed in yeast. Over-expression of CrMTP4 in C. reinhardtii yielded a significant increase in tolerance to Cd toxicity and increased Cd accumulation although tolerance to Mn was not increased. In comparison, the metal tolerance of three chlorophyte microalgae strains (Chlorella luteoviridis, Parachlorella hussii, and Parachlorella kessleri) that had previously been adapted to wastewater growth was examined. In comparison to wild type C. reinhardtii, all three natural strains showed significantly increased tolerance to Cd, Cu, Al and Zn, and furthermore their Cd tolerance and uptake was greater than that of the CrMTP4 over-expression strains. Despite CrMTP4 gene over-expression being a successful strategy to enhance the Cd bioremediation potential of a metal-sensitive microalga, a single gene manipulation cannot compete with naturally adapted strain mechanisms that are likely to be multigenic and due in part to oxidative stress tolerance.

The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga Chlamydomonas reinhardtii, GPD2 and GPD3 were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast gpd1D mutant demonstrated the functionality of both enzymes. C. reinhardtii knockdown mutants for GPD2 and GPD3 showed no difference in growth but displayed significant reduction in TAG concentration compared with the wild type in response to phosphorus or nitrogen starvation. Overexpression of GPD2 and GPD3 in C. reinhardtii gave distinct phenotypes. GPD2 overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, GPD3 overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared with the wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides, and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in GPD3 overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.

The diversity and biological characteristics of eukaryotic communities within acid mine drainage (AMD) sites is less well studied than for prokaryotic communities. Furthermore, for many eukaryotic extremophiles the potential mechanisms of adaptation are unclear. This study describes an evaluation of eight highly acidic (pH 1.6-3.1) and one moderately acidic (pH 5.6) metal-rich acid mine drainage ponds at a disused copper mine. The severity of AMD pollution on eukaryote biodiversity was examined, and while the most species-rich site was less acidic, biodiversity did not only correlate with pH but also with the concentration of dissolved and particulate metals. Acid-tolerant microalgae were present in all ponds, including the species Chlamydomonas acidophila, abundance of which was high in one very metal-rich and highly acidic (pH 1.6) pond, which had a particularly high PO-P concentration. The C. acidophila strain named PM01 had a broad-range pH tolerance and tolerance to high concentrations of Cd, Cu and Zn, with bioaccumulation of these metals within the cell. Comparison of metal tolerance between the isolated strain and other C. acidophila strains previously isolated from different acidic environments found that the new strain exhibited much higher Cu tolerance, suggesting adaptation by C. acidophila PM01 to excess Cu. An analysis of the metabolic profile of the strains in response to increasing concentrations of Cu suggests that this tolerance by PM01 is in part due to metabolic adaptation and changes in protein content and secondary structure.

The Parys Mountain copper mine (Wales, UK) contains a wide range of discrete environmental microniches with various physicochemical conditions that shape microbial community composition. Our aim was to assess the microbial community in the sediments and overlying water column in an acidic mine drainage (AMD) site containing abundant filamentous biogenic growth via application of a combination of chemical analysis and taxonomic profiling using 16S rRNA gene amplicon sequencing. Our results were then compared to previously studied sites at Parys Mt. Overall, the sediment microbiome showed a dominance of bacteria over archaea, particularly those belonging to Proteobacteria (genera Acidiphilium and Acidisphaera), Acidobacteriota (subgroup 1), Chloroflexota (AD3 cluster), Nitrospirota (Leptospirillum) and the uncultured Planctomycetota/CPIa-3 termite group. Archaea were only present in the sediment in small quantities, being represented by the Terrestrial Miscellaneous Euryarchaeota Group (TMEG), Thermoplasmatales and Ca. Micrarchaeota (Ca. Micracaldota). Bacteria, mostly of the genera Acidiphilium and Leptospirillum, also dominated within the filamentous streamers while archaea were largely absent. This study found pH and dissolved solutes to be the most important parameters correlating with relative proportions of bacteria to archaea in an AMD environment and revealed the abundance patterns of native acidophilic prokaryotes inhabiting Parys Mt sites and their niche specificities.

Background Archaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called “alphabet-plasmas”, and collectively dubbed “E-plasma”, were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts. Results Their genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the “E-plasma” in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of “E-plasma”, which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales. Conclusions This study has analysed the metagenome-assembled genome of “E-plasma”, which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle.