Lin-Xing Chen

Contemporary environmental variation determines microbial diversity patterns in acid mine drainage.

A wide array of microorganisms survive and thrive in extreme environments. However, we know little about the patterns of, and controls over, their large-scale ecological distribution. To this end, we have applied a bar-coded 16S rRNA pyrosequencing technology to explore the phylogenetic differentiation among 59 microbial communities from physically and geochemically diverse acid mine drainage (AMD) sites across Southeast China, revealing for the first time environmental variation as the major factor explaining community differences in these harsh environments. Our data showed that overall microbial diversity estimates, including phylogenetic diversity, phylotype richness and pairwise UniFrac distance, were largely correlated with pH conditions. Furthermore, multivariate regression tree analysis also identified solution pH as a strong predictor of relative lineage abundance. Betaproteobacteria, mostly affiliated with the ‘Ferrovum’ genus, were explicitly predominant in assemblages under moderate pH conditions, whereas Alphaproteobacteria, Euryarchaeota, Gammaproteobacteria and Nitrospira exhibited a strong adaptation to more acidic environments. Strikingly, such pH-dependent patterns could also be observed in a subsequent comprehensive analysis of the environmental distribution of acidophilic microorganisms based on 16S rRNA gene sequences previously retrieved from globally distributed AMD and associated environments, regardless of the long-distance isolation and the distinct substrate types. Collectively, our results suggest that microbial diversity patterns are better predicted by contemporary environmental variation rather than geographical distance in extreme AMD systems.

Shifts in microbial community composition and function in the acidification of a lead/zinc mine tailings

In an attempt to link the microbial community composition and function in mine tailings to the generation of acid mine drainage, we simultaneously explored the geochemistry and microbiology of six tailings collected from a lead/zinc mine, i.e. primary tailings (T1), slightly acidic tailings (T2), extremely acidic tailings (T3, T4 and T5) and orange-coloured oxidized tailings (T6). Geochemical results showed that the six tailings (from T1 to T6) likely represented sequential stages of the acidification process of the mine tailings. 16S rRNA pyrosequencing revealed a contrasting microbial composition between the six tailings: Proteobacteria-related sequences dominated T1-T3 with relative abundance ranging from 56 to 93%, whereas Ferroplasma-related sequences dominated T4-T6 with relative abundance ranging from 28 to 58%. Furthermore, metagenomic analysis of the microbial communities of T2 and T6 indicated that the genes encoding key enzymes for microbial carbon fixation, nitrogen fixation and sulfur oxidation in T2 were largely from Thiobacillus and Acidithiobacillus, Methylococcus capsulatus, and Thiobacillus denitrificans respectively; while those in T6 were mostly identified in Acidithiobacillus and Leptospirillum, Acidithiobacillus and Leptospirillum, and Acidithiobacillus respectively. The microbial communities in T2 and T6 harboured more genes suggesting diverse metabolic capacities for sulfur oxidation/heavy metal detoxification and tolerating low pH respectively.

Comparative metagenomic and metatranscriptomic analyses of microbial communities in acid mine drainage

The microbial communities in acid mine drainage have been extensively studied to reveal their roles in acid generation and adaption to this environment. Lacking, however, are integrated community- and organism-wide comparative gene transcriptional analyses that could reveal the response and adaptation mechanisms of these extraordinary microorganisms to different environmental conditions. In this study, comparative metagenomics and metatranscriptomics were performed on microbial assemblages collected from four geochemically distinct acid mine drainage (AMD) sites. Taxonomic analysis uncovered unexpectedly high microbial biodiversity of these extremely acidophilic communities, and the abundant taxa of Acidithiobacillus, Leptospirillum and Acidiphilium exhibited high transcriptional activities. Community-wide comparative analyses clearly showed that the AMD microorganisms adapted to the different environmental conditions via regulating the expression of genes involved in multiple in situ functional activities, including low-pH adaptation, carbon, nitrogen and phosphate assimilation, energy generation, environmental stress resistance, and other functions. Organism-wide comparative analyses of the active taxa revealed environment-dependent gene transcriptional profiles, especially the distinct strategies used by Acidithiobacillus ferrivorans and Leptospirillum ferrodiazotrophum in nutrients assimilation and energy generation for survival under different conditions. Overall, these findings demonstrate that the gene transcriptional profiles of AMD microorganisms are closely related to the site physiochemical characteristics, providing clues into the microbial response and adaptation mechanisms in the oligotrophic, extremely acidic environments.

Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

High-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer’ strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Our study demonstrates the potential of the ‘divide and conquer’ strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.

Correlating Microbial Diversity Patterns with Geochemistry in an Extreme and Heterogeneous Environment of Mine Tailings

ABSTRACT Recent molecular surveys have advanced our understanding of the forces shaping the large-scale ecological distribution of microbes in Earths extreme habitats, such as hot springs and acid mine drainage. However, few investigations have attempted dense spatial analyses of specific sites to resolve the local diversity of these extraordinary organisms and how communities are shaped by the harsh environmental conditions found there. We have applied a 16S rRNA gene-targeted 454 pyrosequencing approach to explore the phylogenetic differentiation among 90 microbial communities from a massive copper tailing impoundment generating acidic drainage and coupled these variations in community composition with geochemical parameters to reveal ecological interactions in this extreme environment. Our data showed that the overall microbial diversity estimates and relative abundances of most of the dominant lineages were significantly correlated with pH, with the simplest assemblages occurring under extremely acidic conditions and more diverse assemblages associated with neutral pHs. The consistent shifts in community composition along the pH gradient indicated that different taxa were involved in the different acidification stages of the mine tailings. Moreover, the effect of pH in shaping phylogenetic structure within specific lineages was also clearly evident, although the phylogenetic differentiations within the Alphaproteobacteria, Deltaproteobacteria, and Firmicutes were attributed to variations in ferric and ferrous iron concentrations. Application of the microbial assemblage prediction model further supported pH as the major factor driving community structure and demonstrated that several of the major lineages are readily predictable. Together, these results suggest that pH is primarily responsible for structuring whole communities in the extreme and heterogeneous mine tailings, although the diverse microbial taxa may respond differently to various environmental conditions.

Biogeochemical Processes Governing Natural Pyrite Oxidation and Release of Acid Metalliferous Drainage

The oxidative dissolution of sulfide minerals (principally pyrite) is responsible for the majority of acid metalliferous drainage from mine sites, which represents a significant environmental problem worldwide. Understanding the complex biogeochemical processes governing natural pyrite oxidation is critical not only for solving this problem but also for understanding the industrial bioleaching of sulfide minerals. To this end, we conducted a simulated experiment of natural pyrite oxidative dissolution. Pyrosequencing analysis of the microbial community revealed a distinct succession across three stages. At the early stage, a newly proposed genus, Tumebacillus (which can use sodium thiosulfate and sulfite as the sole electron donors), dominated the microbial community. At the midstage, Alicyclobacillus (the fifth most abundant genus at the early stage) became the most dominant genus, whereas Tumebacillus was still ranked as the second most abundant. At the final stage, the microbial community was dominated by Ferroplasma (the tenth most abundant genus at the early stage). Our geochemical and mineralogical analyses indicated that exchangeable heavy metals increased as the oxidation progressed and that some secondary sulfate minerals (including jarosite and magnesiocopiapite) were formed at the final stage of the oxidation sequence. Additionally, we propose a comprehensive model of biogeochemical processes governing the oxidation of sulfide minerals.

Predicting taxonomic and functional structure of microbial communities in acid mine drainage

Predicting the dynamics of community composition and functional attributes responding to environmental changes is an essential goal in community ecology but remains a major challenge, particularly in microbial ecology. Here, by targeting a model system with low species richness, we explore the spatial distribution of taxonomic and functional structure of 40 acid mine drainage (AMD) microbial communities across Southeast China profiled by 16S ribosomal RNA pyrosequencing and a comprehensive microarray (GeoChip). Similar environmentally dependent patterns of dominant microbial lineages and key functional genes were observed regardless of the large-scale geographical isolation. Functional and phylogenetic β-diversities were significantly correlated, whereas functional metabolic potentials were strongly influenced by environmental conditions and community taxonomic structure. Using advanced modeling approaches based on artificial neural networks, we successfully predicted the taxonomic and functional dynamics with significantly higher prediction accuracies of metabolic potentials (average Bray–Curtis similarity 87.8) as compared with relative microbial abundances (similarity 66.8), implying that natural AMD microbial assemblages may be better predicted at the functional genes level rather than at taxonomic level. Furthermore, relative metabolic potentials of genes involved in many key ecological functions (for example, nitrogen and phosphate utilization, metals resistance and stress response) were extrapolated to increase under more acidic and metal-rich conditions, indicating a critical strategy of stress adaptation in these extraordinary communities. Collectively, our findings indicate that natural selection rather than geographic distance has a more crucial role in shaping the taxonomic and functional patterns of AMD microbial community that readily predicted by modeling methods and suggest that the model-based approach is essential to better understand natural acidophilic microbial communities.

Microbial communities, processes and functions in acid mine drainage ecosystems

Acid mine drainage (AMD) is generated from the oxidative dissolution of metal sulfides when water and oxygen are available largely due to human mining activities. This process can be accelerated by indigenous microorganisms. In the last several decades, culture-dependent researches have uncovered and validated the roles of AMD microorganisms in metal sulfides oxidation and acid generation processes, and culture-independent studies have largely revealed the diversity and metabolic potentials and activities of AMD communities, leading towards a full understanding of the microbial diversity, functions and interactions in AMD ecosystems. This review describes the diversity of microorganisms and their functions in AMD ecosystems, and discusses their biotechnological applications in biomining and AMD bioremediation according to their capabilities.

Microbial communities evolve faster in extreme environments

Evolutionary analysis of microbes at the community level represents a new research avenue linking ecological patterns to evolutionary processes, but remains insufficiently studied. Here we report a relative evolutionary rates (rERs) analysis of microbial communities from six diverse natural environments based on 40 metagenomic samples. We show that the rERs of microbial communities are mainly shaped by environmental conditions, and the microbes inhabiting extreme habitats (acid mine drainage, saline lake and hot spring) evolve faster than those populating benign environments (surface ocean, fresh water and soil). These findings were supported by the observation of more relaxed purifying selection and potentially frequent horizontal gene transfers in communities from extreme habitats. The mechanism of high rERs was proposed as high mutation rates imposed by stressful conditions during the evolutionary processes. This study brings us one stage closer to an understanding of the evolutionary mechanisms underlying the adaptation of microbes to extreme environments.

Metabolic versatility of small archaea Micrarchaeota and Parvarchaeota

Small acidophilic archaea belonging to Micrarchaeota and Parvarchaeota phyla are known to physically interact with some Thermoplasmatales members in nature. However, due to a lack of cultivation and limited genomes on hand, their biodiversity, metabolisms, and physiologies remain largely unresolved. Here, we obtained 39 genomes from acid mine drainage (AMD) and hot spring environments around the world. 16S rRNA gene based analyses revealed that Parvarchaeota were only detected in AMD and hot spring habitats, while Micrarchaeota were also detected in others including soil, peat, hypersaline mat, and freshwater, suggesting a considerable higher diversity and broader than expected habitat distribution for this phylum. Despite their small genomes (0.64–1.08 Mb), these archaea may contribute to carbon and nitrogen cycling by degrading multiple saccharides and proteins, and produce ATP via aerobic respiration and fermentation. Additionally, we identified several syntenic genes with homology to those involved in iron oxidation in six Parvarchaeota genomes, suggesting their potential role in iron cycling. However, both phyla lack biosynthetic pathways for amino acids and nucleotides, suggesting that they likely scavenge these biomolecules from the environment and/or other community members. Moreover, low-oxygen enrichments in laboratory confirmed our speculation that both phyla are microaerobic/anaerobic, based on several specific genes identified in them. Furthermore, phylogenetic analyses provide insights into the close evolutionary history of energy related functionalities between both phyla with Thermoplasmatales. These results expand our understanding of these elusive archaea by revealing their involvement in carbon, nitrogen, and iron cycling, and suggest their potential interactions with Thermoplasmatales on genomic scale.