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ScienceDirect Microbial communities, processes and functions in acid mine drainage ecosystems Lin-xing Chen1, Li-nan Huang1, Celia Me´ndez-Garcı´a2, Jia-liang Kuang1, Zheng-shuang Hua1, Jun Liu1 and Wen-sheng Shu1 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. Addresses 1 State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, College of Ecology and Evolution, Sun Yat-Sen University, Guangzhou 510275, PR China 2 Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, USA Corresponding author: Shu, Wen-sheng ([email protected])

can dramatically accelerate the acid generation process and therefore play significant roles in material and energy flows of the whole ecosystem [3,6]. Due to the dominance by a few taxa more than a lack of complexity [7,8] and its remarkably simple geochemical features [9], AMD environments have been established as model systems for quantitative analyses of microbial ecology and community function [3,4], and as ideal targets for biogeochemical studies of iron and sulfur cycles [10–12]. Moreover, microorganisms from AMD environments are the major contributors in biomining of low-grade ores [13], and have significant potentials in AMD bioremediation [2]. This review summarizes the taxonomic diversity and metabolic functions of AMD microorganisms and their biotechnological applications in biomining and AMD bioremediation.

The diversity of AMD microorganisms During the last several decades, isolation and cultivation methods [4,9], and 16S rRNA gene and meta-omics based molecular analyses [3,7,14], have drastically enlarged our knowledge of microbial diversity in AMD ecosystems (Figures 1 and 2).

Current Opinion in Biotechnology 2016, 38:150–158 This review comes from a themed issue on Environmental biotechnology Edited by Benardo Gonzalez Ojeda and Regina Wittich

http://dx.doi.org/10.1016/j.copbio.2016.01.013 0958-1669/# 2016 Elsevier Ltd. All rights reserved.

Introduction Acid mine drainage (AMD) is characterized by low pH and high concentrations of metals and sulfate, representing an extreme environment to life [1] as well as a major environmental challenge worldwide [2]. AMD ecosystems include distinct environments of AMD solutions, sediments, and biofilms [3], providing multiple niches for AMD microorganisms. The microorganisms thriving in AMD environments have evolved with distinct mechanisms to the extreme conditions [4,5], by which they Current Opinion in Biotechnology 2016, 38:150–158

Proteobacteria, Nitrospira, Actinobacteria, Firmicutes, Acidobacteria, Aquificae and Candidate division TM7 represent the primary bacterial lineages detected (Figure 1). The most extensively studied taxa are iron- and/or sulfuroxidizing Acidithiobacillus spp. (Acidithiobacillia) [15–17], and iron-oxidizing Leptospirillum spp. (Nitrospira) [18–20]. The lesser known ‘Ferrovum’ spp. (Betaproteobacteria), which have been widely detected as dominant members and suggested as major iron-oxidizing populations in lessrestrictive pH conditions [7,21,22,23], have recently been obtained in pure culture [24]. The majority members in these three genera are validated as autotrophs (Figure 2), fixing carbon from open environments. Multiple heterotrophic acidophiles co-occurring with these carbon-fixers may drive the carbon cycling by consuming organic carbons, including members from Alphaproteobacteria (Acidiphilium, Acidocella, Acidicaldus, Acidomonas, Acidisphaera) [25–27], Gammaproteobacteria (Acidibacter ferrireducens and Metallibacterium scheffleri) [28,29], Actinobacteria (Acidithrix ferrooxidans, Ferrimicrobium acidiphilum and Acidimicrobium ferrooxidans) [6,30], and Acidobacteria [31,32]. Additionally, Sulfobacillus spp. (Firmicutes) were detected as mixotrophs [33–35], while Alicyclobacillus spp. www.sciencedirect.com

Microbiology ecology in AMD Chen et al. 151

Figure 1

BetaGa

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Current Opinion in Biotechnology

The taxonomic and phylogenetic diversity of frequently detected prokaryotic microorganisms in AMD ecosystems including environments of acid solutions, biofilms and sediments (left panel), and their distribution in AMD solutions of different pH ranges (right panel) (data from [7]; a total of 125 AMD samples).

(Firmicutes) were documented as heterotrophs or chemolithoautotrophs in mine environments or AMD treatment plants [2,36]. Some representatives of sulfate-reducing bacteria (SRB) within Gammaproteobacteria, Deltaproteobacteria and Firmicutes have also been detected, including members from the genera of Thermodesulfobium, Syntrophobacter, Desulfurella, Desulfomonile, Desulfovibrio and Desulfosporosinus [37]. Other less frequently detected bacterial taxa include Gallionella ferruginea and the putative heterotrophic growers Thiomonas spp. [38–41]. Archaea, including the phyla of Euryarchaeota, Crenarchaeota and Candidate division Parvarchaeota, typically constitute a minor fraction of AMD communities [3,4,9,14], but have been found dominant in specific mining environments [42–44]. In the Euryarchaeota, members from the iron-oxidizing and cell wall-lacking genus Ferroplasma are the most widely detected [42– 45,46,47–50]. Importantly, Ferroplasma spp. have been found to dominate the later stages of the acidification processes of mine wastes [43,44], indicating their critical www.sciencedirect.com

roles in AMD generation. Another cell wall-lacking genus Acidiplasma was also isolated and detected with ironoxidation and reduction activities [51]. Besides, multiple Thermoplasmatales archaeon genomes (alphabet plasmas) have been reconstructed from metagenomic datasets, and one of those (G-plasma) was cultured as Cuniculiplasma divulgatum to represent a new family Cuniculiplasmataceae [52]. Within the Crenarchaeota, the iron and/or sulfur oxidizers from genera Acidianus, Metallosphaera, Stygiolobus, Sulfolobus and Sulfurisphaera have been mostly identified as extreme thermophiles inhabiting AMD ecosystems [9]. The poorly studied archaeal group ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms) in Candidate division Parvarchaeota was firstly discovered in the acidophilic biofilms from the Richmond Mine at Iron Mountain, California [53,54]. Closely related gene sequences have been recovered in other AMD environments [42,55], however their ecological roles have not been clearly revealed, due to their relatively low activities [23,42,54]. The filterable ARMAN spp. were initially speculated to be aerobic based on proteomic data Current Opinion in Biotechnology 2016, 38:150–158

152 Environmental biotechnology

lfu r

itr og ar en bo n C

N

Acidithiobacillus caldus DSM 22753

Acidianus brierleyi DSM 1651

Acidithiobacillus caldus ATCC 51756

Acidiplasma aeolicum DSM 18409

Acidithiobacillus ferrivorans DSM 22755

Acidiplasma cupricumulans DSM 16551

Acidithiobacillus ferrooxidans ATCC 23270

“Ferroplasma acidarmanus” fer1

Acidithiobacillus ferrooxidans ATCC 53993

“Ferroplasma sp. Type II”

Acidithiobacillus thiooxidans DSM 17318

Ferroplasma acidiphilum

Acidithiobacillus ferriphilus DSM 100412

Metallosphaera sedula DSM 535

Acidithiobacillus ferridurans ATCC 33020

Picrophilus torridus DSM 9790

Acidithrix ferrooxidans DSM 28176

Picrophilus oshimae DSM 9789

Acidibacter ferrireducens DSM 27237

Sulfolobus acidocaldarius DSM 639

Acidiphilium cryptum JF-5

Sulfolobus solfataricus P2 Sulfolobus tokodaii JCM10545

Acidiphilium multivorum AIU301

Stygiolobus azoricus DSM 6296

“Ferrovum myxofaciens” P3G

Thermoplasma acidophilum DSM 1728

“Ferrovum” sp. FKB7 Thiomonas delicata DSM 17897 Desulfomonile tiedjei DSM 6799 Desulfovibrio longus DSM 6739 Leptospirillum ferriphilum DSM 17947

Bacteria

Acidiphilium angustum ATCC 35903

Thermoplasma volcanium GSS1

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O xi d R ati ed o n O ucti xi on da R ti ed o u n Fi ctio xa n R tio ed n u Fi ctio xa n tio n

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Thermogymnomonas acidicola JCM 13583 “Thermoplasmatales archaeon A-plasma” “Thermoplasmatales archaeon C-plasma” “Thermoplasmatales archaeon D-plasma”

“Leptospirillum ferrodiazotrophum”

“Thermoplasmatales archaeon E-plasma”

Leptospirillum ferrooxidans ATCC 29047

“Thermoplasmatales archaeon I-plasma”

Leptospirillum group IV UBA BS

Cuniculiplasma divulgatum JCM 30642

“Leptospirillum rubarum”

Candidatus Micrarchaeum acidiphilum ARMAN-2

Alicyclobacillus disulfidooxidans ATCC 51911

Candidatus Parvarchaeum acidiphilum ARMAN-4

Sulfobacillus acidophilus DSM 10332

Candidatus Parvarchaeum acidiphilum ARMAN-5

Sulfobacillus thermosulfidooxidans DSM 9293 Desulfosporosinus sp. aSRB1 (FK) Desulfosporosinus sp. aSRB2 (FK) Acidimicrobium ferrooxidans DSM 10331

Experiment confirmed with feature With genes, while not experiment confirmed

Ferrimicrobium acidiphilum DSM 19497 Acidobacterium capsulatum ATCC 51196 Hydrogenobaculum acidophilum DSM 11251 Metallibacterium scheffleri DSM 24874

Experiment confirmed without feature No information, or not analyzed Current Opinion in Biotechnology

The metabolic characteristics of representative prokaryotic microorganisms detected in AMD ecosystems, including their capabilities in the cycling of iron, sulfur, nitrogen and carbon, which are significant for AMD microorganisms. The information is based on cultivation-dependent approaches and/or predicted from -omics data.

[54], while later found to exclusively inhabit the anoxic areas of biofilms by fluorescence in situ hybridization (FISH) analysis and could only be enriched using anoxic culturing techniques [56]. The detected eukaryotic microorganisms in AMD environments included Archaeplastida, Stramenophiles, Alveolates, Rhizaria, Excavata, Opisthokonta, and algae and fungi [57–59]. Although the current knowledge of AMD eukaryotic microorganisms is still limited, while their significant roles, including those in the whole ecosystem function and AMD bioremediation, should not be omitted (see below).

From diversity to ecosystem function: processes and functional networks The successful isolation and functional validation of AMD acidophiles, and the application of approaches Current Opinion in Biotechnology 2016, 38:150–158

including meta-omics [40,42,46], taxa-specific gene chips [60,61], FISH [56,62] and DNA-stable isotope probing (DNA-SIP) [35], have rapidly advanced our understanding of the microbial roles, distributions and interactions in AMD ecosystems (Figures 2 and 3). The oxidation of iron and sulfur is vital for acidophilic chemolithoautotrophic microbes to gain energy [4]. The oxidation of Fe2+ to Fe3+ is a process fueled by the oxidative environment and driven by the activities of a wide array of indigenous microorganisms, such as members of Leptospirillum, ‘Ferrovum’, Acidithiobacillus, Ferroplasma, Sulfobacillus and Sulfolobus [4,6,9]. The mechanisms for energy conservation and associated genes in species belonging to this functional group have been extensively reported [12]. As the primary oxidant in low pH AMD environments [4], obtained Fe3+ can oxidize acid-insoluble metal sulfides (e.g., FeS2, MoS2) to generate thiosulfate, and acid-soluble www.sciencedirect.com

Microbiology ecology in AMD Chen et al. 153

Figure 3

sunlight darkness

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Scheme displaying the main element cycling operating in AMD ecosystems in all identifiable environments (acid solution, sediment, and biofilm) and involved taxa (see legend). Dissolution of pyrite (FeS2) present in the sediments fuels AMD generation. Redox reactions involving iron dominate in acid solution and at early biofilm stages as compared to mature biofilms. Oxidation of reduced inorganic sulfur compounds (RISCs) occurs across environments and gains importance in the mature biofilm. Autotrophic carbon and nitrogen fixation take place closer to the air/water interface and heterotrophs are responsible for biofilm degradation at higher depths, where anaerobic respiration of sulfate occurs, contributing to generation of RISCs. Degrading biofilm is the biofilm sink to the sediment–solution interface, which degrades under microaerobic and anaerobic conditions. Colors reflect the elemental nature of the transformations depicted: red, Fe; yellow, sulfur; green, nitrogen; brown, carbon; blue, hydrogen. Abbreviations: VFA, volatile fatty acid; SRB, sulfate reducing bacteria.

metal sulfides (e.g., FeS, ZnS) to generate elemental sulfur via intermediary polysulfides [63]. Other reduced inorganic sulfur compounds (RISCs) such as sulfite and tetrathionate, may also be generated. These, together with the thiosulfate and elemental sulfur, can be oxidized to sulfate by sulfuroxidizers with enzymes encoded by multiple genes, which (and the associated mechanisms) have been reviewed recently [11]. Furthermore, the reduction of sulfate with the generation of sulfide has been detected in AMD ecosystems [64], thus completing the sulfur cycle in this extreme environment. This happens in the mineral-loaded sediments with a massive occurrence of SRBs where organic carbons are available [33,65], resulting in higher pH and lower levels of redox potential and dissolved metals. Likewise, Fe3+ is susceptible to biological reduction to Fe2+ by bacteria (e.g., Acidiphilium spp., Acidithiobacillus spp.) and archaea (e.g., Ferroplasma spp., Acidiplasma aeolicum) under anaerobic conditions. Such processes have been documented in both natural settings [29,36] and laboratory conditions [47,51]. www.sciencedirect.com

Organic carbon and nitrogen are limited in AMD ecosystems [4]. Although exogenous inputs of these resources are possible, the existence of microbial species capable of carbon and nitrogen fixation coupled with iron and sulfur oxidation will ensure a steady supply of these elements into the system [18,23,42,66], indicating a vital role of these organisms for the growth and maintenance of communities. Notably, the essential function of nitrogen fixation is executed by a limited number of low abundance taxa [2346], and removal of these keystone species will likely result in the collapse of the entire community [18], an important implication for AMD prevention. Also, the acid-tolerance algae and fungi could provide sufficient low molecular weight organic carbon resources for the heterotrophs [59], such as SRBs and Fe3+ reducer, thus serve as an important role in completing the cycling of iron and sulfur in AMD ecosystems. Acidophilic biofilms represent the most extensively studied environment in AMD ecosystems [3]. These biofilms Current Opinion in Biotechnology 2016, 38:150–158

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can vary substantially in their morphologies due to differences in the environment chemistry, mirroring their changes at the functional level (Figure 3). Thin subaerial biofilms floating in AMD solutions rely actively on Fe2+ and RISCs oxidation, especially at early developmental stages [19,67]. In contrast, thicker and more mature microbial growths possess a more heterotrophic metabolism that appears related to increasing proportions of Thermoplasmatales archaea [68,69]. A restricted set of microbes, particularly At. ferrooxidans [70,71], Leptospirillum spp. (Group II and III) [72], and ‘Ferrovum’ spp. [23], have been shown to contribute to the turnover of extracellular polymeric substances, therefore contributing to the metabolism of the ubiquitous carbon source that allows the establishment of a more heterotrophy-inclined community in time. It has been suggested that archaea inhabiting mature and degrading AMD biofilms, with strong evidence for Thermoplasmatales and ARMAN, would possess a heterotrophic metabolism, whereas the aerobic iron-oxidizing Leptospirillum may have a limited anaerobic capacity for the consumption of biofilm carbon [36]. Additionally, mixed acid fermentation have also been proposed to take place in

biofilms [31,61], the excreted acetate from the fermentation process could serve as an electron donor for the heterotrophic SRBs and Fe3+ reducers, and may also negatively affect the growth of chemolithotrophic microorganisms (e.g., Acidithiobacillus spp., Leptospirillum spp.) through iron and sulfur oxidation inhibiting [73]. AMD acidophiles also have evolved with multiple mechanisms for adaptation of the high metal concentrations, including converting them into less toxic forms (through precipitation, oxidation, or reduction), or pumping them into the periplasm or out of the cells [74]. Besides, the fungi in AMD ecosystems are capable to absorb heavy metals into their cells or onto the extracellular polysaccharide [59], thus reducing the solubility of heavy metals, which is benefit for the growth of other indigenous microorganisms.

Biotechnological applications of AMD microorganisms The knowledge about microorganisms in AMD ecosystems, as summarized above, provides the basic clues for

Figure 4

Model reactor

Air Fe oxidizers

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AMD (pH=2.23) High levels of Fe2+, Fe3+,Zn2+,Mn2+, Cu2+,SO42-

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Schematic representation of an integrated reactor system for AMD bioremediation using AMD-related microorganisms, including sections for pH adjustment and heavy metals recovery. The specific role of modules are as follows: module 1, Fe2+ in the AMD solutions is oxidized to Fe3+ by iron oxidizers with the input of air; module 2 and module 3, two sequential modules for sufficient precipitation of Fe3+ as schwertmannite; module 4 and module 5, SRBs feeding with organic carbon resources reduce sulfate to generate H2S, which is used for the precipitation of Cu2+, Zn2+, Mn2+. Please note that the excess H2S should be absorbed from into the air. Current Opinion in Biotechnology 2016, 38:150–158

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Microbiology ecology in AMD Chen et al. 155

their biotechnological applications in biomining and AMD bioremediation. Biomining refers to the extraction of metals from sulfidic ores and concentrates using the capacities of acidophilic chemolithotrophic microorganisms in accelerating the oxidative dissolution of sulfide minerals [13], and has been reviewed recently [75]. As the most widely applied biomining operation, heap leaching is mediated by microbial consortia of acidophiles, including Fe2+ oxidizing autotrophs, H2SO4 generating autotrophs, and also heterotrophs and mixotrophs [13,75]. Integrated reactor systems based on the activities of ironoxidizers and SRBs have recently been used to lower the levels of acidity and to recover heavy metals in AMD solutions (e.g., [76]). In an unpublished work by Liu et al., AMD from DaBaoShan polymetallic ore of Guangdong, China [42], was conducted for bioremediation using an integrated reactor systems. As illustrated in Figure 4, influent Fe2+ is firstly oxidized to Fe3+ and then precipitated as schwertmannite with the input of alkaline materials (e.g., NaOH). Next, sulfate is reduced by the SRBs inhabiting the artificial biofilms in the system, and H2S generated is used for the precipitation of Cu2+, Zn2+ and Mn2+ as metal sulfides. In general, while biomining takes full advantage of ironand sulfur-oxidizers to the extreme [13,75], the activities of sulfate reducers are vital in the AMD bioremediation systems [2,76]. It should be noted that, in both biomining operations and bioremediation systems, the microbial communities vary between different sections, with clearly defined and specific roles for the populated microorganisms. Furthermore, in any single biological unit, a microbial consortium with similar features, not a single taxon, is responsible for the system functions [75]. The microbial consortium for a specific function in the system, may comprise several taxa [75], and/or different genotypes of a single taxa as speculated from natural AMD ecosystems [3], with differences in metabolic potentials, likely due to the multiple micro-niches generated. Biomining has been well established and widely applied [75], while it is not the case for AMD bioremediation technique using AMD indigenous microorganisms [76]. Though many attempts have been performed for AMD bioremediation, and at least two commercial processes have been documented successful (see [2] for details), however, more environmentally benign and economical options are needed. As a potential AMD bioremediation operation, integrated bioreactor systems have been demonstrated to remediate the test AMD solutions with high performance (Figure 4; [76], unpublished data Liu et al.), however, further efforts, including in-depth investigations of the microbial processes therein and isolation of www.sciencedirect.com

naturally occurring acidophilic SRBs and construction of efficient artificial consortia, are needed to address the multiple challenges towards their full-scale applications.

Conclusions Engineered biological systems mirror in many ways the interactions and processes of large-scale ecosystems [77]. The development of molecular approaches and their wide applications in the studies of natural AMD communities have resulted in remarkable insights into the diversity and function of these extraordinary organisms and have provided significant clues for biotechnological applications of AMD microorganisms. Meanwhile, recent high throughput sequencing-based investigations have detected a large number of rare taxa in acidic environments [7,8]. These low abundance species may play a crucial role in functional stability over time and upon perturbations. Future extensive meta-omics and single cell genomics will provide novel insights into these lesser-known, but potentially important taxa in AMD communities. Additionally, the complex ecological interactions between different taxa including viruses, prokaryotes, eukaryotic microbes, deserve more attention. On the other hand, the reductive processes in AMD ecosystems (including those of sulfur and iron cycling), as well as the community diversity and dynamics of the associated microorganisms, which are particularly crucial for AMD bioremediation, merit further studies. Ultimately, an ecologically sound and efficient use of AMD microbes will benefit from a thorough knowledge of the ecology of all community members populating AMD ecosystems.

Acknowledgements We thank the three anonymous reviewers for their valuable comments on the manuscript. This work was supported by the National Natural Science Foundation of China (U1201233, U1501232, 31370154), the Guangdong Province Key Laboratory of Computational Science and the Guangdong Province Computational Science Innovative Research Team.

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