Li and Li Environmental Microbiome (2021) 16:9 Environmental Microbiome https://doi.org/10.1186/s40793-021-00380-3

RESEARCH ARTICLE Open Access Metagenome-assembled genomes infer potential microbial metabolism in alkaline sulphidic tailings Wenjun Li1,2 and Xiaofang Li1*

Abstract Background: Mine tailings are hostile environment. It has been well documented that several microbes can inhabit such environment, and metagenomic reconstruction has successfully pinpointed their activities and community structure in acidic tailings environments. We still know little about the microbial metabolic capacities of alkaline sulphidic environment where microbial processes are critically important for the revegetation. Microbial communities therein may not only provide soil functions, but also ameliorate the environment stresses for plants’ survival. Results: In this study, we detected a considerable amount of viable bacterial and archaeal cells using fluorescent in situ hybridization in alkaline sulphidic tailings from Mt Isa, Queensland. By taking advantage of high-throughput sequencing and up-to-date metagenomic binning technology, we reconstructed the microbial community structure and potential coupled iron and nitrogen metabolism pathways in the tailings. Assembly of 10 metagenome-assembled genomes (MAGs), with 5 nearly complete, was achieved. From this, detailed insights into the community metabolic capabilities was derived. Dominant microbial species were seen to possess powerful resistance systems for osmotic, metal and oxidative stresses. Additionally, these community members had metabolic capabilities for sulphide oxidation, for causing increased salinity and metal release, and for leading to N depletion. Conclusions: Here our results show that a considerable amount of microbial cells inhabit the mine tailings, who possess a variety of genes for stress response. Metabolic reconstruction infers that the microbial consortia may actively accelerate the sulphide weathering and N depletion therein. Keywords: Sulphidic tailings, Metagenomics, Binning, Community genomics, Sulphide oxidation

Introduction coupled weathering of sulphide and carbonate wastes Mine tailings are milled residue wastes of ore processing. [1]. This makes mine tailings a rather hostile environ- Those of metal mines, typically Pb/Zn/Cu/Ni, always ment to microbes, while still a considerable amount contain abundant sulphides that are mostly pyrite based, cells, though with a relatively low diversity compared e.g. FeS2. Due to the high reactivity of sulphides, high with normal soils, had been detected therein [1]. The levels of metals and salinity can be generated by the contribution of microbial activities to the weathering of sulphides has long been recognized in mine tailings. At

* Correspondence: [email protected] the acid mine drainage (AMD) site of Iron Mountain, 1Hebei Key Laboratory of Soil Ecology, Key Laboratory for Agricultural Water Calif., Thiobacillus ferrooxidans and Leptospirillum fer- Resource, Center for Agricultural Resources Research, Institute of Genetics rooxidans were found to be dominant in the community and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China and contribute to the generation of AMD [2]. In a cop- Full list of author information is available at the end of the article per bioleaching heap, sulfurophilic and iron-ophilic

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acidophilic bacteria were found to actively colonize the gypsum and kaolinite, and contain approximately 1300, mineral surface [3]. These microbes show adaptability to 1800 and 2900 mg/kg of residue Cu, Pb and Zn, respect- extremely acidic environment and play an important role ively. The 16S-based amplicon sequencing reveals that the in the sustainability of this system. With the advent of dominant microbial species in the tailings include Rubro- the modern molecular tools, community genomics of bacter spp. of the Actinobacteria, Truepera spp. of the microbial consortia in acid mine environments has been Deinococcus-Thermus, and Thioalkalivibrio spp. and Thio- well explored [4, 5], while that of alkaline mine tailings bacillus spp. of the Proteobacteria. No substantial changes has not been extensively studied. Alkaline mine environ- were detected of the microbial community structure in ment is normally subjected to revegetation which re- summer and in winter [6], and dominant species changed quires the establishment of soil function including little in spite of significant increases in diversity detected microbial diversity [6]. along with the revegetation efforts [15]. Microbial community networks related to sulphide weathering in neutralized tailings have been determined Fluorescent in situ hybridization (FISH) [7, 8], yet these studies are based on mainly 16S rRNA Cells from the tailings samples were firstly enriched by a gene detection and then possible functions of the com- sucrose density centrifugation [17], followed by the munity members are inferred from physiological infor- standard FISH procedure for soil with minor modifica- mation of known related pure cultures. This approach is tions [18, 19]. To obtain sufficient microbial cells from useful but is highly limited and speculative with regard the tailings, quadruplicates with 5 g of tailings each were to approximating the community metabolic capabilities used for cell enrichment. Oligonucleotide probes were related to the in situ elemental cycling and adaptation used for detecting bacteria (mixed EUB338) and archaea strategies. In contrast, metagenome-based analyses en- (ARCH915) [20]. The hybridized samples were observed able reconstruction of nearly complete genomes of mi- within 24 h of performing the FISH and images were crobial community members. Such analyses provides taken using a ZEISS LSM 510 META Confocal Micro- detailed insights of metabolic capabilities and enables es- scope. For enumeration of microorganisms, from each timating the possible contributions of populations to the sample a total of 30 confocal acquisitions were taken, community processes [5]. Metagenomic approaches have and 21 quality images were used to determine the cell been successfully used to resolve the microbial biogeo- abundances using DAIME 1.3 [21]. chemical potential in saline sea water [9, 10], and to re- construct microbial metabolism in AMD. It is found DNA extraction and sequencing that acidophilic bacteria community in AMD has a mi- DNA extraction was done using the PowerSoil® DNA crobial diversity higher than expected [11, 12]. Recently, Isolation Kit (MO BIO Laboratories, Inc.), after cell en- draft genomes acquired through metagenomics have richment using the previously described protocol [17]. A been used to understand the bacterial colonization of total of seven DNA samples of tailings samples were the infant gut [13] and microbial systems in hypersaline subjected to shotgun sequencing. The sequencing was lakes [14]. done at the Institute for Molecular Bioscience, The Uni- In this study, we analyzed metagenomes of alkaline versity of Queensland, using Illumina MiSeq with sulphidic tailings from a tailings storage facility of north- paired-end 300 bp reads. The detailed sequencing west Queensland. Microbial metabolic capabilities for method is described in our previous study [16]. stress resistance and elemental cycling were implied through the annotation of 10 metagenome-assembled Bioinformatics genomes (MAGs) from the tailings metagenomes. The For the purpose of high quality assembly of the meta- results may provide further understanding on biogeo- genome, the seven metagenomes were pooled together chemistry of alkaline sulphidic tailings and valuable in- and analyzed using the flexible pipeline MetaWRAP [22] ference for tailings revegetation practice. for genome-resolved metagenomic data analysis. The as- sembly module was used for sequence assembly. The as- Materials and methods sembly contigs obtained were all longer than 1000 bp. Sampling The workflow of MaxBin2 in MetaWRAP was used for Sampling sites and methods have been described in our contig encapsulation. MetaWRAP provides three assem- previous studies [15, 16]. Briefly, base metal mine tail- bly pipelines, namely Concoct, Metabat2 and Maxbin2, ings were sampled from a storage facility Mt Isa, north- and after comparison Maxbin2 was chose for subsequent west Queensland. The microbial community structure analysis [23]. Then the obtained bins were further ana- based on 16S rRNA gene sequencing related to revegeta- lyzed for species annotation. Species annotation was per- tion has been determined and described elsewhere. The formed in the annotate_bins module, based on the tailings mainly comprise of quartz, dolomite, pyrite, NCBI_nt and NCBI_tax databases. Taxator-tk was used Li and Li Environmental Microbiome (2021) 16:9 Page 3 of 10

for the classification of each contig, and a bin as a whole was estimated to annotate species. CheckM was used for the quality assessment of the assembled bins (MAGs; https://ecogenomics.github.io/CheckM/). For gene anno- − tation, a cutoff E value of 10 6 and an identity of 70% were used. Metawrap was run on a 42-core, 192 GB RAM server. Bins were then used for microbial meta- bolic pathway analysis using the online analysis platform KEGG [24].

Results and discussion The environment The tailings studied are basically neutral/alkaline in pH and of a high level of residue heavy metals, salinity and sulphidic minerals. Salinity and metal release in sulphi- dic tailings is mostly from the coupled weathering of sul- phides and carbonates. The microbially-mediated and chemical-mediated weathering of sulphides would have both occurred in the amended and revegetated tailings [15]. The microbial processes may become as important Fig. 1 A representative FISH image (magnification, × 100) of the as the chemical weathering processes in neutral/alkaline bacterial (green, FITC dyes) and archaeal cells (red, Cy3 dyes) sulphidic tailings, where the redox potential of oxygen detected in the tailing and ferric is reduced by the high pH and ferrous can be oxidized more easily with diverse electron-acceptors (e.g. nitrate) [25]. Moreover, increased nutrients supplied The community genomics from the organic matter amendment and plant roots, as The analysis of 5 Gb of sequenced DNA obtained from indicated by our previous studies, is very likely resulting the tailing samples resulted in 10 genomic bins belong- in increased activities of the sulphide weathering micro- ing to eight genera (Table 1). Five of the 10 MAGs, organisms [6]. Rubrobacter sp., Truepera sp., Thioalkalivibrio sp., Acid- imicrobium sp. and Rhodomicrobium sp., had high com- Cell abundance in the tailings pleteness of > 90% and a contamination rate of 1.35– Cell abundance in the tailings was enumerated using 4.87%. Our previous studies based on 16S rRNA ampli- conventional FISH. We examined samples by FISH that con sequencing showed that these 5 genera on average were prepared as either sterilized raw tailings, the raw accounted for > 12% of the total communities in the tail- tailings or a cell enrichment obtained from the tailings ings sampled. These 5 MAGs were of high quality, which by sucrose density gradient centrifugation. Strong back- were binned with an N50 of 9307–32,314 bp, with the ground fluorescence was observed and direct observa- longest contigs ranging between 51,994–209,767 bp and tion of cell fluorescence was impossible for raw tailings the number of coding sequences ranging between 2963 materials. Background fluorescence is a common prob- and 4168. Five other bins were obtained of a lower qual- lem for cell enumeration in soil-like materials using ity but with a completeness > 50%. These results enabled FISH, and can be strong in metal tailings due to the the metabolic capacities of the communities to be deter- presence of abundant uranium, lead and molybdenum mined and these capacities could be related to the tail- minerals. ings’ bio-weathering. All the microbial genetic features The FISH images taken on tailings samples prepared discussed here were based on the annotation of contigs by cell enrichment were of good quality and suitable for with a length > 8000 bp. Restricting the analyses to large cell enumeration (Fig. 1). The bacterial cell number in contigs would serve to minimize system errors such as the tailings was estimated to be approximately 107 cells/ those caused by sequencing and bioinformatic assembly. g tailings, assuming the cell extraction efficiency to be This metagenomic-based analysis allows a comprehen- 10%. Bacteria made up the major component of the cells sive estimation of the community metabolic networks and Archaea were only scarcely detected. We did not de- [5]. Additionally, the sequencing depth obtained here tect any difference of the cell abundances between the was much greater than that of previous metagenomic tailings with and without revegetation. The bacterial and studies of other tailings communities [26], which in this archaeal cell abundances in these tailings is close to instance enabled good estimations of the community those reported for other base metal tailings [7, 20]. metabolic capabilities. Li and Li Environmental Microbiome (2021) 16:9 Page 4 of 10

Table 1 General properties of the binned population genomes

Bin Closest species Bin size N50 Longest contig Bin GC content Number of Number of Annotated (bp) (bp) completeness (%) CDS RNAs (%) T1 Rubrobacter 4,197,005 32, 20,9767 94 66.1 4168 57 42 xylanophilus 314 T2 Truepera radiovictrix 3,255,015 12, 53,587 90 69.3 3046 35 41 637 T3 Thioalkalivibrio sp. 3,135,801 16, 69,403 91 64.1 2923 44 47 696 T5 2,249,954 7700 41,708 51 65.8 1988 22 15 T10 3,250,127 1063 13,563 78 62.0 2510 41 35 T4 Acidimicrobium 3,035,868 37, 201,471 95 68.8 2983 49 39 ferrooxidans 225 T8 Rhodomicrobium 3,603,927 9307 51,994 94 64.0 3429 59 41 vannielii T9 Desulfuromonas 1,678,559 1063 17,628 63 38.3 1385 32 15 acetoxidans T17 Thiobacillus 3,570,600 1305 8554 50 60.1 2903 44 21 denitrificans T18 Erythrobacter litoralis 4,900,514 848 9004 60 64.3 3638 56 36

Key populations from the near complete MAGs, CO2 fixation, in the form of a minimum set of cbbLSX Rubrobacter sp., Thioalkalivibrio sp. and Thiobacillus [34] (Fig. 2). The findings imply these genera can utilize sp., were identified to have prominent capacities for ac- atmospheric carbon sources, and this may greatly in- quiring energy and stress resistance in the tailing envir- crease their survival ability in the oligotrophic tailings onment (Table 2). Rubrobacter sp. had a wide spectrum environment. of genes for utilizing polysaccharides and aromatic com- pounds, such as the pca [27], sal [28], cat [29] and bpa Key functional genes/pathways related to the [27] operons. This kind of capability would be particu- environment larly important considering tailings are an oligotrophic The ability of microbes to cope with salinity and metal environment [15], and in this instance, the organic (loid) stress in the tailings was evident in the annotated amendment for revegetation in the form of woodchips MAGs. Various genes for osmotic stress resistance were would have caused an increase in content of polysaccha- detected in the MAGs of Rubrobacter sp., Truepera sp., rides and chitin [30]. However, it is worth noting that Thioalkalivibrio sp., and Acidimicrobium sp.. These in- the N content of the tailings and woodchips was very cluded bet and pro systems that code for the choline low, which would have impeded the decomposition of monooxygenase/betaine aldehyde dehydrogenase path- the woodchips in the long run [31]. Our recent studies way [27] and for the L-proline glycine betaine ABC report a very slow decomposition of litter amendment in transport system [28], respectively. Genes coding for re- the tailings. Nonetheless, the utilization of diverse or- sistance to all the metals Zn, Cd, As, Cu and Pb were ganic molecules such as polysaccharides and aromatic detected in all genera. Impressively, multiple copies of compounds may be a vital survival strategy for these mi- czc, encoding for Co-Zn-Cd resistance, were frequently crobes after depleting easily labile and soluble organic detected in one contig (Fig. 3). All the MAGs contained carbon. both the pco and cop systems for Cu resistance, and 6 Thioalkalivibrio sp., Acidimicrobium sp. and Thioba- out of the 8 genera had both the ACR3 and ars systems cillus sp. may have found niches within the tailings by for As resistance (Table 2). The high frequency of these consuming sulphides and respiring on nitrates, as in- systems emphasizes the importance of metal resistance ferred by the presence of sulfur-oxidizing and denitrifi- for microbial survival in the tailings [16]. cation genes in their binned MAGs. Interestingly, Sulphidic metal tailings are an environment where Rubrobacter sp. and Acidimicrobium sp. were found to multiple stresses will occur. This includes those of oxi- have cox operons for CO oxidation, inferring an ability dative damage (such as from free Fe2+ [32]), salinity [29], to respire on CO. CO utilization has been documented moisture content changes [34], and extremely high levels for many microbes residing in saline environments, such of heavy metals [33]. The Fenton reaction of intracellular as in seawater [32] and saline soils [33]. In addition, four Fe2+ by superoxide is fatal to cells [32]. We detected out of the eight genera were found to contain genes for genes for ferroxidase (Fig. 4) and superoxide dismutases Table 2 Genetic features detected in the binned genomes from the Mt Isa mine tailings Li and Li Features Rubrobacter Truepera Thioalkalivibrio Acidimicrobium Rhodomicrobium Desulfuromonas Thiobacillus Erythrobacter xylanophilus radiovictrix sp. ferrooxidans vannielii acetoxidans denitrificans litoralis niomna Microbiome Environmental Carbon cbb √√ √ √ metabolism cox √√√ pca √ sal √ √ cat √ √ FAHF √√√√

bph √√√ (2021)16:9 malE √√√ Sulfur sox √√ √ metabolism Blr3520 √ √ dsr √√ √ Nitrogen nos √ √ metabolism nor √ √ nar √√√ √√ nir √√√ √ nap nas √√ √ √ nrf NaT √√ √ √ NiT √ √√√ nif √ urt √ Metal resistance czc √√√√√ √√ cus √√ √ √ √ cop √√√√√ √ √√ pco √√√√√ √ √√ mer √√√√√ ACR3 √√ √ √√ ars √√√ √ √√ ae5o 10 of 5 Page Iron acquisition pit √√√√ √ SDP √√√ efu √ Table 2 Genetic features detected in the binned genomes from the Mt Isa mine tailings (Continued) Li and Li Features Rubrobacter Truepera Thioalkalivibrio Acidimicrobium Rhodomicrobium Desulfuromonas Thiobacillus Erythrobacter xylanophilus radiovictrix sp. ferrooxidans vannielii acetoxidans denitrificans litoralis niomna Microbiome Environmental Oxidative stress GT √√ √ √√ Glo √√√√√ √√ SAM √√ Fr √√√√√ √ √√ NO √√ √ SOD √√√ √ √ √√

Osmotic stress mdo √√ √√ (2021)16:9 bet √√√√ √ opu √√ SOX √√ pro √√√√ √ Mobility msh √ √ flg/fli √√√√√ mre √√√√√ che √√ √ √ Notes: cbb RuBisCo genes for carbon fixation, cox carbon monoxide dehydrogenase genes, pca Protocatechuate pathway for aromatic compound catabolism, sal Salicylate pathway for aromatic compound catabolism, cat Catechol pathway for aromatic compound catabolism, FAHF Gentisate pathway for aromatic compound catabolism, bph Biphenyl pathway for aromatic compound catabolism, malE Maltose ABC transporter for polysaccharides metabolism, sox Sulfur oxidation genes, Blr3520 Conserved hypothetical genes for sulfur oxidation, dsr dissimilatory sulfite reductase genes, nos Nitrous oxide reductase genes, nor Nitric oxide reductase genes, nar Respiratory nitrate reductase genes, nir Nitrite reductase genes, nap periplasmic nitrate reductase genes, nas assimilatory nitrate reductase genes, nrf Cytochrome c nitrite reductase genes, NaT Nitrate ABC transporter genes, NiT Nitrite ABC transporter genes, urt Urea ABC transporter, czc Cobalt-zinc-cadmium resistance genes, cus Cation efflux genes, cop Copper resistance genes, pco Copper multicopper resistance genes, mer Mercury resistance genes, ACR3 Arsenic resistance genes, ars Arsenic resistance genes, pit Ferric ion transporter genes, SDP High affinity iron transporter siderophore genes, efu Ferrous ion transporter genes, GT Glutathione S-transferase genes for oxidative resistance, Glo Methylglyoxal detoxification genes, SAM SAM-dependent mehtyltransferase genes, Fr Ferroxidase genes, NO Oxidative stress response genes, SOD Superoxide dismutase genes, mdo Glucans biosynthesis genes, bet Choline pathway for osmotic stress response, opu Choline transporter genes, SOX Sarcpsome oxidase genes, pro L-proline glycine betaine transporter genes, msh mannose-sensitive haemagglutinin (MSHA)-like pilus genes for cell attachment, flg/fli flagellar genes, mre rod-shape determining genes, che chemotaxi genes ae6o 10 of 6 Page Li and Li Environmental Microbiome (2021) 16:9 Page 7 of 10

Fig. 2 A Neighbor-Joining tree showing the phylogenetics of the genes for ribulose bisphosphate carboxylase large chain of carbon fixation recovered from the binned genomes in this study. The tree was constructed using MEGA 6.06 based on the sequence alignment using the CLUSTAL method with default parameters

Fig. 3 Gene clusters for metal resistance found in the binned genomes from the Mt Isa metal mine tailings. HMT, hypothetical metal transporter gene; czc, cadmium-zinc-cobalt resistance gene; H, hypothetical gene; GLU, ferredoxin-dependent glutamate synthase; ccdA, antitoxin gene; ars, arsenic resistance genes; mer, mercury resistance genes; MDM, hypothetical metal-related genes; tetR, tetracycline repressor gene; dsb, thiol:disulfide interchange genes; cad, cadmium resistance genes; cop, copper resistance genes; chr, chromium resistance genes. More details can be found in the notes of Table 2 Li and Li Environmental Microbiome (2021) 16:9 Page 8 of 10

Fig. 4 A Neighbor-Joining tree showing the phylogenetics of the ferroxidase genes recovered from the binned genomes in this study. The tree was constructed using MEGA 6.06 based on the sequence alignment using the CLUSTAL method with default parameters as well as genes for Fe uptake systems (e.g. Fur, pit)in of the microbial consortia, which may be vital for the almost all the tailings MAGs. Likely, these genes are survival of the community via inter-species cooperation providing these microorganisms with defenses against (Fig. 5). Two microbial pathways for sulphide oxidation, oxidative stress. Other protection mechanisms like the direct contact pathway and indirect pathway [37, methylglyoxal detoxification genes [35] and glutaredoxin 38], may exist in the tailings (Fig. 5). Thioalkalivibrio sp., genes [36] were also detected in most of the MAGs. Acidimicrobium sp. and Thiobacillus sp. are well known sulfur oxidizers able to respire on sulphides [39, 40], and Sulphide oxidation and salinity generation in this study dsr (organized as dsrABCKJOPR) and sox KEGG annotation successfully reconstructed the meta- (organized as soxXYZABH) genes for sulphur oxidation bolic capacity of sulphide oxidation and nitrogen cycling were found in these three MAGs from the tailings.

Fig. 5 A conceptual diagram depicting the microbial mediated sulphide oxidation resulting salinity and N depletion in the tailings. DMO, direct microbial oxidation; IO, inorganic oxidation; IMO, indirect microbial oxidation Li and Li Environmental Microbiome (2021) 16:9 Page 9 of 10

Thioalkalivibrio sp. and Thiobacillus sp. were both metagenomic data. All authors contributed to the interpretation of the found to harbor novel conserved genes (Blr3520 genes) results and revision of the manuscript. The author(s) read and approved the final manuscript. annotated for sulfur oxidation as well. In addition, msh operons encoding mannose-sensitive haemagglutinin Funding (MSHA)-like pilus for cell attachment to substrates [41] This project is financially supported by the National Natural Science were detected in the Thioalkalivibrio sp. genome. This in- Foundation of China (No. 41877414), UQ Postdoctoral Fund (ID UQ605715), dicated the ability of Thioalkalivibrio sp.toattachtopyr- and Mount Isa Mines Ltd. (formerly Xstrata Queensland Ltd.).. ite surfaces for oxidation activities. Microbes can colonize Declarations and etch pyrite surfaces directly and oxidize the released sulfur, which was recently observed during ferric- Competing interests catalyzed pyrite oxidation [38]. The features detected in The authors declare no competing interests. the abovementioned genomes coincide with this model. It Author details is thus suggested that in the oxic layer of the tailings 1Hebei Key Laboratory of Soil Ecology, Key Laboratory for Agricultural Water Thioalkalivibrio sp., Acidimicrobium sp. and Thiobacillus Resource, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang sp. may colonize and oxidize sulphides particles directly, 050021, China. 2University of Chinese Academy of Sciences, Beijing 100049, causing mineral dissolution and contributing to the ele- China. vated levels of salinity and metals in the pore water. 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