Microbiome of highly polluted coal mine drainage from Onyeama, Nigeria, and its potential for sequestrating toxic heavy metals

Geochemistry and ecotoxicology of AMD

AMD systems are an important source of metal/metalloid pollution to the receiving hydrosphere with devastating consequences on the biological drivers of affected ecosystems. Environmental menaces of AMD have not been exhaustively reported worldwide. Scanty information exists across Africa and many developing economies. The homogenised mixture of detached biofilm and AMD samples from a derelict coal mine at three sampling periods were assayed for geochemical delineation and analysed for pollution intensity against reference background geochemical values. The measured values of the physical properties and contents of selected HMs in drains from a coal mine in Nigeria were as presented in Supplementary Table A.1. Virtually all the measured parameters exceeded the permissible limits of WHO guidelines for potable water. The AMD water was acidic (pH = 3.1 ± 0.265), and contained characteristic anions that are common to AMD including dissolved sulphides (1.37 ± 0.233 mg l⁻¹), sulphates (313.0 ± 15.9 mg l⁻¹), carbonate (253.0 ± 22.4 mg l⁻¹) and nitrate (86.6 ± 41.0 mg l⁻¹) above the allowable limits of WHO. Although the acidic pH of AMD in the present study compares well with those associated with mines in Russia¹⁴, more extreme acidic pH values have been reported in other climes. Negative pH values of − 1.56 and − 3.6 were observed in AMD from Iberian Pyrite Belt²⁰ and Richmond Mine at Iron Mountain, USA²¹, respectively. The values of physicochemical parameters associated with the AMD from Onyeama were similar to data reported for other mine wastewaters in Nigeria²² and elsewhere⁴. It is known that sulphide minerals, in presence of water and oxygen, oxidise to sulphate as observed in the elevated sulphate concentration (313 ± 15.9 mg l⁻¹) in the present study. The low pH observed in the AMD is due to the formation of sulphuric acid from sulphate in presence of protons (H⁺). This consequently causes the leaching of metal/metalloid ions into the drains. The concentrations of dissolved organic matter in AMD tends to be relatively low (< 20 mg l⁻¹)²⁰, but the total organic carbon of the AMD samples from the Onyeama coal mine was 25.7 ± 5.96 mg l⁻¹ signifying oligotrophic conditions. Moreover, carbonate (253 ± 22.4 mg l⁻¹) in the AMD indicated alkali stemming of acidic pH from the characteristic < 2 associated with freshly formed AMD to > 3.0 as presently observed.

A comprehensive assessment of HMs is pivotal to evaluating the potential of AMD to mitigate the degree of pollution in receiving environments. The contents of toxic metals and metalloid measured from the AMD water sample were extremely high, ranging from the Cr content (3.87 ± 3.87 mg l⁻¹) to that of Pb (326.0 ± 26.8 mg l⁻¹) (Supplementary Table A.1). Concentrations of Pb in AMD recorded in this study was higher than 12 mg l⁻¹ associated with Iron Mountains’ AMD in the United States²¹ and 30 mg l⁻¹ in Sao Domingo mine’s AMD in Portugal²³. Other metals contained in the AMD include Cd (95.0 ± 5.12 mg l⁻¹), Co (27.3 ± 9.25 mg l⁻¹), Ni (28.8 ± 13.4 mg l⁻¹), As (56.7 ± 14.7 mg l⁻¹), and Fe (39.7 ± 22.3 mg l⁻¹). All the metals/metalloids were apparently at toxic concentrations when compared with the WHO permissible limit and the values obtained from the unpolluted surface water located several kilometres away from the mine. Pb and Cd that connoted the highest concentrated HMs in the AMD have no metabolic importance other than rendering havoc to biota^8,24 in ecosystems the AMD emptied. These non-metabolic HMs exacerbate ecophysiologies of the receiving milieus with anticipated degrees of public health. Such health consequences include mutagenicity, genotoxicity, neurotoxicity etc⁵. This was reportedly the case with surface waters juxtaposing with AMD from the Onyeama coal mine reportedly enriched with TOC and toxic concentrations of HMs²⁵. Similarly, AMDs have reportedly remained one of the major point sources for anthropogenic HMs pollution of waters globally^2,11,12.

The awful impact of metals/metalloid poised AMD on the receiving water quality is better modelled via integrating multivariate data into pollution indexes and the functionalities of the ecosystems (Table 1). The added HMs in the AMD from coal mine as determined by contamination factor (CF) was at least 397 (± 223) factors for Fe and up to 2.97 (± 0.16) × 10⁶ factors for Cd (Supplementary Table A.2). It implies an inordinate tendency of the AMD to contaminate water bodies. The added HMs was in the order: Cd > Co > Pb > As > Ni > Cr > Fe (Table 1). Enrichment of five HMs was exceptionally high (Cd > Co > Pb > As > Ni), while Cr and Fe were very high and moderately enriched the AMD water, respectively. The astronomically high contamination and enrichment factors of the AMD signified the enrichment potentials the AMD portends on receiving surface waters. The AMD from the Onyeama coal mine has been reportedly impacting the water qualities of rivers within the location²⁵. It is assumed that the extremely high concentrations of toxic metals/metalloids in the AMD dilutes out upon discharges into nearby rivers, contaminating the surface water and raising the bioavailable metals/metalloids beyond safe thresholds. Further reports of toxic metals/metalloids enrichment of surface waters via inflow of AMDs from other mines in Nigeria²⁶ and other climes^3,4,27 are worrisome and oblige mitigations.

The HMs-enriched environments inadvertently exert ecotoxicity unto the drivers of the ecosystems. The level of HMs accumulation to the organic matter in the AMD, through geo-accumulation (I_geo) index of Fe (7.60 ± 0.779) to Cd (20.9 ± 0.075) (Supplementary Table A.2), was very severe and in a similar order to CF. It possibly implies organic matter in the AMD harbours the mobile toxic metal/metalloid concentrations and make them available to the food web²⁸. Thus, biomagnification of the toxic metals/metalloids along the trophic level becomes palpable and a challenge to the biota of any surface water receiving the AMD and to public health^21,28. Ecological risk assessments define and categorise the pollution status of ecosystems with the HMs contained in the AMD. Based on the potential ecological risk factor (Er), Cd exerted an extremely high-risk index (36.3 ± 1.96 × 10⁶), and none of the metals/metalloids exercised less than 1000 risk index (Supplementary Table A.2). All the HMs/metalloid contained in the AMD posed very high ecological risks and could be categorised in the order of Cd > Co > Pb > As > Ni > Cr > Fe. The modified potential ecological risk factor (MEr), however, stipulated that five HMs posed a very high risk in the order: Cd > Co > Pb > As > Ni, whereas Cr and Fe were determined to be of considerate and low risks, respectively. The HMs exerted high risk to the AMD ecosystem as calculated by ecological risk quotient (RQ) in the order: Pb > Cd > As > Ni > Co > Fe > Cr. The ecological risk index of all the HMs as a whole was very high (375,000 ± 22,400) index as stipulated by the modified potential ecological risk index (Table 1). The prodigiously high ecological risks indexes of the HMs/metalloid in the AMD indicated grave danger the AMD would portend on the surface- and ground-waters.

Microbial community structure of AMD from Onyeama coal mine

A total of 26,160 and 40,403 valid (filtered) sequence reads were obtained for bacteria and eukarya, respectively, after a quality check of biofilm-water amplicon sequence data. The valid sequences were clustered into 2036 and 1002 operational taxonomic units (OTUs) of bacteria and eukarya domains of life, respectively, as presented in Table 2. Microbial community structures are sensitive descriptors of ecological stressors pivotal to understanding ecosystem functions²⁹. The number of clustered high quality, non-chimeric sequences as OTUs based on UCLUST and CD-HIT against the sequence reads was depicted as asymptotic rarefaction curves (Supplementary Fig. A.1). The curves revealed that higher numbers of OTUs were delineated from valid sequence reads of 16S rRNA genes, unlike the lesser number of OTUs obtained from valid sequence reads of ITS2 region located between 5.8S and 28S rRNA genes of eukaryotes. The OTU richness observed in the rarefaction curves established coverage of the majority of species and was further validated with the richness and diversity estimations presented in Table 2. Despite the higher number of valid sequence reads obtained from the amplified ITS2 (40,403) than that of 16S rRNA genes (26,160), the observed OTUs were more in 16S rRNA genes (2036) than those of ITS2 (1002). More than 99.8% and about 98.5% of the microbial community in AMD from the Onyeama coal mine represented eukarya and bacteria OTUs, respectively, based on estimated Good’s library coverage. The coverage degree of the MiSeq sequencing corroborated the rarefaction curves. Furthermore, the estimated OTU richness (based on higher values obtained from ACE, Chao1 and JackKnife indexes) showed that bacterial phylotypes were richer than those of eukarya. Alpha diversity indexes (NPShannon, Shannon, and inverse Simpson) phylogenetic diversity index revealed that bacteria in the AMD were more diverse than eukarya OTUs.

Taxonomy and phylogeny of microbial OTUs in AMD from coal mine

The taxonomic composition and relative abundances of the AMD microbiome, as shown in Fig. 1, revealed that the bacterial community spanned 10 phyla whose sequence reads were at least 1% (Fig. 1a). Whereas the eukarya domain of life (with sequence reads ≥ 1%) found in the AMD include Fungi, Plantae and Animalia kingdoms (Fig. 1b). Ascomycota, unclassified Fungi phylum (Fungi_p), Basidiomycota, and Mucoromycota represented Fungi kingdom, while Ciliophora and Arthropoda phyla were Animalia and Chlorophyta phylum epitomised Plantae kingdom. Association of the domain Eukarya (comprising Alveolates, Chlorophyta and Fungi as observed in this study) with AMD is reported to a lesser extent when compared with Bacteria³⁰. The Fungi, largely represented by Ascomycota and Basidiomycota, are primarily found in sub-surface low-pH biofilms thriving in AMD³¹. While the Alveolates are suggested to have acted as primary/secondary consumers, the amoebae were secondary grazers in the AMD ecosystem^29,32. Fungi taxa must have participated in carbon cycling as the main decomposers in the microbial community of the AMD. The taxonomic composition and relative abundance of phyla regarded as ‘Others’ (sequence reads < 1%) were presented in Supplementary Table A.3.

Among the phyla dominating the Bacteria domain of life in the Onyeama AMD were highly diverse classes that are known with AMDs. Low abundance Firmicutes and Actinobacteria lineages have also been previously characterized in AMD amplicon sequence data sets^6,10). Previous studies revealed that oligotrophic Leptospirillum and archaea dominated the early stage of biofilm development in AMD³³. Acidophilic copiotrophic heterotrophs comprising surprisingly wide diversity (physiology and phylogeny) with prevailing metabolic traits are known to succeed the early colonisers³⁴. Interestingly, families in the Class Bacteroidia, including Porphyromonadaceae (12.4%), Prolixibacteraceae (1.6%), and unclassified GU454901 (1.2%), were dominant in the coal AMD as previously reported¹⁰. In Eukarya, dominant classes in the kingdom Fungi spread among Ascomycota (Eurotiomycetes, 33.8%; unclassified Ascomycota class, 18.1%; Dothideomycetes, 3.7%; Sordariomycetes, 3.3%; and Saccharomycetes, 1.3%), unclassified Fungi (4.0%), Mucoromycota (Umbelopsidomycetes, 1.2%), and Basidiomycota (Tremellomycetes, 1.2%, and Agaricomycetes, 1.0%). Dominant Animalia in the AMD comprised sub-kingdom Alveolata and Metazoa represented by unclassified Ciliophora (12.6%) and unclassified Arthropoda_c (8.4%), respectively. Metabolic CO₂ from protozoan respiration is assumed to further increase the level of dissolved inorganic carbon contributing to carbonate concentration that curtailed acidic pH in the microenvironment³². Unclassified Chlorophyta class (5.5%) was the only taxonomic class of Phylum Chlorophyta belonging to kingdom Plantae that formed part of dominant Eukarya in the AMD.

The phylogeny, based on evolutionary history, of bacterial OTUs, was deduced via the Neighbor-Joining method as an unrooted phylogenetic tree (Fig. 2). The bacterial OTUs have a relative abundance ≥ 1% of the total valid sequence reads. The dominant bacterial OTUs aligned into three clades was based on monophyletic and polyphyletic grouping. The OTUs have not been reported as dominating bacteria communities in AMD biofilm development other than transforming AMD³⁵. The relative abundance of the bacterial OTUs is presented in Supplementary Table A.4. Species of Paludibacter are acidophilic and have been associated with the reduction of sulphate and Fe³⁺ in an AMD-impacted site³⁵. Furthermore, the Rubrivivax gelatinosus group use NiFe hydrogenase to stem H⁺ to hydrogen. Whereas, Novosphingobium flavum group degrade coal hydrocarbons and generate hydrogen using formate dehydrogenase enzyme³⁶. Moreover, Thauera selenatis is known for using selenate or other metals as the preferred electron acceptor for respiration. Dechloromonas species are famous for their denitrifying role in the extreme ecosystem³⁷. Nevertheless, the dominant eukaryotic OTUs (sequence reads ≥ 0.5%) spread across Fungi (7 OTUs), Animalia (3 OTUs) and Plantae (3 OTUs) as presented along with their corresponding counts and ratio (see Supplementary Table A.5). The evolutionary relatedness of representative sequences (based on the abundance) of the OTUs was calculated and delineated as unrooted phylogenetic trees (Fig. 3). It is important to note that majority of the dominant OTUs delineated as Eukarya in the AMD were unclassified, and their role in carbon fluxes cannot be ascertained for now.

Sequestration of toxic HMs/metalloid from simulated AMD and actual AMD from coal mine

Fortification of rich or semi-rich culture broth with toxicants of interest is a common approach to selecting competent microorganisms³⁸. After culture enrichment, 26,373 valid reads from non-chimeric sequences were clustered into 95 OTUs of bacteria. Alpha diversity of the enriched culture revealed low species richness based on estimated values via ACE, Chao1 and JackKnife (Table 2). Estimated diversity further depicted poorly diverse bacteria species in enrichment culture via phylogenetic diversity valued at just 182. This also corroborated the estimates of other diversity indexes (NPShannon, Shannon, and inverse Simpson) presented in Table 2. Culture enrichment was biased towards a few OTUs that the culture conditions supported. On the contrary, a richer and more diverse community was observed in the coal AMD as depicted in the alpha diversity indexes. After clustering the valid sequence reads of 16S rRNA gene sequencing from clone library into OTUs, the consortium of bacteria containing 7 dominant groups of OTUs was observed to be involved in toxic metal sequestration of AMD. The taxonomy and counts at inoculation and post-incubation comprised two taxonomic classes of bacteria (Supplementary Table A.6), whose evolutionary relatedness was depicted as an unrooted phylogenetic tree (Fig. 4). The classes with their OTUs include ϒ-Proteobacteria (Acinetobacter pittii group, Enterobacteriaceae group, unclassified FWNZ species, and Pseudomonas citronellolis group), and Bacilli (Sporosarcina koreensis group, Bacillus cereus group, and Exiguobacterium aurantiacum group). The bacteria (particularly Acinetobacter pittii, Pseudomonas citronellolis, and Bacillus cereus) have been involved in the degradation of indole, and a heterocyclic aromatic compound found in coal, via an attack on either/or both the carbocyclic and N-heterocyclic rings³⁹. Studies involving Acinetobacter⁴⁰, Enterobacteriaceae¹⁷, Pseudomonas⁴¹, Sporosarcina^7,42, Bacillus⁴², and Exiguobacterium⁴⁰ OTUs for sequestration of toxic metals/metalloids in environmental media have been reported. Bioaccumulation of Cd, Co, and Zn was reported for Sporosarcina sp. G3 as sequestration strategy, whereas Cr and Hg were reduced via redox-active enzymatic activities to innocuous forms⁴³.

Urease-producing bacteria instigate insoluble metal-carbonate micro-precipitation through urease activity¹⁶. The growth-time courses and urease activities of the bacteria consortium in simulated AMD were presented as curves (Fig. 5). It was observed that the impact of high concentrations of HMs cocktails was not pronounced beyond the early 6 h post-inoculation, which was regarded as the lag phase. The bacteria consortium might have activated necessary genes needed to tolerate and sequester the metals/metalloids toxicity during the lag phase without cell multiplications. Afterwards, the bacteria consortium grew steadily with the production of urease, based on increasing measurement of urease activity, as incubation continued. At 30 h post-inoculation, 245.3 (± 23.7) U ml⁻¹ activity of urease was observed in broth without a toxic metal cocktail. However, more urease activity (255 ± 7.6 U ml⁻¹) by the bacteria consortium was observed in medium amended with low concentrations of metal cocktails unlike lesser activities of 235 (± 7.6) U ml⁻¹ and 193.7 (± 10.7) U ml⁻¹ associated with medium and high metal concentrations, respectively. As the growth remains stationary and pH further increased to > 8.2, urease activities were at least 253 U ml⁻¹ in all the cultures. Although urease activities at acidic pH have been reported in acid-tolerant human pathogens¹⁹, the findings in this report were assumedly the first amongst bacterial strains from AMD-polluted environments. The urease activities at acidic pH compared favourably with activities at alkaline pH in previous studies^7,16,42,44. Moreover, the pH of the culture system kept increasing, alleviating the acidity condition that initially prevailed in the AMD system.

Interestingly, urease activity was observed in low quantity at acidic pH, unlike higher activity when the pH inclined towards alkaline (Fig. 5). It is proposed that urea finds its way into Onyeama coal mine drains through runoff from agricultural soils fortified with urea fertilizers and animal manures, which are common agricultural practices in Nigeria. The products of urea hydrolysis might have equilibrated in water to form bicarbonate, ammonium and hydroxyl ions that serially increased the culture pH. Ultimately, the bicarbonate equilibrium might have shifted to form carbonate ions (HCO₃⁻ + H⁺ + 2NH₄⁺ + 2OH⁻ ↔ CO₃²⁻ + NH₄⁺  + 2H₂O) that enhanced the metal-carbonate micro-precipitation (Me²⁺  + Cell → Cell-Me²⁺ + CO₃²⁻ → Cell-MeCO₃). The gradual increase in pH could have further indulged the formation of CO₃²⁻ from HCO₃⁻, leading to metal-CO₃ precipitation around cells and in culture media. Bicarbonates enrichment with inherent ammonia production was thought to have provided additional acid neutralization of the AMD. The growth kinetics after the presumed lag phase in the early 6 h to late exponential phase at 18 h showed that a low concentration of HMs cocktails did not have an impact on the growth of the bacteria consortium. Consequently, the bacteria consortium exhibited excellent sequestration of multi-component toxic HMs in both the simulated toxic metal-rich AMD and the actual AMD obtained from the Onyeama coal mine (Table 3).

The bacteria consortium displayed more than 94% efficiency of Cd and Pb sequestration in natural AMD, while 100% efficiency was observed in all the simulated AMD treatments (Table 3). Low performance was found with Ni and As, but not less than 70% sequestration efficiency was observed in all treatments. Efficient sequestrations of toxic metals, up to 100% removal efficiency of most toxic metals, observed with the bacteria consortium were similar to findings in a previous study¹³. Mixed-bacterial cultures are known to be able to perform more complex tasks and survive in more unstable environments than a monoculture. Nevertheless, 89.3–98% removal efficiencies of Ni, Pb, Co, and Cd from solution have been reportedly achievable with urease-producing Sporosarcina koreensis⁴⁵. Similarly, Bacillus sp. KK1 reportedly mitigated lead-contaminated mines tailings containing mobile Pb (1050 mg kg⁻¹) to form insoluble precipitates of PbS and PbSiO₃³⁴. Growth-dependent sequestration of HMs cocktails by the bacteria consortium was adduced to be via precipitation. The weight of the precipitates was evaluated to be proportional to concentrations of HMs cocktail present. The bacteria consortium was observed to drive the formation of as much as 15.6 (± 0.92) mg ml⁻¹ precipitates (Table 3) that were assumed to be in form of HMs-carbonates in TGYM supplemented with high concentrations of HMs cocktail within 24 h post-inoculation. In natural AMD bio-stimulated with urea and seeded with bacteria consortium for 24 h, 10.5 (± 0.52) mg ml⁻¹ HMs precipitates was observed unlike 8.57 (± 2.52) mg ml⁻¹ precipitates obtained from natural AMD toxic metals sequestration without urea fortification. It appeared that the quantity of toxic metal precipitate was proportional to quantities of available toxic metals, which corresponded to the number of heterogeneous nucleation sites on the surface of the bacterial cells. Omoregie et al.⁴² reported a relatively similar quantum of precipitation as CaCO₃ with species of ureolytic Firmicutes isolated from limestone caves. As such, there was no correlation between urease activity and quantum of toxic metal precipitation since there is a likelihood that other metabolic activities may be linked to urease activities. Nevertheless, the bioremediation strategies demonstrated in the present study exhibited excellent toxic metal sequestrations unlike insignificant (p > 0.05) natural attenuation process of the autochthonous community without augmentation with bacteria consortium and stimulation with nutrients (as presented in Table 3).

In conclusion, AMD from the Onyeama coal mine is a point source of pollution to the surrounding environments because of its richness in anions and toxic metals/metalloids. It has a high potential of enriching the receiving hydrosphere with toxic metals/metalloids and exerts severe ecological risks (Er > 320) with Cd and Pb wielding a huge critical risk index (38.1 ± 2.18 × 106) on the biological elements of the ecosystems. The dominance of Proteobacteria (50.8%), Bacteroidetes (18.9%), Ascomycota (60.8%), and Ciliophora (12.6%) characterised the microbial community of the AMD, where unclassified OTUs occurred mostly among the species. Enrichment of the AMDs skewed the bacterial community as depicted in the alpha diversity indexes against that of coal AMD leading to the selection of bacteria consortium with an excellent potential of stemming the toxicants in the AMD. The bacteria consortium efficiently removed toxic metals/metalloids (> 70%) through precipitation and simultaneously neutralised AMD acidity. The bacteria consortium exhibited appreciable urease activity (> 190 U ml⁻¹), through which the precipitation was assumed possible via the formation of metal/metalloid-carbonates. The bacteria consortium is suggested to be a sustainable biotechnological candidate in designing a bioremediation strategy for decommissioning AMD before discharge into the surrounding environment.

Source: Ecology - nature.com