Microbial diversity at the moderate acidic stage in three different sulfidic mine tailings dumps generating acid mine drainage

In freshly deposited sulfidic mine tailings the pH is alkaline or circumneutral. Due to pyrite or pyrrhotite oxidation the pH is dropping over time to pH values <3 at which acidophilic iron- and sulfur-oxidizing prokaryotes prevail and accelerate the oxidation processes, well described for several mine waste sites. The microbial communities at the moderate acidic stage in mine tailings are only scarcely studied. Here we investigated the microbial diversity via 16S rRNA gene sequence analysis in eight samples (pH range 3.2–6.5) from three different sulfidic mine tailings dumps in Botswana, Germany and Sweden. In total 701 partial 16S rRNA gene sequences revealed a divergent microbial community between the three sites and at different tailings depths. Proteobacteria and Firmicutes were overall the most abundant phyla in the clone libraries. Acidobacteria, Actinobacteria, Bacteroidetes, and Nitrospira occurred less frequently. The found microbial communities were completely different to microbial communities in tailings at <pH 3 described in the literature.     

Microbial diversity of mine water at Zhong Tiaoshan copper mine, China

Microbial diversity of mine water at Zhong Tiaoshan copper mine, Shanxi province, China, was analyzed using a culture-independent 16S rRNA gene (rDNA) based on cloning approach. A total of 59 Operational Taxonomic Units (OTUs) were obtained from 226 clones from all three samples (8 OTUs from sample SX1, 25 from SX2 and 26 from SX3). 46 of them were representative OTUs and were sequenced. 93.5% of the total clones had sequences that were less than 5% difference from those in the nucleic acids database. The percentage of overlapping OTUs among samples was from 12.1% to 35.3%. Phylogenetic analysis indicated that 60.62% of the clones were affiliated with members of the Proteobacteria (alpha -3.10%, beta -24.78%, gamma -31.41%, delta -1.33%), whereas 29.20% of the clones were closely related to the Nitrospira (Leptospirillum ferrooxidans 20.80%, Leptospirillum ferriphilum 0.88% and Leptospirillum group III 7.52%, respectively). The rest clones were affiliated with the Firmicutes (2.65%) and the Bacteroidetes (7.52%). The results of Principal Component Analysis (PCA) based on the percentages of OTUs and biogeochemical data revealed that biogeochemical properties affected the diversity of microbial communities in mine water. Especially, the pH value, temperature and different concentrations of elements such as lead, zinc, sulfur, iron and copper seemed to be key factors affecting the composition and structure of microbial communities in this study.     

Archaeal diversity in acid mine drainage from Dabaoshan Mine, China

Three acid mine drainage (AMD) samples collected from Dabaoshan Mine (Guangdong Province, China) were studied. In addition to physicochemical analyses, the diversity and community structures of the archaeal communities in these samples were described at the genetic level by amplified ribosomal DNA restriction analysis (ARDRA). Nine different ARDRA patterns were obtained from 146 clones and were studied as operational taxonomic units (OTUs), which were re-amplified and sequenced. Sequence data and phylogenetic analysis showed that most of the clones belonged to the Thermoplasmatales, and that archaea belonging to the Sulfolobales were absent. Only 1 OTU attributed to Ferroplasma was found and was observed to be abundant in all 3 samples. Eight OTUs were related to 2 new undefined groups in the Thermoplasmatales. Of the 8 OTUs, the clones in 2 similar units were isolated from samples collected from an abandoned sulfide mine (Huelva, Spain) and those in 5 similar units were isolated from samples collected from a closed copper mine (Tonglushan, China). These diversities were characterized by the reciprocal of Simpson's index (1/D) and correlated with the concentrations of ferrous ions and toxic ions in the AMD samples. The high temperature of the sampling sites was one of the factors that could explain why archaea belonging to the Thermoplasmatales were abundant in the analyzed AMD samples while those belonging to the Sulfolobales were absent.     

Biosorption of metal and salt tolerant microbial isolates from a former uranium mining area. Their impact on changes in rare earth element patterns in acid mine drainage

The concentration of metals in microbial habitats influenced by mining operations can reach enormous values. Worldwide, much emphasis is placed on the research of resistance and biosorptive capacities of microorganisms suitable for bioremediation purposes. Using a collection of isolates from a former uranium mining area in Eastern Thuringia, Germany, this study presents three Gram-positive bacterial strains with distinct metal tolerances. These strains were identified as members of the genera Bacillus, Micrococcus and Streptomyces. Acid mine drainage (AMD) originating from the same mining area is characterized by high metal concentrations of a broad range of elements and a very low pH. AMD was analyzed and used as incubation solution. The sorption of rare earth elements (REE), aluminum, cobalt, copper, manganese, nickel, strontium, and uranium through selected strains was studied during a time course of four weeks. Biosorption was investigated after one hour, one week and four weeks by analyzing the concentrations of metals in supernatant and biomass. Additionally, dead biomass was investigated after four weeks of incubation. The maximum of metal removal was reached after one week. Up to 80% of both Al and Cu, and more than 60% of U was shown to be removed from the solution. High concentrations of metals could be bound to the biomass, as for example 2.2 mg/g U. The strains could survive four weeks of incubation. Distinct and different patterns of rare earth elements of the inoculated and non-inoculated AMD water were observed. Changes in REE patterns hint at different binding types of heavy metals regarding incubation time and metabolic activity of the cells.     

Isolation and identification of a Candida digboiensis strain from an extreme acid mine drainage of the Lignite Mine,Gujarat

An extremely acidic mine drainage (AMD) water sample was collected in 1998 and 2008 from Panandhro lignite mine, Gujarat, India. The yeast isolated from this sample was identified using mini API identification system, as a member of genus Candida. The major cellular fatty acids detected by FAME from the isolate are C_16:0 and C_{18:2 cis 9,12}/C_18:0α as 25.23 and 19.5%, respectively. The isolate was identified as Candida digboiensis by 18S rRNA gene sequence analysis and designated as Candida digboiensis SRDyeast1. Phylogenetic analysis using D1/D2 variable domains showed that the closest relative of this strain is Candida blankii with 3% divergence. This organism has been reported for the first time from the lignite mine AMD sample, and for cellular fatty acid analysis. This yeast is able to survive in the AMD sample preserved at 10–42 °C temperature since last 10 years along with iron oxidizing microorganisms. It can grow in the presence of 40% glucose, 10% NaCl and in the pH range of 1 to 10. The isolate is capable of producing enzymes like protease and lipase. This isolate differs from the type strain Candida digboiensis in as many as six physiological and metabolic characteristics. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

First insights to the microbial communities in the plant process water of the multi-metal Kevitsa mine

Metallurgical processes demand large quantities of water. However, in many locations, water is becoming scarce and process water recycling is needed. Closing water loops can be challenging due to build-up of flotation chemicals, metal ions and microorganisms in the recycled water affecting the flotation performance. Here, we have characterized the microbial communities over a 2-month period in different locations of the multi-metal Kevitsa mine in Northern Finland, by microbiome sequencing, enumeration of bacteria, archaea and fungi by qPCR, and cultivation. The microbial communities showed high diversity, but were dominated by Alpha- and Gammaproteobacteria. In addition, various fungal taxa were detected, whereas the archaeal taxa were only sparsely detected from the sequence data. The number of bacterial 16S rRNA gene copies in Process water and Ni thickener overflow varied between 0.5–3.3 × 10⁵ mL⁻¹, whereas the Flotation tailings showed two orders of magnitude lower amounts. Fungi were present at 3.0 × 10²–8.1 × 10⁴ 5.8S rRNA gene copies mL⁻¹ in all samples, while the number of archaea fluctuated between 8.8 × 10¹–3.2 × 10⁵ 16S rRNA gene copies mL⁻¹. The number of all microbial groups were generally lower in September than in August. When tested on 8 different cultivation media, the microorganisms generally responded positively to organic carbon, and were also shown to oxidize thiosulfate, which may indicate that build-up of organic flotation chemicals and sulfur species from the ore may cause the microbial numbers to increase. This study is part of the H2020 ITERAMS project (Grant agreement# 730480), which strives to improve the recycling of water and minimize the environmental impact of mines.     

Cross-correlating analyses of mineral-associated microorganisms in an unsaturated packed bed flow-through column test; cell number,activity and EPS

In heap bioleaching and waste-rock dumps, complex microbial communities exist in the flowing and interstitial liquid phases and mineral surface-associated biofilms, often embedded in extracellular polymeric substances (EPS). Microbial activity in the interstitial phase and mineral ore surface facilitates mineral degradation, resulting in either metal recovery or acidic, metal -bearing drainage from sulfidic waste-rock. Determining microbial presence and activity through microorganisms leaving the heap or dump has severe limitations. Hence, increasingly the ore-bed is sampled to quantify and characterise this. Here, methods for cell detachment and quantification, microbial activity measurement on the mineral surface and evaluation of EPS, quantitatively and biochemically, were refined and validated to assess microbial presence, using mineral coated beads in continuous flow-through columns. Number of wash steps required were assessed over increasing colonisation times over 30 days. Microbial cells colonising the mineral surface, pre- and post-washing were visualised by scanning electron microscopy (SEM) and their activity quantified by isothermal microcalorimetry (IMC). Using IMC, detachment and enumeration of detached cells, we demonstrated that 6–8 washes provided a reliable estimation of mineral-associated microorganisms, with less than 10% of cells or microbial activity associated with the surface following treatment. This allowed consolidated refinement of the protocol using traditional detachment method, SEM and IMC to provide correlative data. Extraction of EPS in a complete flow-through system is reported for the first time and the biochemical composition was similar to those reported under batch bioleaching conditions.     

Five novel acid-tolerant oligotrophic thiosulfate-metabolizing chemolithotrophic acid mine drainage strains affiliated with the genus Burkholderia of Betaproteobacteria and identification of two novel soxB gene homologues

Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958^T (96-98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8^T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417^T (99.6%) and Burkholderia vietnamiensis LMG 10929^T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958^T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.