Macroscopic and Microscopic Morphological Features of Stromatolites Related To Activity of Eukaryote-Dominated Biofilms in an Acid Mine Drainage Environment: Biosignatures and Understanding Preservation of Stromatolites as Trace Fossils

Euglena-, diatom-, and algae-dominated biofilms are the principal producers of iron-rich biolaminates that result in biosedimentary structures, or stromatolites, in an acid mine drainage (AMD) environment in Indiana. These structures are considered trace fossils because they are produced by organism-sediment interactions and record physicochemical conditions of the environment. Our purpose was to link the biofilm types to specific micro- and micromorphological features and the physicochemical conditions under which they were formed. Analyses revealed that Euglena-dominated biofilm produced thin, porous microlaminae by trapping, binding, and relocating AMD precipitates as the biofilm kept pace with chemical sedimentation. More massive microlaminae were produced by high rates of chemical sedimentation brought on by increased discharge and dilution of acidity. Diatom- and algae-dominated biofilms produced thick, mm–cm-scale, porous, spongelike micro- to macrolaminae through oxygenic photosynthesis and/or metal uptake in extracellular polymeric substances, which promoted mineral precipitation on cell walls to create a rigid, porous structure. The variations in biolaminate textures within the stromatolites record seasonal changes in the microbial populations and physicochemical conditions of the AMD environment. These iron-rich stromatolites represent trace fossils that record morphological biosignatures of eukaryote-dominated microbial biofilms and may serve as appropriate proxies in the search for similar evidence of eukaryotic life in other iron-rich paleoenvironments, such as those on early Earth and Mars.     

云南东川黄铜矿酸性浸矿废水中微生物群落分析

利用限制性片段长度多态性和16s rRNA PCR扩增及其序列分析来研究中国云南东川黄铜矿酸性浸矿废水(AMD)中微生物群落结构。基于建立16s rRNA克隆文库的分子方法常常被用来研究AMD中微生物生态。一小部分的rRNA被PCR扩增,克隆,然后用限制性片段长度多态性来筛选出其不同种类。在三个样地中,共挑选了357个克隆子,经限制性酶切分析后聚为100个可操作分类单元(OTU)。一个OTU代表各个样地中出现的同一种酶切图谱。有58个OTU被测序分析,结果在基因数据库中比对分析,其系统发育分析表明它们聚类为六大类:γ-proteobacteria,Acidobacteria,Clostridia,Actinobacteria,Nitrospira,α-pro- teobacteria。其中,γ-proteobacteria为最大的群落,在三个样地DCK-Ⅰ,DCN-Ⅰand DC-Ⅱ中分别占36%,57%,62%的比例。但与浸矿相关的微生物如At.ferrooxidans;L ferrooxidans等却发现的很少,仅在两个样地中各发现四个相关的OTUs。在这个AMD环境中浸矿相关的微生物都不是优势种群。     

Toward a Solution for Acid Mine Drainage Treatment: Role of Electron Donors in Sulfate Reduction at Low pH

Abstract

Sediment historically impacted by acid mine drainage was exposed to different initial pH and electron donors to investigate the effect that both conditions had on the performance and fingerprint of the community from naturally acidic sediments. Batch experiments were fed with either acetate, lactate, or glycerol at initial pH of 5, 4, or 3, under sulfate-reducing conditions. The performance results indicated that sulfide production efficiency was above 85% in the treatments fed with lactate and glycerol at pH 5 and 4. However, acetate consumption efficiency was greater than 85% only in the treatments with acetate at pH 5 and lactate at pH 5 and 4. Glycerol fed treatments successfully produced sulfide even at initial pH?=?3. Sulfide production rates were related to the initial pH in treatments fed with lactate and acetate and independent of the pH in the glycerol fed treatments. 16S rRNA gene T-RFLP analysis of the enriched communities indicated that the initial pH could explain the differences of the microbial community fingerprint obtained after 90?days. This study points out the fact that acidic stress is a heavy burden for the development of sulfate-reducing microorganisms, especially for those that use acetate as substrate.     

Microbial Community Composition in Acid Mine Drainage Lake of Xiang Mountain Sulfide Mine in Anhui Province,China

Acid mine drainage (AMD) lake of Xiang Mountain in Anhui Province, China, was characterized by acidic waters (pH around 2.8) containing high concentrations of soluble metals and sulfate. To investigate the function and dynamics of this extreme ecosystem, four water samples were collected from the lake in the fall of 2010. The acidophilic community structure was analyzed by molecular approaches, and bacterial and archaeal clone libraries of 16S rRNA genes were constructed. In contrast to dominance of chemolithotrophic acidophiles in typical AMD environments, autotrophic iron/sulfur-oxidizing bacteria were detected in only one sample with low abundance. Unexpectedly, the Cyanobacteria group was the predominant in all four samples (54.9%?77%). Chemoheterotrophs Acidiphilium and Acidisphaera were also abundant. These two heterotrophic groups contain bacteriochlorophyll that can perform photosynthesis, an advantage to grow and survive in such oligotrophic acidic environments. Only two clone sequences related to Legionella (2.8% of the total clones) were recovered from one sample in sharp contrast to its higher abundance (12.7%) in the summer of 2009. All archaeal sequences were affiliated to the phylum Crenarchaeota. The results of statistical analysis suggested that the water chemistry of the AMD lake controlled microbial composition of the AMD ecosystem.     

Laboratory Scale Bioremediation of Acid Mine Water Drainage from a Disused Tin Mine

Real acidic mine-water drainage was seeded with Acidithiobacillus ferrooxidans to catalyse the removal of iron contained therein. The addition of At. ferrooxidans increased metal precipitation kinetics and decreased the water iron content by ~70%. Supplementing non-sterile mine water with a bacterial growth medium accelerated metal removal by indigenous micro-organisms both at the 500 ml shake-flask and 5 l bioreactor scale.     

封闭环境下酸性矿坑水中微生物生态多样性的研究

铜绿山铜矿是世界开采时间最长的矿井之一,在开采过程中有许多矿井被废弃,许多废弃的矿井内产生了大量的对环境有害的酸性矿坑水.酸性矿坑水取自铜绿山铜矿某废弃矿井,利用限制性酶切片断多样性分析(RFLP分析)对酸性矿坑水中的微生物生态多样性进行了研究.研究表明,酸性矿坑水呈酸性,相对于其他极端与非极端生态环境,酸性矿坑水中的细菌与古菌的群落多样性较低.RFLP分析与系统发育分析表明,酸性矿坑水中细菌主要由A.fcrrooxidans(属于gamma-Proteobacteria)和L.ferrooxidans(属于Nitospira)成;古菌主要由Thermoplasma相关古菌组成.在这种封闭环境的酸性矿坑水中首次发现了类似于产甲烷古菌的克隆片断,其占古菌种群的四分之一左右.本研究将促进对酸性矿坑水中细菌及古菌群落组成及其对酸性矿坑水产生的作用的研究.     

A Novel Community of Acidophiles in an Acid Mine Drainage Sediment

A sediment sample (pH 2.5) was collected at an acid mine drainage site in Anhui, China. The present acidophilic microbial community in the sediment was studied with a 16S rRNA gene clone library. Small-subunit rRNA genes were PCR amplified, cloned and screened by amplified rDNA restriction analysis (ARDRA). Subsequently, 10 different clones were identified and they were affiliated with Acidobacteria, β/γ-Proteobacteria, δ-Proteobacteria, Nitrospira, Candidate division TM7, and Low G + C Gram-positives. Phylogenetic analysis of 16S rRNA gene sequences revealed a diversity of acidophiles in the sediment that were mostly novel. Unexpectedly, 16S rRNA gene sequences affiliated with δ-Proteobacteria were found to constitute more than 60% of clone library. To our knowledge, this is the first occasion that bacteria of δ-Proteobacteria have been found dominant in the acidic habitat. Anaerobic sulfate- or metal reduction is the predominant physiological trait of bacteria of this subdivision. The high sulfate, ferric iron and the presence of bioavailable carbon in the anaerobic microenvironment may result in the dominance of bacteria of δ-Proteobacteria.     

Sulfate Reduction Based Bioprocesses for the Treatment of Acid Mine Drainage and the Recovery of Metals

Biological sulfate reduction is increasingly replacing chemical unit processes in mining biotechnology. Sulfate reducing bacteria (SRB) can be used for treating ground‐ and surface waters contaminated with acid mine drainage (AMD), and for recovering metals from wastewater and process streams. Biologically produced H₂S precipitates metals as metal sulfides, while biogenic bicarbonate alkalinity neutralizes acidic waters. This paper reviews various passive and active SRB‐based alternatives as well as some process design aspects, such as reactor types, process configurations, and choices of substrates for sulfate reduction. The latest developments of using various low‐cost substrates together with new bioprocess designs are increasing the uses and applications of SRB‐based bioreactors in AMD control and selective metal recovery.     

Diversity of Prokaryotic Communities Indigenous to Acid Mine Drainage and Related Rocks from Baiyin Open-Pit Copper Mine Stope,China

Metagenomic approach permits us to obtain the latent resources from culturable and unculturable microorganisms in ecosystem. In this study, high-throughput sequencing was practiced to comprehensively probe prokaryotic community within extreme acidic environment of Baiyin open-pit mine stope, which varied in pH and other physicochemical parameters. Bioinformatics analysis was further accomplished to process millions of Illumina reads and analyzed alpha and beta diversities, and prokaryotic community profile in different samples obtained from the acidic mine stope. Diversity indices such as ACE, Chao, Shannon, and Simpson were varied among samples. Both taxon richness and evenness were significantly higher in the solid samples than that of the water samples. Taxonomic diversity was unexpectedly higher within confined pit ecosystem. Most of the sequences were assigned to phyla Proteobacteria, Firmicutes, and Acidobacteria. In archaea, Euryarchaeota and Thaumarchaeota were major phyla reported, however, archaea occupied very little share in the metagenome. At class level, variation in community structure was higher within samples. Among iron- and sulfur-related acidophiles, 30.8% of the sequences were unidentified at genera level, while the remaining were dominated by sulfur and/or iron oxidizing Acidithiobacillus and heterotrophic Acidiphilum related groups. The community profile of solid and water groups was different and metagenomic biomarkers were higher in solid, while acidophiles and archaea were reported only in water group by using LEfSe. Among samples, community structure and abundance was varied in terms of OTUs abundance, which clearly indicates spatial variation and proposed the influence of physicochemical and geochemical properties on phylogenetic diversity. This study offers numerous treasured datasets for better understanding the community composition under the influence of geochemical and physicochemical factors and possible novelty in terms of taxonomic/phylogenetic diversity in acidic ecosystem.     

Chemical Characterisation of Organic Electron Donors For Sulfate Reduction For Potential Use in Acid Mine Drainage Treatment

The production of acid mine drainage (AMD) containing high amounts of sulfate, heavy metals and low pH is of increasing concern. AMD is highly corrosive and results in economic and environmental problems. Organic electron donors for sulfate reduction were chemically characterised for potential use in AMD treatment. This was done in a process to develop a correlation between chemical composition and the capacity to drive sulfate reduction. Potential organic electron donors for sulfate reduction were chemically characterised in terms of dry matter content, ash content, total Kjeldahl nitrogen, lignin content, cellulose content, crude fat, crude fibre, in vitro digestibility, water-soluble carbohydrates, total non-structural carbohydrates and starch content. The chemical composition of the organic electron donors was then compared to results obtained from pilot plant studies where the organic electron donors for sulfate reduction were evaluated in terms of sulfate reduction. The chemical composition of the carbon source severely impacted its capacity to drive sulfate reduction and may be used to assist in predicting the sulfate reduction capacity of a carbon source. Organic electron donors for sulfate reduction high in protein content and low in lignin content or high in carbohydrate and crude fat content increased the capacity of a carbon source to drive sulfate reduction. The higher the fibre content of a carbon source, the lower the capacity to drive sulfate reduction. No correlation could be drawn between % dry matter, % ash content and sulfate reduction for the organic electron donors tested. Chemical characterisation can be used to assist in predicting sulfate reduction capacity of organic electron donors.     

Spatial Variation and Fractionation of Bed Sediment-Borne Copper,Zinc, Lead,and Cadmium in a Stream System Affected by Acid Mine Drainage

An investigation was conducted to examine the spatial variation and fractionation of bed sediment-borne Cu, Zn, Pb, and Cd in a stream system affected by acid mine drainage. The pH had a major control on the spatial variation pattern of soluble, exchangeable, and carbonate-bound Cu, Zn, and Cd. There was a prominent concentration peak of carbonate-bound, oxide-bound, and organic-bound metals at the 29 km station, as controlled by the abundance of organic C, carbonate C, and oxides of manganese and iron. In general, the residual fraction was the dominant form for all four investigated metals. It was likely that oxide-Mn played a more important role in binding Zn and Cd than oxide-Fe did. In contrast, Cu had a higher affinity for iron hydrous oxides than for manganese oxide. Pb had a higher affinity for oxides of iron and manganese than for carbonates and organic matter. The presence of organic-bound metals in both the acidic upstream reach and non-acidic downstream reach suggests that the binding of these metals by organic matter was not markedly affected by pH, while the correspondence of organic C peak and organic-bound metal peaks at the 29 km station indicates a strong control by organic matter abundance on the quantity of organic-complexed metals.     

Impacts of Mine Drainage and Other Nonpoint Source Pollutants on Aquatic Biota in the Upper Powell River System,Virginia

The Clinch-Powell River system of Virginia and Tennessee, USA, is among the most biologically diverse ecosystems in the world, and has been identified as a conservation priority of national importance. Other researchers have attributed declines in Powell River freshwater mussel populations to coal mining-related activities. The objectives of this paper are to synthesize the results of several studies aimed at assessing acid mine drainage (AMD) impacts in the Powell River watershed, and to describe the relative roles of AMD, nutrient loading, and urban runoff in structuring the benthic macroinvertebrate communities of the Powell River. Impacts ranged from acute toxicity due to the combination of acid and metals, acute aluminum toxicity in circumneutral pH waters, and physical impacts from solid ferrihydrite, to decreased clam growth due to urban runoff. These findings support the concern that AMD negatively impacts aquatic biota in the Powell River watershed in a variety of ways, and emphasize the importance of taking measures to properly reclaim areas that are producing mine drainage. However, past-mining related pollutants are not the only source of aquatic-community impact in these watersheds, as nutrient loading and urban runoff both were associated with substantial changes in community structure and clam growth.     

Effects of Bioavailable Heavy Metal Species,Arsenic, and Acid Drainage from Mine Tailings on a Microbial Community Sampled Along a Pollution Gradient in a Freshwater Ecosystem

Sediment and water samples representing a pollution gradient in a long, narrow lake polluted at one end by heavy metals, arsenic, and acid drainage from mine tailings, together with samples from an unpolluted reference lake, were analyzed to determine effects of pollutants on the microbial community of the polluted lake. Ribosomal ribonucleic acid, fatty acid, and phospholipid analyses, along with assays of CO₂ production, denitrification, and enzyme activities, were performed to characterize the microflora; and environmental conditions were defined by various physicochemical analyses, including determination of bioavailable metal species. Mine waste pollution fostered the growth of Holophagal Acidobacteria, green sulphur bacteria, and α-Proteobacteria but inhibited numerous other types of microorganisms, reducing the overall productivity, biomass, and biodiversity of the microflora. The beneficial effects imply toleration of pollutants, suppression of competing or antagonistic species, and utilization of biogenic sulphide; and the toxic effects are attributable to bioavailable metals, arsenic, and sulphuric acid produced by oxidation of sulphides. The bioavailability and toxicity of sediment-bound metals were evidently increased by acidification, elevation of sediment Eh, and inhibition of metal-immobilizing bacteria by pollutants but were decreased by metal-scavenging oxyhydroxides, sulphide, and organic matter. Metal toxicity also depended on specific metal properties (e.g., electronegativity), providing a basis for inferring mechanisms of toxicity and oxidation states of metals and explaining differences in relative toxicity. The pollutants harmed the ecosystem as a whole by inhibiting microorganisms that performed crucial ecological functions, notably oxygen-releasing photosynthesis, decomposition and humification of organic matter, nutrient recycling, and control of metal availability.     

Microcosm-based Study of the Attenuation of an Acid Mine Drainage-Impacted Site through Biological Sulfate and Iron Reduction

Acid mine drainage is a widespread environmental problem and is characterized by elevated proton, sulfate, and dissolved iron concentrations. To understand the driving forces behind the attenuation of AMD, we compared microcosms using sediment and groundwater collected at Davis Mine, Massachusetts, a site where both generation and attenuation of AMD occur. A shift in key geochemical parameters over time was due to the natural microbial population, which was supported by detailed molecular biology results. The attenuation of AMD was stimulated through amendment with glycerol, with nitrogen and phosphorous, or with algae extract, but not by wood chips, suggesting the addition of different organic electron donors as a bioremediation strategy.     

Palatability of Leaves Conditioned in Streams Affected by Mine Drainage: A Feeding Experiment with Gammarus Pulex (L.)

Both the absence of leaf shredding macroinvertebrates and low microbial activity are of major importance in determining slow and incomplete leaf decay in extremely acidic (pH<3.5) mining streams. These streams are affected by a heavy ochre deposition causing the formation of massive iron plaques on leaf surfaces that hinder microbial exploitation. An investigation was carried out to determine whether iron plaques and leaf conditioning status (acid conditioned with and without iron plaques, neutral conditioned, unconditioned) affect the feeding preference of the shredder Gammarus pulex (L.). Leaf respiration rates and fungal biomass (ergosterol contents) were measured to determine microbial colonization. Neutral conditioned leaves had significantly higher microbial colonization than acid conditioned leaves with iron plaques. Notwithstanding, leaves of both conditioning types were consumed at high rates by G. pulex. The microbial colonization had no influence on feeding preference in the experiment. It is presumed that iron adsorbed organic material caused the high palatability of leaves with iron plaques. The results indicate that the large deposits of leaves coated with iron plaques will be available to the stream food web when water quality will be restored to neutral as planed in scenarios for the future development of mining streams.     

Advances In Biotreatment of Acid Mine Drainage and Biorecovery of Metals: 2. Membrane Bioreactor System for Sulfate Reduction

Several biotreatmemt techniques for sulfate conversion by the sulfate reducing bacteria (SRB) have been proposed in the past, however few of them have been practically applied to treat sulfate containing acid mine drainage (AMD). This research deals with development of an innovative polypropylene hollow fiber membrane bioreactor system for the treatment of acid mine water from the Berkeley Pit, Butte, MT, using hydrogen consuming SRB biofilms. The advantages of using the membrane bioreactor over the conventional tall liquid phase sparged gas bioreactor systems are: large microporous membrane surface to the liquid phase; formation of hydrogen sulfide outside the membrane, preventing the mixing with the pressurized hydrogen gas inside the membrane; no requirement of gas recycle compressor; membrane surface is suitable for immobilization of active SRB, resulting in the formation of biofilms, thus preventing washout problems associated with suspended culture reactors; and lower operating costs in membrane bioreactors, eliminating gas recompression and gas recycle costs. Information is provided on sulfate reduction rate studies and on biokinetic tests with suspended SRB in anaerobic digester sludge and sediment master culture reactors and with SRB biofilms in bench-scale SRB membrane bioreactors. Biokinetic parameters have been determined using biokinetic models for the master culture and membrane bioreactor systems. Data are presented on the effect of acid mine water sulfate loading at 25, 50, 75 and 100 ml/min in scale-up SRB membrane units, under varied temperatures (25, 35 and 40 °C) to determine and optimize sulfate conversions for an effective AMD biotreatment. Pilot-scale studies have generated data on the effect of flow rates of acid mine water (MGD) and varied inlet sulfate concentrations in the influents on the resultant outlet sulfate concentration in the effluents and on the number of SRB membrane modules needed for the desired sulfate conversion in those systems. The pilot-scale data indicate that the SRB membrane bioreactors systems can be applied toward field-scale biotreatment of AMD and for recovery of high purity metals and an agriculturally usable water.     

Assessment of trace element pollution and human health risks associated with cultivation of mine soil: A case study in the Iberian Pyrite Belt

A risk-based corrective action (RBCA) approach was conducted to assess the potential health risks associated with occupational and environmental exposures to trace elements in cultivated mine soil, reporting a site-specific environmental health and safety case study in the Spanish sector of the Iberian Pyrite Belt. The median concentrations of As in soil (580 mg kg^?1), Cu (635 mg kg^?1), Pb (2100 mg kg^?1), and Zn (270 mg kg^?1) largely exceeded the regional geochemical baseline, reaching values above which adverse health effects may potentially occur. The results of the RBCA analysis suggest the possibility that a median carcinogenic risk (9.3E-04) may be associated to arsenic exposure by ingestion and dermal contact pathways. In addition, the median hazard index was more than four times higher than the acceptable risk level, with As (hazard quotient value of 3.3) being also the largest single contributor to the overall non-carcinogenic risk. However, no detrimental health effects are expected to occur through inhalation of soil particles in people living near the source zone. Preventive measures should be applied to reduce surface soil exposure in the light of the results achieved.     

Cultivation-dependent and cultivation-independent characterization of the microbial community in acid mine drainage associated with acidic Pb/Zn mine tailings at Lechang, Guangdong, China

Cultivation-based and molecular approaches were used to characterize the phylogenetic composition and structure of the microbial community in an extremely acidic (pH 2.0) acid mine drainage (AMD) associated with Pb/Zn mine tailings that were undergoing vigorous acid generation. Acidophilic bacteria were isolated and enumerated on solid media, and were found to be restricted to isolates related to Acidithiobacillus ferrooxidans and Acidiphilium cryptum. By contrast, cloning and phylogenetic analysis of 16S rRNA genes revealed that, although low in total taxonomically distinct groups, the tailings AMD ecosystem harbored a wide range of phylogenetically diverse microbes. Of the 141 clones examined, 104 were phylogenetically affiliated with the recently discovered, iron-oxidizing Leptospirillum group III within the Nitrospira. It thus appears that iron serves as the major electron donor in this habitat. Thirty clones were affiliated with the Proteobacteria, half of which belonged to organisms related to Alphaproteobacteria species capable of ferric iron reduction. Other clones were grouped with Betaproteobacteria and Gammaproteobacteria (six clones each), and even with Deltaproteobacteria (three clones), a subdivision with anaerobic sulfate or metal (iron) reduction as the predominant physiological trait of its members. Finally, four clones were clustered within the Firmicutes and the Acidobacteria. Approximately half of the sequence types representing the majority of the total clones fell into lineages that are poorly represented by cultured organisms or have thus far been represented by only a few environmental sequences. Thus, the present study extends our knowledge of the biodiversity of microorganisms populating highly acidic AMD environments.     

Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions

Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N-methyl-D-aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringer's solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 +/- 0.9 to 48 +/- 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200-400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound-induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D-serine (100 micrograms intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.