Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sand horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the horizons used for the radioactive waste disposal were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10⁴ cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of the disposal site. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the deep repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Anaerobic transformation of carbon monoxide by microbial communities of Kamchatka hot springs

Carbon monoxide (CO) is one of the common gaseous compounds found in hot volcanic environments. It is known to serve as the growth substrate for a number of thermophilic prokaryotes, both aerobic and anaerobic. The goal of this work was to study the process of anaerobic transformation of CO by microbial communities inhabiting natural thermal environments: hot springs of Uzon Caldera, Kamchatka. The anaerobic microbial community of Treshchinny Spring (80°C, pH 6.5) was found to exhibit two peaks of affinity for CO (K _S1 = 54 nM and K _S2 = 1 μM). The actual rate of anaerobic CO transformation by the microbial community of this spring, calculated after obtaining the concentration dependence curve and extrapolated to the natural concentration of CO dissolved in the hot spring water (20 nM), was found to be 120 μmol l⁻¹ of sediment day⁻¹. In all the hot springs studied, more than 90% of the carbon of ¹⁴CO upon anaerobic incubation was recovered as ¹⁴CO₂. From 1 to 5% of ¹⁴CO was transformed to volatile fatty acids (VFA). The number of microorganisms capable of anaerobic CO oxidation determined by dilution-to-extinction method reached 10⁶ cells ml⁻¹ of sediment. CO-transforming anaerobic thermophilic microorganisms isolated from the springs under study exhibited hydrogenogenic type of CO oxidation and belonged to the bacterial genera Carboxydocella and Dictyoglomus. These data suggest a significant role of hydrogenogenic carboxydotrophic prokaryotes in anaerobic CO transformation in Uzon Caldera hot springs.     

Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sandy horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the production horizons of the radioactive waste disposal site were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10(4) cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of production horizons. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the underground repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository production horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Growth and Reproduction of Microorganisms Under Extremely Alkaline Conditions

An aerobic and an anaerobic strain of bacteria were isolated from two extremely alkaline springs. Growth and reproduction of both microorganisms were demonstrated above pH 11.0.     

热泉微生物驱动的氮循环研究进展及展望

热泉微生物是驱动热泉氮(N)循环的主导力量,开展热泉生态系统中驱动氮循环微生物种群构成及其与环境响应的研究,对于探索热泉中氮的生物地球化学循环、生命进化、生物修复等方面都具有重要的理论和应用价值。本文综合阐述了热泉生态系统驱动氮循环的功能微生物(如固氮菌、氨氧化菌、厌氧氨氧化菌、反硝化菌、异化硝酸盐还原菌)在系统发育学上的分布、功能基因的相对丰度、活性及其与环境因子(如温度、pH)的相关性等方面的研究现状和亟待解决的问题。并展望了热泉生境中驱动氮循环微生物未来的研究方向。     

Ecology and habitats of extremophiles

This review describes the main natural extreme environments, characterized by high temperature, high and low pH and high salinity, that can be colonized by microorganisms. The environments covered are: freshwater alkaline hot springs; acidic solfatara fields; anaerobic geothermal mud and soils; acidic sulphur and pyrite areas; carbonate springs and alkaline soil; and soda and highly saline lakes. The community structure, in terms of available energy sources and representative autotrophic and heterotrophic microorganisms, is discussed for each type of habitat.The authors are with the Department of Biotechnology. Technological Institute of Iceland, Keldnaholt, 112 Reykjavik, Iceland and with the Institute of Biology, University of Iceland, Reykjavik, Iceland     

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10³ CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.     

Bacterial Diversity in the Hyperalkaline Allas Springs (Cyprus), a Natural Analogue for Cementitious Radioactive Waste Repository

The biogeochemical gradients that will develop across the interface between a highly alkaline cementitious geological disposal facility for intermediate level radioactive waste and the geosphere are poorly understood. In addition, there is a paucity of information about the microorganisms that may populate these environments and their role in biomineralization, gas consumption and generation, metal cycling, and on radionuclide speciation and solubility. In this study, we investigated the phylogenetic diversity of indigenous microbial communities and their potential for alkaline metal reduction in samples collected from a natural analogue for cementitious radioactive waste repositories, the hyperalkaline Allas Springs (pH up to 11.9), Troodos Mountains, Cyprus. The site is situated within an ophiolitic complex of ultrabasic rocks that are undergoing active low-temperature serpentinization, which results in hyperalkaline conditions. 16S rRNA cloning and sequencing showed that phylogenetically diverse microbial communities exist in this natural high pH environment, including Hydrogenophaga species. This indicates that alkali-tolerant hydrogen-oxidizing microorganisms could potentially colonize an alkaline geological repository, which is predicted to be rich in molecular H₂, as a result of processes including steel corrosion and cellulose biodegradation within the wastes. Moreover, microbial metal reduction was confirmed at alkaline pH in this study by enrichment microcosms and by pure cultures of bacterial isolates affiliated to the Paenibacillus and Alkaliphilus genera. Overall, these data show that a diverse range of microbiological processes can occur in high pH environments, consistent with those expected during the geodisposal of intermediate level waste. Many of these, including gas metabolism and metal reduction, have clear implications for the long-term geological disposal of radioactive waste.     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

Biodiversity of the microbial mat of the Garga hot spring

Background

Microbial mats are a good model system for ecological and evolutionary analysis of microbial communities. There are more than 20 alkaline hot springs on the banks of the Barguzin river inflows. Water temperature reaches 75 °C and pH is usually 8.0–9.0. The formation of microbial mats is observed in all hot springs. Microbial communities of hot springs of the Baikal rift zone are poorly studied. Garga is the biggest hot spring in this area.

Results

In this study, we investigated bacterial and archaeal diversity of the Garga hot spring (Baikal rift zone, Russia) using 16S rRNA metagenomic sequencing. We studied two types of microbial communities: (i) small white biofilms on rocks in the points with the highest temperature (75 °C) and (ii) continuous thick phototrophic microbial mats observed at temperatures below 70 °C. Archaea (mainly Crenarchaeota; 19.8% of the total sequences) were detected only in the small biofilms. The high abundance of Archaea in the sample from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. In the microbial mats, primary products were formed by cyanobacteria of the genus Leptolyngbya. Heterotrophic microorganisms were mostly represented by Actinobacteria and Proteobacteria in all studied samples of the microbial mats. Planctomycetes, Chloroflexi, and Chlorobi were abundant in the middle layer of the microbial mats, while heterotrophic microorganisms represented mostly by Firmicutes (Clostridia, strict anaerobes) dominated in the bottom part. Besides prokaryotes, we detect some species of Algae with help of detection their chloroplasts 16 s rRNA.

Conclusions

High abundance of Archaea in samples from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. Metagenomic analysis of microbial communities of the microbial mat of Garga hot spring showed that the three studied points sampled at 70 °C, 55 °C, and 45 °C had similar species composition. Cyanobacteria of the genus Leptolyngbya dominated in the upper layer of the microbial mat. Chloroflexi and Chlorobi were less abundant and were mostly observed in the middle part of the microbial mat. We detected domains of heterotrophic organisms in high abundance (Proteobacteria, Firmicutes, Verrucomicrobia, Planctomicetes, Bacteroidetes, Actinobacteria, Thermi), according to metabolic properties of known relatives, which can form complete cycles of carbon, sulphur, and nitrogen in the microbial mat. The studied microbial mats evolved in early stages of biosphere formation. They can live autonomously, providing full cycles of substances and preventing live activity products poisoning.

     

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya

Soil samples collected from two hot springs, Soldhar and Ringigad, both located in the Garhwal region of Uttaranchal Himalaya were analysed for their physical, chemical and microbial components. The alkaline pH, total absence of carbon and nitrogen, and high temperature were features common to soil samples from both sites. The Soldhar samples contained higher amounts of Cu, Fe and Mn. Ringigad soil was devoid of Cu, but had much higher phosphate. While the optimum incubation temperature for isolating the maximum microbial counts from soil samples from the two sites was 50 degrees C, microbial growth in broth was also observed when incubated at 80 degrees C. Microscopic examination revealed three types of microbial populations, i.e., bacteria, yeast and filamentous organisms. The soil samples were found to be dominated by spore forming rods. Out of 58 aerobic isolates, 53 were gram positive bacilli. Gram positive anaerobic oval rods were also observed up to 60 degrees C. Soil dilution plates revealed the presence of antagonistic and phosphate solubilizing populations.     

The Microbial Ecology of a Hyper-Alkaline Spring,and Impacts of an Alkali-Tolerant Community During Sandstone Batch and Column Experiments Representative of a Geological Disposal Facility for Intermediate-Level Radioactive Waste

Naturally occurring hyper-alkaline springs and associated hyper-alkaline environments may have components that are analogous to a cement-based deep geological disposal facility (GDF) for intermediate level radioactive waste (ILW). Such high pH environments could give insights into the biogeochemical processes that could occur in the region of a GDF environment after the ingress of GDF-derived groundwater leads to the formation of a hyper-alkaline plume in the surrounding rock mass. This study focuses on the microbial community composition found at a highly alkaline spring near Buxton, Derbyshire, England, and the variation in community structure across spatially separated sample points of contrasting pH values (ranging from pH 7.5–13). Communities containing alkaliphilic and alkalitolerant bacteria were observed across the site by PCR amplification and 16S rRNA gene pyrosequencing and included members of the families Comamonadaceae and Xanthomonadaceae. At pH 13, the sequence library was dominated by Gammaproteobacteria of the families Pseudomonadaceae and Enterobacteriaceae. Bacterial communities from the site demonstrated the ability to reduce Fe(III) in microcosm experiments up to pH 11.5, suggesting the potential to reduce other metals and radionuclides of relevance to cement-encapsulated intermediate level radioactive waste (ILW) disposal. In laboratory column flow-through experiments, microbial communities present at the field site were also able to colonize crushed sandstone. Bacterial community composition varied between columns that had been supplied with alkali surface waters from the site amended with carbon (lactate and acetate, as proxies for products of cellulose degradation from ILW), and control columns that were not supplied with added carbon. Members of the family Clostridiaceae dominated the sequence library obtained from the carbon amended column inlet (45.8% of library), but became less dominant at the outlet (20.8%). Members of the family Sphingomonadaceae comprised 11.8% of the sequence library obtained from the control column inlet, but were not present in sediments collected from the column outlet, whereas the relative abundance of members of the family Comamonadaceae increased from the column inlet (35.2%) to the column outlet (57.2%). The spatial variation in community composition within the columns is indicative of discrete biogeochemical zonation in these flow-through systems.     

Microbial Community Structure and Arsenic Biogeochemistry in an Acid Vapor-Formed Spring in Tengchong Geothermal Area,China

Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73–0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41–95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.     

Putative anaerobic activity in aerated composts

It has been suggested that anaerobic microenvironments develop in aerobic composts, regardless of the aeration system used, and that anaerobic activity is responsible for odor generation and nitrogen losses. This study was designed to measure levels of microorganisms capable of anaerobic growth in two aerated composts: municipal solid waste, a relatively nutrient-rich compost, and pulp and paper-mill solid waste, which is relatively nutrient-poor. Anaerobic microorganisms were isolated from both composts at mesophilic and thermophilic temperatures. The majority of the anaerobic mesophiles were facultative anaerobes, whereas facultative, anaerobic thermophiles varied from 0 to 100%. Serially-diluted samples were spot-plated onto various media to preserve microbial consortia. Levels of aerobic and anaerobic exoenzyme production on spot-plates were similar on cell-wall, starch, and casein media. Although microbial levels on spread plates indicate that aerobes are present in much higher numbers than anaerobes (in 47 of 56 subsamples, 90% of the population were aerobes), microbial growth levels and exoenzyme production on spot-plates indicate that anaerobes may be responsible for a large portion (greater than or equal to 72%) of the metabolic activity in anaerobic microenvironments of aerobic composts.     

Simultaneous nitrogen and carbon removal in a packed A/O reactor: effect of C/N ratio on microbial community structure

In this research, a novel packed anoxic/oxic moving bed biofilm reactor (MBBR) was established to achieve high-organic matter removal rates, despite the carbon/nitrogen (C/N) ratio of 2.7–5.1 in the influent. Simultaneous nitrification–denitrification (SND) was investigated under a long sludge retention time of 104 days. The system exhibited excellent performance in pollutant removal, with chemical oxygen demand and total nitrogen (TN) enhanced to 93.6–97.4% and 34.4–60%, respectively. Under low C/N conditions, the nitrogen removal process of A/O MBBR system was mainly achieved by anaerobic denitrification. The increase of C/N ratio enhanced SND rate of the aerobic section, where dissolved oxygen was maintained at the range of 4–6 mg/L, and resulted in higher TN removal efficiency. The microbial composition and structures were analyzed utilizing the MiSeq Illumina sequencing technique. High-throughput pyrosequencing results indicated that the dominant microorganisms were Proteobacteria and Bacteroidetes at the phylum level, which contributes to the removal of organics matters. In the aerobic section, abundances of Nitrospirae (1.12–29.33%), Burkholderiales (2.15–21.38%), and Sphingobacteriales (2.92–11.67%) rose with increasing C/N ratio in the influent, this proved that SND did occur in the aerobic zone. As the C/N ratio of influent increased, the SND phenomenon in the aerobic zone of the system is the main mechanism for greatly improving the removal rate of TN in the aerobic section. The C/N ratio in the aerobic zone is not required to be high to exhibit good TN removal performance. When C/NH₄⁺ and C/TN in the aerobic zone were higher than 2.29 and 1.77, respectively, TN removal efficiency was higher than 60%, which means that carbon sources added to the reactor could be saved. This study would be vital for a better understanding of microbial structures within a packed A/O MBBR and the development of cost-efficient strategies for the treatment of low C/N wastewater.

     

Microbial diversity and autotrophic activity in Kamchatka hot springs

Microbial communities of Kamchatka Peninsula terrestrial hot springs were studied using molecular, radioisotopic and cultural approaches. Analysis of 16S rRNA gene fragments performed by means of high-throughput sequencing revealed that aerobic autotrophic sulfur-oxidizing bacteria of the genus Sulfurihydrogenibium (phylum Aquificae) dominated in a majority of streamers. Another widely distributed and abundant group was that of anaerobic bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). Archaea of the genus Vulcanisaeta were abundant in a high-temperature, slightly acidic hot spring, where they were accompanied by numerous Nanoarchaeota, while the domination of uncultured Thermoplasmataceae A10 was characteristic for moderately thermophilic acidic habitats. The highest rates of inorganic carbon assimilation determined by the in situ incubation of samples in the presence of ¹⁴C-labeled bicarbonate were found in oxygen-dependent streamers; in two sediment samples taken from the hottest springs this process, though much weaker, was found to be not dependent on oxygen. The isolation of anaerobic lithoautotrophic prokaryotes from Kamchatka hot springs revealed a wide distribution of the ability for sulfur disproportionation, a new lithoautotrophic process capable to fuel autonomous anaerobic ecosystems.

     

样品磷化氢含量与某些微生物群落及酶活的关系

探讨了样品中磷化氢含量与不同微生物群落及几种酶活性的关系。结果表明,样品的磷化氢含量与厌氧微生物总量、有机磷细菌、反硝化细菌、碱性磷酸酶活性及脱氢酶活性呈显著的正相关关系,其相关系数(R2)分别达到了0.93,0.90,0.69,0.79和0.82;而与好氧异养微生物总量、无机磷细菌、硫酸盐还原菌、酸性磷酸酶的关系不大。此结果说明,相关类群的微生物在磷化氢的产生中可能起重要的作用。     

Bacterial Diversity and Functional Activity of Microbial Communities in Hot Springs of the Baikal Rift Zone

In this study the bacterial diversity of thermophilic microbial mats (40 to 65°C) in three alkaline hot springs of the Baikal Rift Zone (BRZ) was determined through pyrosequencing of 16S rRNA gene libraries. Significant diversity of bacterial species was found in the biomats of the hot springs with total number of detected phylotypes of 607. The highest share of the microbial community was represented by the phyla Chloroflexi (Seya Spring, 76.4%), Deinococcus-Thermus (Alla Spring, 45.1%), Nitrospira (Alla Spring, 36.1%), Cyanobacteria (Tsenkher Spring, 33.1%), and Proteobacteria (Tsenkher Spring, 22.6%), but their ratio varied significantly in different springs. A comparison of the biodiversity and composition of microbial communities between hot springs showed a decrease in biodiversity with increasing temperature. A large number of sequences showed a low degree of similarity with cultivated representatives in public databases. Microbial communities showed intensive rates of production and destruction of organic compounds, as revealed by the quantitative assessment of their functional activity.     

Analysis of the diversity of aerobic,thermophilic endospore-forming bacteria in two Algerian hot springs using cultural and non-cultural methods

Terrestrial hot environments are important resources for isolation of thermophilic microorganisms. Few studies have been made on microbial diversity of Algerian geothermal sites. This paper reports the diversity of thermophilic, aerobic endospore-forming bacteria from water and sediment samples taken from Hammam Ouled Ali and Hammam Debagh, two hot springs with a wide range of temperatures and a very rich mineral composition, located in the region of Guelma, north-east of Algeria using culture-dependent and culture-independent approaches Sequences of the V4 region of the 16S rRNA gene from environmental DNA extracted from sediment samples were analyzed and a set of isolates from water and sediment have been characterized by phenotypic and molecular methods. Phylogenetic surveys using environmental DNA sequences indicated that three families dominated the two hot springs: Planococcaceae, Bacillaceae, and Paenibacillaceae. Phenotypic characterization revealed the morphological, biochemical, and physiological properties of these microorganisms, all of which exhibited a range of common extracellular enzymatic activities. Amplified ribosomal DNA restriction analysis (ARDRA) was used to cluster isolates into different phylotypic groups and phylogenetic analysis of 16S rRNA gene sequences of selected isolates showed that all were closely related to four genera of thermophilic Bacilli: Bacillus, Anoxybacillus, Geobacillus, and Brevibacillus. Our results provide important insights into the microbial ecology of Guelma hot springs. They showed that the phylogenetic diversity of bacterial communities within the two studied hot springs was mostly aerobic, with the presence of taxonomic groups of great biotechnological interest. Bioprospection of thermozymes and other biomolecules within these communities will probably provide a data basis for their industrial exploitation.     

Activity of Microorganisms in Acid Mine Water I. Influence of Acid Water on Aerobic Heterotrophs of a Normal Stream

Comparison of microbial content of acid-contaminated and nonacid-contaminated streams from the same geographical area indicated that nonacid streams contained relatively low numbers of acid-tolerant heterotrophic microorganisms. The acid-tolerant aerobes survived when acid entered the stream and actually increased in number to about 2 × 10³ per ml until the pH approached 3.0. The organisms then represented the heterotrophic aerobic microflora of the streams comprised of a mixture of mine drainage and nonacid water. A stream which was entirely acid drainage did not have a similar microflora. Most gram-positive aerobic and anaerobic bacteria died out very rapidly in acidic water, and they comprised a very small percentage of the microbial population of the streams examined. Iron- and sulfur-oxidizing autotrophic bacteria were present wherever mine water entered a stream system. The sulfur-oxidizing bacteria predominated over iron oxidizers. Ecological data from the field were verified by laboratory experiments designed to simulate stream conditions.