Comparison of Bacteria and Archaea communities in municipal solid waste, individual refuse components, and leachate

Refuse decomposition in landfills is a microbially mediated process that occurs primarily under anaerobic conditions. Because of limited moisture conditions, hydraulic transport as a means of cellular translocation within the landfill appears limited, especially during the initial stages of decomposition. Thus, microbial communities within the incoming refuse serve as a primary source of facultative and obligate anaerobic microorganisms that initiate refuse decomposition. Fresh residential refuse was collected five times over 26 months, and microbial communities in these samples were compared with those in individual refuse components and decomposed refuse. Bacterial and archaeal community structures were determined using T-RFLP. The Bacterial microbial community richness was correlated (r(2) = 0.91) with seasonal differences in ambient air temperature. Analysis of the results shows that fresh refuse is most likely not the source of methanogens in landfills. Microbial communities in the solid and leachate phases were different, indicating that both matrices must be considered when characterizing microbial diversity within a landfill.     

Microbial Diversity in the Deep Marine Sediments from the Qiongdongnan Basin in South China Sea

The continental shelf and slope in the northern South China Sea is well known for its prospect of oil/gas/gas-hydrate resources. To study microbial communities and their roles in carbon cycling, a 4.9-m sediment core was collected from the Qiongdongnan Basin on the continental slope of the South China Sea during our cruise HY4-2005-5 in 2005. Geochemical, mineralogical, and molecular phylogenetic analyses were carried out. Sulfate concentration in pore water decreased with depth. Abundant authigenic carbonates and pyrite were observed in the sediments. The bacterial community was dominated by aerobic and facultative organisms. Bacterial clone sequences belonged to the Gamma-, Alpha-, Deltaproteobacteria and Firmicutes group, and they were related to Fe(III) and/or Mn(IV) reducers, sulfate reducers, aromatic hydrocarbon degraders, thiosulfate/sulfite oxidizers, and denitrifiers. Archaeal clone sequences exhibited greater overall diversity than the bacterial clones with most sequences related to Deep-Sea Archaeal Group (DSAG), Miscellaneous Crenarchaeotic Group (MCG), and Uncultured Euryarchaeotic Clusters (UECs). Archaeal sequences related to Methanosarcinales, South African Gold Mine Euryarchaeotic Group (SAGMEG), Marine Benthic Group-D (MBG-D) were also present. Most of these groups are commonly present in deep-sea sediments, particularly in methane/organic-rich or putative methane hydrate-bearing sediments.     

Nitrogen removal through different pathways in an aged refuse bioreactor treating mature landfill leachate

In this study, an aged refuse bioreactor was constructed to remove nitrogen in a mature landfill leachate. The nitrogen removal efficiency and the microbial community composition in the bioreactor were investigated. The results showed that the aged refuse bioreactor removed more than 90 % of total nitrogen in the leachate under the nitrogen loading rate (NLR) of 0.74 g/kg (vs) day, and the total nitrogen removal rate decreased to 62.2 % when NLR increased up to 2.03 g/kg (vs) day. Quantitative polymerase chain reaction results showed that the average cell number of ammonia-oxidizing bacteria in the bioreactor was 1.58?×?10⁸ cells/g, which accounted for 0.41 % of total bacteria. The number of anammox bacteria in the reactor was 1.09?×?10⁸ cells/g, which accounted for 0.27 % of total bacteria. Isotopic ¹⁵N tracing experiments showed that nearly 10 % of nitrogen was removed by anammox. High-throughout 454 pyrosequencing revealed that the predominant bacteria in the bioreactor were Proteobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, and Gemmatimonadetes, including various nitrifiers and denitrifiers with diverse heterotrophic and autotrophic metabolic pathways, supporting that nitrogen was removed through different pathways in this aged refuse bioreactor.     

五年填埋龄准好氧和厌氧填埋体及陈垃圾的理化特性

研究了5年填埋龄准好氧和厌氧填埋体的沉降量、温度和填埋气产生特性,并分析了填埋处理陈腐垃圾的理化性质.结果表明：5年填埋龄准好氧填埋体的表面沉降量和减容率均显著高于厌氧填埋体,同时渗滤液回灌比清水回灌更容易引起垃圾填埋体表面的不均匀沉降.厌氧填埋体的平均温度（25.6 ℃）略高于准好氧填埋体（24.8 ℃）,但差异不显著.准好氧填埋体不同时期的O₂浓度分别高于厌氧填埋体,而CH₄浓度则显著低于厌氧填埋体.5年填埋龄准好氧和厌氧填埋体陈垃圾中厨余物和纸类等易降解有机物含量显著降低,塑料、玻璃、砖瓦和竹木等含量则升高.其中,陈垃圾中有机质与营养元素含量均高于典型南方土壤;除厌氧填埋体陈垃圾中Cr含量超标外,其余重金属含量均未超出《土壤环境质量标准（GB 15618—1995）》的三级标准要求.     

Effects of in situ nitrogen removal on degradation/stabilization of MSW in bioreactor landfill

The effects of in situ nitrogen removal on degradation of municipal solid waste (MSW) in bioreactor landfill system were investigated. The in situ nitrogen removal bioreactor landfill (NBL) consisted of fresh-refuse filled, methanogenic and nitrifying reactors was operated. The two-phase bioreactor landfill (BL) comprised of fresh-refuse filled and methanogenic reactors was used as control. The methanogenic and nitrifying reactors were all loaded with aged refuse whose placement time was 6-7 yr. Furthermore, the nitrifying reactor was in situ aerated. The results showed that the degradation of fresh-refuse was delayed and CH4 production also was reduced in the in situ nitrogen removal bioreactor landfill. It was feasible to perform in situ ammonia nitrification in aged refuse. Moreover, the efficiency of oxygen utility was high during the in situ nitrification because of the porous characteristic of aged refuse. Supplementing only 8.5mg O2 mg(-1)Nd(-1) to aged refuse could make ammonia removed completely. However, aeration did not accelerate the further stabilization of aged refuse.     

Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures

Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.     

In situ nitrogen removal in phase-separate bioreactor landfill

The feasibility of in situ nitrogen removal in phase-separate bioreactor landfill was investigated. In the experiment, two sets of bioreactor landfill systems, namely conventional two-phase and in situ nitrogen removal bioreactor landfills, were operated. The in situ nitrogen removal bioreactor landfill (NBL) was comprised of a fresh-refuse filled reactor (NBLF), a methanogenic reactor (NBLM) and a nitrifying reactor (NBLN), while the two-phase bioreactor landfill (BL) used as control was comprised of a fresh-refuse filled reactor (BLF) and a methanogenic reactor (BLM). Furthermore, the methanogenic and nitrifying reactors used aged refuse as bulk agents. The results showed that in situ nitrogen removal was viable by phase-separation in the bioreactor landfill. In total 75.8 and 47.5 g of nitrogen were, respectively, removed from the NBL and the BL throughout the experiment. The methanogenic reactor used the aged refuse as medium was highly effective in removing organic matter from the fresh leachate. Furthermore, the aged refuse was also suitable to use as in situ nitrification medium. The degradation of fresh refuse was accelerated by denitrification in the initial stage (namely the initial hydrolyzing stage) despite being delayed by denitrification in a long-term operation.     

Performance and bacterial compositions of aged refuse reactors treating mature landfill leachate

Aged landfill leachates become more refractory over time and difficulty to treat. Recently, aged refuse bioreactors show great promise in treating leachates. In this study, aged refuse bioreactors were constructed to simulate landfill leachate degradation process. The characteristics of leachate were: COD_cr, ∼2200 mg/L; BOD₅, ∼280 mg/L; total nitrogen, ∼2030 mg/L; and ammonia, ∼1900 mg/L. Results showed that bioreactor could remove leachate pollutants effectively at hydraulic loading of 20 L/m³ d. The removal rate reduced when hydraulic loading doubled or temperature lowered. Effluent recirculation could alleviate the temperature effect. Combining aged refuse and slag biofilters could treat leachate more efficiently. Pyrosequencing analysis indicated that bacteria from Pseudomonas, Lysobacter, Bacillus and δ-proteobacter, Flexibacteraceae were more abundant in the samples. The Shannon index decreased at lower temperature, while evenness and equitability increased with recirculation. We suggest that filter medium and temperature may be the main factors for shaping bacterial community structure.     

Microbial Community Structure in Three Deep-Sea Carbonate Crusts

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as α-, γ-, and δ- Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative δ ¹³C values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.     

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Distance-Decay and Taxa-Area Relationships for Bacteria,Archaea and Methanogenic Archaea in a Tropical Lake Sediment

The study of of the distribution of microorganisms through space (and time) allows evaluation of biogeographic patterns, like the species-area index (z). Due to their high dispersal ability, high reproduction rates and low rates of extinction microorganisms tend to be widely distributed, and they are thought to be virtually cosmopolitan and selected primarily by environmental factors. Recent studies have shown that, despite these characteristics, microorganisms may behave like larger organisms and exhibit geographical distribution. In this study, we searched patterns of spatial diversity distribution of bacteria and archaea in a contiguous environment. We collected 26 samples of a lake sediment, distributed in a nested grid, with distances between samples ranging from 0.01 m to 1000 m. The samples were analyzed using T-RFLP (Terminal restriction fragment length polymorphism) targeting mcrA (coding for a subunit of methyl-coenzyme M reductase) and the genes of Archaeal and Bacterial 16S rRNA. From the qualitative and quantitative results (relative abundance of operational taxonomic units) we calculated the similarity index for each pair to evaluate the taxa-area and distance decay relationship slopes by linear regression. All results were significant, with mcrA genes showing the highest slope, followed by Archaeal and Bacterial 16S rRNA genes. We showed that the microorganisms of a methanogenic community, that is active in a contiguous environment, display spatial distribution and a taxa-area relationship.     

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs,Western Siberia,Russia

The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.     

Consuming un-captured methane from landfill using aged refuse bio-cover

A novel simulated bio-cover was developed to facilitate the biological methane oxidation process using aged refuse and aged sludge from landfill. It was found that 78.7% and 66.9% of CH₄ could be removed, with the aged refuse: aged sludge (w/w%) ratio of 7:3 and 6:4 in bio-cover system, respectively. The maximum CH₄ removal rate could reach 100%, when the aged refuse with the disposal time more than 14 years were applied in bio-cover. Some controlled factors for the methanotrophic activity, i.e. moisture, Eh and organic matter content, were also investigated. It was found that CH₄ oxidation rate increased greatly, when the moisture content and organic matter were increased from 6.0%, 4.8% to 8.0%, 9.5%, respectively. The optimum conditions for this bio-cover system was found to be as follows: aged refuse: aged sludge ratio of 7:3, the moisture content of 8-9%, Eh of 104-108 mV and organic matter of 9.5%.     

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model

Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.     

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.     

A meta-analysis of the microbial diversity observed in anaerobic digesters

In this study, the collective microbial diversity in anaerobic digesters was examined using a meta-analysis approach. All 16S rRNA gene sequences recovered from anaerobic digesters available in public databases were retrieved and subjected to phylogenetic and statistical analyses. As of May 2010, 16,519 bacterial and 2869 archaeal sequences were found in GenBank. The bacterial sequences were assigned to 5926 operational taxonomic units (OTUs, based on ?97% sequence identity) representing 28 known bacterial phyla, with Proteobacteria (1590 OTUs), Firmicutes (1352 OTUs), Bacteroidetes (705 OTUs), and Chloroflexi (693 OTUs) being predominant. Archaeal sequences were assigned to 296 OTUs, primarily Methanosaeta and the uncharacterized WSA2 group. Nearly 60% of all sequences could not be classified to any established genus. Rarefaction analysis indicates that approximately 60% of bacterial and 90% of archaeal diversity in anaerobic digesters has been sampled. This analysis of the global bacterial and archaeal diversity in AD systems can guide future studies to further examine the microbial diversity involved in AD and development of comprehensive analytical tools.     

Microbial biodiversity of thermophilic communities in hot mineral soils of Tramway Ridge, Mount Erebus, Antarctica

Tramway Ridge, located near the summit of Mount Erebus in Antarctica, is probably the most remote geothermal soil habitat on Earth. Steam fumaroles maintain moist, hot soil environments creating extreme local physicochemical differentials. In this study a culture-independent approach combining automated rRNA intergenic spacer analysis (ARISA) and a 16S rRNA gene library was used to characterize soil microbial (Bacterial and Archaeal) diversity along intense physicochemical gradients. Statistical analysis of ARISA data showed a clear delineation between bacterial community structure at sites close to fumaroles and all other sites. Temperature and pH were identified as the primary drivers of this demarcation. A clone library constructed from a high-temperature site led to the identification of 18 novel bacterial operational taxonomic units (OTUs). All 16S rRNA gene sequences were deep branching and distantly (85–93%) related to other environmental clones. Five of the signatures branched with an unknown group between candidate division OP10 and Chloroflexi . Within this clade, sequence similarity was low, suggesting it contains several yet-to-be described bacterial groups. Five archaeal OTUs were obtained and exhibited high levels of sequence similarity (95–97%) with Crenarchaeota sourced from deep-subsurface environments on two distant continents. The novel bacterial assemblage coupled with the unique archaeal affinities reinvigorates the hypotheses that Tramway Ridge organisms are relics of archaic microbial lineages specifically adapted to survive in this harsh environment and that this site may provide a portal to the deep-subsurface biosphere.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.