Detergent-resistant α-amylase derived from Anoxybacillus karvacharensis K1 and its production based on whey.

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5'-dithio-bis-[2-nitrobenzoic acid (DNTB), ß-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme's activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 µmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with ß-glucosidase for 24 h.

Mining thermophiles for biotechnologically relevant enzymes: evaluating the potential of European and Caucasian hot springs.

The development of sustainable and environmentally friendly industrial processes is becoming very crucial and demanding for the rapid implementation of innovative bio-based technologies. Natural extreme environments harbor the potential for discovering and utilizing highly specific and efficient biocatalysts that are adapted to harsh conditions. This review focuses on extremophilic microorganisms and their enzymes (extremozymes) from various hot springs, shallow marine vents, and other geothermal habitats in Europe and the Caucasus region. These hot environments have been partially investigated and analyzed for microbial diversity and enzymology. Hotspots like Iceland, Italy, and the Azores harbor unique microorganisms, including bacteria and archaea. The latest results demonstrate a great potential for the discovery of new microbial species and unique enzymes that can be explored for the development of Circular Bioeconomy.Different screening approaches have been used to discover enzymes that are active at extremes of temperature (up 120 °C), pH (0.1 to 11), high salt concentration (up to 30%) as well as activity in the presence of solvents (up to 99%). The majority of published enzymes were revealed from bacterial or archaeal isolates by traditional activity-based screening techniques. However, the latest developments in molecular biology, bioinformatics, and genomics have revolutionized life science technologies. Post-genomic era has contributed to the discovery of millions of sequences coding for a huge number of biocatalysts. Both strategies, activity- and sequence-based screening approaches, are complementary and contribute to the discovery of unique enzymes that have not been extensively utilized so far.

tRNA modification profiles in obligate and moderate thermophilic bacilli.

Transfer RNAs (tRNAs) are the most ancient RNA molecules in the cell, modification pattern of which is linked to phylogeny. The aim of this study was to determine the tRNA modification profiles of obligate (Anoxybacillus, Geobacillus, Paragebacillus) and moderate (Bacillus, Brevibacillus, Ureibacillus, Paenibacillus) thermophilic aerobic bacilli strains to find out its linkage to phylogenetic variations between species. LC-MS was applied for the quantification of modified nucleosides using both natural and isotopically labeled standards. The presence of m2A and m7G modifications at high levels was determined in all species. Relatively high level of i6A and m5C modification was observed for Paenibacillus and Ureibacillus, respectively. The lowest level of Cm modification was found in Bacillus. The modification ms2i6A and m1G were absent in Brevibacillus and Ureibacillus, respectively, while modifications Am and m22G were observed only for Ureibacillus. While both obligate and moderate thermophilic species contain Gm, m1G and ms2i6A modifications, large quantities of them (especially Gm and ms2i6A modification) were detected in obligate thermophilic ones (Geobacillus, Paragebacillus and Anoxybacillus). The collective set of modified tRNA bases is genus-specific and linked to the phylogeny of bacilli. In addition, the dataset could be applied to distinguish obligate thermophilic bacilli from moderate ones.

Anoxybacillus karvacharensis sp. nov., a novel thermophilic bacterium isolated from the Karvachar geothermal spring in Nagorno-Karabakh.

Twelve thermophilic Anoxybacillus strains were isolated from sediment and water samples from a Karvachar hot spring located in the northern part of Nagorno-Karabakh. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, one of the isolates, designated strain K1T, was studied in detail. The cells are straight, motile rods that are 0.2-0.4×2.3-7.2 µm in size. The strain is a Gram-stain-positive, moderately thermophilic facultative anaerobe with an optimum growth temperature of 60-65 °C and a growth temperature range of 45-70 °C. Growth of strain K1T was observed at pH 6-11 (optimum, pH 8-9) and was inhibited in the presence of NaCl concentrations above 2.5 % (optimum, 1-1.5 %). The isolate could utilize a wide variety of carbon sources, including d-arabinose, d-ribose, d-galactose, d-fructose, d-mannitol, maltose, aesculin, melibiose, sucrose, trehalose, raffinose, amidone, glycogen, turanose, d-lyxose, d-tagatose, potassium gluconate and 2-keto-gluconate. The strain was able to hydrolyse starch, casein and gelatin, was positive for oxidase and catalase, and reduced nitrate to nitrite, but was negative for H2S production. Production of urease and indole was not observed. The major cellular fatty acids were C15 : 0 iso, C16 : 0 and C17 : 0 iso (52.5, 13.6 and 19.6 % of total fatty acids, respectively). Strain K1T shares >99 % 16S rRNA sequence similarity and a genomic average nucleotide identity value of 94.5 % with its closest relative, Anoxybacillus flavithermus DSM 2641T, suggesting that it represents a separate and novel species, for which the name Anoxybacillus karvacharensis sp. nov. is proposed. The type strain of Anoxybacillus karvacharensis is K1T (=DSM 106524T=KCTC 15807T).

Microbial Diversity of Terrestrial Geothermal Springs in Armenia and Nagorno-Karabakh: A Review.

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8-70 °C and pH range 6.0-7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960-2090 m above sea level and are located on the Alpide (Alpine-Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus-Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91-97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.

Antimicrobial and antioxidant potentials of non-cytotoxic extracts of corticolous lichens sampled in Armenia.

Due to wide range of secondary metabolites, lichens were used from antiquity as sources of colorants, perfumes and medicaments. This research focuses on exploring the antioxidant, antimicrobial and cytotoxic activities of methanol, ethanol, acetone extracts and aqueous infusions of corticolous lichens sampled from Armenia. Methanol, ethanol and acetone extracts from all tested lichens were active against Gram-positive bacterial strains. The most effective solvent to retrieve antimicrobial compounds was methanol. Aqueous infusions of tested lichens didn't show any significant antibacterial and antifungal activity. The highest antimicrobial activity was observed for methanol extract of Ramalina sinensis. The minimum inhibitory concentration of methanol extract of Ramalina sinensis were 0.9-1.8 mg mL- 1. Pseudevernia furfuracea demonstrated antifungal activity (Ø 12 mm). Methanol extract of Parmelia sulcata demonstrated largest 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity (71 %). The cytotoxicity was measured on human HeLa (cervical carcinoma) cell lines using microculture tetrazolium test assay. The IC50 values estimated for methanol extracts of Peltigera praetextata, Evernia prunastri, Ramalina sinensis and Ramalina farinacea species in HeLa cell line were within 1.8-2.8 mg mL- 1 and considered as non-cytotoxic. Obtained results suggest that studied lichens can be prospective in biotechnologies as alternative sources of antimicrobial and antioxidant substances.

Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1.

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.

Onshore soil microbes and endophytes respond differently to geochemical and mineralogical changes in the Aral Sea.

There is limited knowledge about how microbiome develops along the geochemical and mineralogical change in onshore soils derived from continuous desiccation of lakes. In this study, geochemistry and mineralogy were studied in the Aral Sea onshore soils exposed in different periods (from the 1970s to 2018), followed by microbial analyses on the studied soils and the aboveground organs of dominant plants. The soils exhibited an increasing gradient of total soluble salts (TSS: 0.4-0.5 g/L to 71.3 g/L) and evaporite minerals (e.g., gypsum, halite) from the farshore to the nearshore. In the studied soils, microbial diversity decreased with increasing TSS, and microbial community dissimilarities among samples was positively correlated with the contents of gypsum and calcite minerals. Among the measured environmental variables, minerals contributed most to the observed microbial variation. In contrast, the endophytic microbial communities in the aboveground organs of dominant plants were not related to any of the measured variables, indicating that they differed from their soil counterparts with respect to their responses to geochemical and mineralogical variations in soils. In summary, these results help us understand the response of onshore soil microbiome to the decline of lake water caused by continuous desiccation.

Draft Genome Sequence of Escherichia coli Strain Tj, Isolated from the Varzob River in Tajikistan.

The 4.6-Mbp draft genome sequence of Escherichia coli strain Tj, isolated from the Varzob River in Tajikistan, is presented. This strain possesses four prophage elements related to Shigella phage SfV, E. coli O157:H7-specific phage ϕV10, lambdoid phage HK225, and coliphage Ayreon. It contains a gene encoding a hemolysin E toxin.

Geothermal springs in Armenia and Nagorno-Karabakh: potential sources of hydrolase-producing thermophilic bacilli.

In recent years, scientists have increasingly focused on the microbial diversity of high-altitude hot springs to explore the biotechnological applications of extremophiles. In this regard, a total of 107 thermophilic bacilli were isolated from 9 high-altitude mineralized geothermal springs (of temperatures ranging from 27.5 to 70 °C) located within the territory of Armenia and Nagorno-Karabakh. The isolated bacilli were phylogenetically profiled and studied for their potential to produce extracellular hydrolytic enzymes (protease, amylase, and lipase). The identification of isolates based on 16S rRNA gene sequences revealed their relationship to members of more than 22 distinct species, of 8 different genera, namely Aeribacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Parageobacillus, Paenibacillus and Ureibacillus. Bacillus licheniformis, Parageobacillus toebii and Anoxybacillus flavithermus were found to be the most abundant species in the springs that were studied. Some of the isolated bacilli shared less than 91-97% sequence identity with their closest match in GenBank, indicating that Armenian geothermal springs harbor novel bacilli, at least at the species level. 71% of the isolates actively produced at least one or more extracellular proteases, amylases, or lipases. In total, 22 strains (28.6%) were efficient producers of all three types of thermostable enzymes.

A glimpse of the prokaryotic diversity of the Large Aral Sea reveals novel extremophilic bacterial and archaeal groups.

During the last five decades, the Aral Sea has gradually changed from a saline water body to a hypersaline lake. Microbial community inhabiting the Aral Sea has been through a succession and continuous adaptation during the last 50 years of increasing salinization, but so far, the microbial diversity has not been explored. Prokaryotic diversity of the Large Aral Sea using cultivation-independent methods based on determination of environmental 16S rRNA gene sequences revealed a microbial community related to typical marine or (hyper) saline-adapted Bacteria and Archaea. The archaeal sequences were phylogenetically affiliated with the order Halobacteriales, with a large number of operational taxonomic units constituting a novel cluster in the Haloferacaceae family. Bacterial community analysis indicated a higher diversity with representatives belonging to Proteobacteria, Actinobacteria and Bacteroidetes. Many members of Alphaproteobacteria and Gammaproteobacteria were affiliated with genera like Roseovarius, Idiomarina and Spiribacter which have previously been found in marine or hypersaline waters. The majority of the phylotypes was most closely related to uncultivated organisms and shared less than 97% identity with their closest match in GenBank, indicating a unique community structure in the Large Aral Sea with mostly novel species or genera.

Insights into the Bacterial Diversity of the Acidic Akhtala Mine Tailing in Armenia Using Molecular Approaches.

The impact of the heavy metal contamination and acidity on the bacterial community was studied in samples collected from the Akhtala copper mine tailing using molecular approaches. The bacterial community structure analysis by PCR-DGGE fingerprinting revealed an abundance of Firmicutes, Acidobacteria, and Proteobacteria in different layers of the Akhtala tailing. 454 pyrotag sequence analyses revealed that a significant part of the sequences originated from Proteobacteria (49%) and Bacteroidetes (43%). Bacterial taxa are distributed also in phyla Saccharibacteria (2%), Verrucomicrobia (1.5%), Gammatimonadetes (1%), and some minor additional bacterial groups. The main primary producers in the Akhtala tailing appear to be obligate autotrophic Thiobacillus and Sulfuritalea species. Representatives of Lutibacter and Lysobacter genera are the most abundant acid-tolerant heterotrophs in the studied tailing. The presence of a large number of yet-uncultivated species emphasizes the importance of the future exploration of the tailing as an important source of novel bacteria.

Production and characterization of exopolysaccharides by Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains isolated from an Armenian geothermal spring.

The thermal ecosystems, including geothermal springs, are proving to be source of thermophiles able to produce extracellular polysaccharides (EPSs). Among the sixteen thermophilic bacilli isolated from sediment sampled from Arzakan geothermal spring, Armenia, two best EPSs producer strains were identified based on 16S rRNA gene sequence analysis and phenotypic characteristics, and designated as Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains. EPSs production was investigated under different time, temperature and culture media's composition. The highest specific EPSs production yield (0.27 g g-1 dry cells and 0.22 g g-1 dry cells for strains G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively) was observed after 24 h when fructose was used as sole carbon source at 65 °C and pH 7.0. Purified EPSs displayed a high molecular mass: 5 × 105 Da for G. thermodenitrificans ArzA-6 and 6 × 105 Da for G. toebii ArzA-8. Chemical composition and structure of the biopolymers, determined by GC-MS, HPAE-PAD and NMR, showed that both the two EPSs are heteropolymers composed by mannose as major monomer unit. Optical rotation values [α] D25 °C of the two EPSs (2 mg ml-1 H2O) were - 142,135 and - 128,645 for G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively.

Bacilli community of saline-alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods.

The bacterial community composition in the A horizon of a natural saline-alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline-alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7-99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline-alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

BACKGROUND: Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. RESULTS: In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. CONCLUSIONS: The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Draft Genome Sequence of Thermus scotoductus Strain K1, Isolated from a Geothermal Spring in Karvachar, Nagorno Karabakh.

The 2,379,636-bp draft genome sequence of Thermus scotoductus strain K1, isolated from geothermal spring outlet located in the Karvachar region in Nagorno Karabakh is presented. Strain K1 shares about 80% genome sequence similarity with T. scotoductus strain SA-01, recovered from a deep gold mine in South Africa.

Novel Methanotrophs of the Family Methylococcaceae from Different Geographical Regions and Habitats.

Terrestrial methane seeps and rice paddy fields are important ecosystems in the methane cycle. Methanotrophic bacteria in these ecosystems play a key role in reducing methane emission into the atmosphere. Here, we describe three novel methanotrophs, designated BRS-K6, GFS-K6 and AK-K6, which were recovered from three different habitats in contrasting geographic regions and ecosystems: waterlogged rice-field soil and methane seep pond sediments from Bangladesh; and warm spring sediments from Armenia. All isolates had a temperature range for growth of 8-35 °C (optimal 25-28 °C) and a pH range of 5.0-7.5 (optimal 6.4-7.0). 16S rRNA gene sequences showed that they were new gammaproteobacterial methanotrophs, which form a separate clade in the family Methylococcaceae. They fell into a cluster with thermotolerant and mesophilic growth tendency, comprising the genera Methylocaldum-Methylococcus-Methyloparacoccus-Methylogaea. So far, growth below 15 °C of methanotrophs from this cluster has not been reported. The strains possessed type I intracytoplasmic membranes. The genes pmoA, mxaF, cbbL, nifH were detected, but no mmoX gene was found. Each strain probably represents a novel species either belonging to the same novel genus or each may even represent separate genera. These isolates extend our knowledge of methanotrophic Gammaproteobacteria and their physiology and adaptation to different ecosystems.

Microbial diversity in an Armenian geothermal spring assessed by molecular and culture-based methods.

The phylogenetic diversity of the prokaryotic community thriving in the Arzakan hot spring in Armenia was studied using molecular and culture-based methods. A sequence analysis of 16S rRNA gene clone libraries demonstrated the presence of a diversity of microorganisms belonging to the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, Firmicutes, Bacteroidetes phyla, and Cyanobacteria. Proteobacteria was the dominant group, representing 52% of the bacterial clones. Denaturing gradient gel electrophoresis profiles of the bacterial 16S rRNA gene fragments also indicated the abundance of Proteobacteria, Bacteroidetes, and Cyanobacteria populations. Most of the sequences were most closely related to uncultivated microorganisms and shared less than 96% similarity with their closest matches in GenBank, indicating that this spring harbors a unique community of novel microbial species or genera. The majority of the sequences of an archaeal 16S rRNA gene library, generated from a methanogenic enrichment, were close relatives of members of the genus Methanoculleus. Aerobic endospore-forming bacteria mainly belonging to Bacillus and Geobacillus were detected only by culture-dependent methods. Three isolates were successfully obtained having 99, 96, and 96% 16S rRNA gene sequence similarities to Arcobacter sp., Methylocaldum sp., and Methanoculleus sp., respectively.

An integrated study reveals diverse methanogens, Thaumarchaeota, and yet-uncultivated archaeal lineages in Armenian hot springs.

Culture-independent and enrichment techniques, with an emphasis on members of the Archaea, were used to determine the composition and structure of microbial communities inhabiting microbial mats in the source pools of two geothermal springs near the towns of Arzakan and Jermuk in Armenia. Amplification of small-subunit rRNA genes using "universal" primers followed by pyrosequencing (pyrotags) revealed highly diverse microbial communities in both springs, with >99 % of pyrosequences corresponding to members of the domain Bacteria. The spring in Arzakan was colonized by a photosynthetic mat dominated by Cyanobacteria, in addition to Proteobacteria, Bacteroidetes, Chloroflexi, Spirochaeta and a diversity of other Bacteria. The spring in Jermuk was colonized by phylotypes related to sulfur, iron, and hydrogen chemolithotrophs in the Betaproteobacteria and Epsilonproteobacteria, along with a diversity of other Bacteria. Analysis of near full-length small subunit rRNA genes amplified using Archaea-specific primers showed that both springs are inhabited by a diversity of methanogens, including Methanomicrobiales and Methanosarcinales and relatives of Methanomassiliicoccus luminyensis, close relatives of the ammonia-oxidizing archaeon (AOA) "Candidatus Nitrososphaera gargensis", and the yet-uncultivated Miscellaneous Crenarchaeotal Group and Deep Hydrothermal Vent Crenarchaeota group 1. Methanogenic enrichments confirmed the predicted physiological diversity, revealing methylotrophic, acetoclastic, and hydrogenotrophic methanogenesis at 45 and 55 °C, but not 65 °C. This is one of only a few studies combining cultivation-independent and -dependent approaches to study archaea in moderate-temperature (37-73 °C) terrestrial geothermal environments and suggests important roles for methanogenic archaea and AOA in the carbon and nitrogen biogeochemical cycles in these environments.

Cultivation and characterization of thermophilic Nitrospira species from geothermal springs in the US Great Basin, China, and Armenia.

Despite its importance in the nitrogen cycle, little is known about nitrite oxidation at high temperatures. To bridge this gap, enrichment cultures were inoculated with sediment slurries from a variety of geothermal springs. While nitrite-oxidizing bacteria (NOB) were successfully enriched from seven hot springs located in US Great Basin, south-western China, and Armenia at ≤ 57.9 °C, all attempts to enrich NOB from > 10 hot springs at ≥ 61 °C failed. The stoichiometric conversion of nitrite to nitrate, chlorate sensitivity, and sensitivity to autoclaving all confirmed biological nitrite oxidation. Regardless of origin, all successful enrichments contained organisms with high 16S rRNA gene sequence identity (≥ 97%) with Nitrospira calida. In addition, Armenian enrichments also contained close relatives of Nitrospira moscoviensis. Physiological properties of all enrichments were similar, with a temperature optimum of 45-50 °C, yielding nitrite oxidation rates of 7.53 ± 1.20 to 23.0 ± 2.73 fmoles cell(-1) h(-1), and an upper temperature limit between 60 and 65 °C. The highest rates of NOB activity occurred with initial NO2 - concentrations of 0.5-0.75 mM; however, lower initial nitrite concentrations resulted in shorter lag times. The results presented here suggest a possible upper temperature limit of 60-65 °C for Nitrospira and demonstrate the wide geographic range of Nitrospira species in geothermal environments.