Nikolay Pimenov

Sulfate Reduction Potential in Sediments in the Norilsk Mining Area, Northern Siberia

Abstract The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a 35SO4 2− tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm−3 day−1. Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 107 cells ml−1. The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28°C vs. 4°C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4°C, but they were also found in mesophilic enrichments.

COMPOSITION AND ACTIVITIES OF MICROBIAL COMMUNITIES INVOLVED IN CARBON,SULFUR, NITROGEN AND MANGANESE CYCLING IN THE OXIC/ANOXIC INTERFACE OF THE BLACK SEA

Own and literature data on the structure and functional activities of microbial communities in the Black Sea chemocline are reviewed. Bacterial numbers in the oxic/anoxic interface increase by an order of magnitude compared to above-lying waters. The dark carbon dioxide fixation rate increases too and often does not correspond to the maximum in total cell numbers. Carbon isotope measurements of particulate organic carbon indicate that bacterial chemosynthesis (rates are between 9.6 and 25 mmol C m −2 d −1 ) is the main source of organic matter in the Black Sea chemocline and accounts for 20-50% of total primary production. The increased dark CO2 fixation rates in the chemocline reflect a mixed signal derived from CO2 fixation of a number of lithoautotrophic bacteria involved in sulfide oxidation, but also with methanotrophs, methanogens and with sulfate reduc- ers. Chemolithoautotrophic bacteria related to Thiobacillus and Thiomicrospira and heterotrophic sulfur oxidizing Rhizobiaceae strains are probably the main sulfide oxidizing bacteria. Shewanella species using Mn (Fe) oxyhydroxides as an alternative electron acceptor to dissolved oxygen may play a role in sulfide oxidation too. Anoxygenic photosynthesis mediated by green sulfur bacteria re- lated to Chlorobium accounts for not more than 13% of the total sulfide flux (Overmann and Manske, this volume). Anammox bacteria together with deni- trifiers may be mainly responsible for the inorganic nitrogen loss in the interface. Below the interface, sulfate reduction, methane oxidation and methanogenesis co-occur. Highest sulfate reduction rates are observed below the interface down to 300 m; however this process is detected throughout the entire water column. Several lines of evidence (isolates, rate measurements, molecular fingerprinting and biomarkers) suggest that both, aerobic and anaerobic methane oxidation oc- cur at the Black Sea oxic/anoxic interface and in anoxic waters. ANME-1 and

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 (KS1xa0=xa054xa0nM and KS2xa0=xa01xa0μM). 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 (20xa0nM), was found to be 120xa0μmolxa0l−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 106xa0cellsxa0ml−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.

2 In Situ Activity Studies in Thermal Environments

Publisher Summary The chapter discusses the essentials of in situ method and its advantages and limitations in application to natural thermal environments: terrestrial hot springs and hydrothermal sediments, shallow-water and deep-sea submarine hot vents, and deep-subsurface thermal habitats. There are several independent approaches to in situ investigations of microbial activities in different types of environments. The use of collecting chambers, for example, allowed methane flux rates to be determined in shallow aquatic habitats. Experiments with radioactively labelled substrates allow their microbiological transformations to be traced over very short exposition periods. Rate estimation of microbial processes in different ecosystems by radioisotopic methods has been widely used. Radioactive substrates (containing 14 C, 35 S, 3 H, etc.) are added to isolated natural samples that are to be incubated for a short period of time at the sampling site or under laboratory conditions closest to those in situ . Radioisotopic methods are widely used to study large-scale microbial processes. The rates obtained for such ecosystems as oceanic and fresh water sediments, soils, swamps and marshes make it possible to estimate annual production and consumption rates of major biogenic elements.

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.

Microbial life in Bourlyashchy, the hottest thermal pool of Uzon Caldera, Kamchatka

Bourlyashchy is the largest and hottest pool in the Uzon Caldera, located in the territory of Kronotsky Nature Reserve, Kamchatka, Russia, with sediment surface temperatures at the margins ranging from 86 to 97xa0°C, and pH from 6.0 to 7.0. The microbial communities of the pool water and sediments were studied comprehensively from 2005 to 2014. Radioisotopic tracer studies revealed the processes of inorganic carbon assimilation, sulfate reduction, lithotrophic methanogenesis and potentially very active process of acetate oxidation to CO2. The total number of microbial cells in water was different in different years ranging from 5.2 to 7.0xa0×xa0106; in sediments, it changed from year to year between 6.3xa0×xa0106 and 1.75xa0×xa0108, increasing with a decrease in temperature. FISH with Archaea- and Bacteria-specific probes showed that the share of Bacteria differed with year, changing from 34 to 71xa0%. According to 16S rRNA gene pyrosequencing data, lithoautotrophs (Aquificales and Thermoproteales) predominated in water samples, while in sediments they shared the niche with organotrophic Crenarchaeota, Korarchaeota, and bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). The majority of organisms in water belonged to cultivated orders of prokaryotes; the only large uncultured group was that representing a novel order in class Thermoprotei. In sediments, unclassified Aquificeae comprised a significant part of the bacterial population. Thus, we showed that the hottest of the terrestrial hot pools studied contains numerous and active microbial populations where Bacteria represent a significant part of the microbial community, and planktonic and sediment populations differ in both composition and function.

Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island, Russian Far East

Aerobic methane oxidation has been mostly studied in environments with moderate to low temperatures. However, the process also occurs in terrestrial thermal springs, where little research on the subject has been done to date. The potential activity of methane oxidation and diversity of aerobic methanotrophic bacteria were studied in sediments of thermal springs with various chemical and physical properties, sampled across the Kunashir Island, the Kuriles archipelago. Activity was measured by means of the radioisotope tracer technique utilizing 14C-labeled methane. Biodiversity assessments were based on the particulate methane monooxygenase (pmoA) gene, which is found in all known thermophilic and thermotolerant methanotrophs. We demonstrated the possibility of methane oxidation in springs with temperature exceeding 74xa0°C, and the most intensive methane uptake was shown in springs with temperatures about 46xa0°C. PmoA was detected in 19 out of 30 springs investigated and the number of pmoA gene copies varied between 104 and 106 copies per ml of sediment. Phylogenetic analysis of PmoA sequences revealed the presence of methanotrophs from both the Alpha- and Gammaproteobacteria. Our results suggest that methanotrophs inhabiting thermal springs with temperature exceeding 50xa0°C may represent novel thermophilic and thermotolerant species of the genera Methylocystis and Methylothermus, as well as previously undescribed Gammaproteobacteria.

Gammaproteobacterial Methanotrophs Dominate Cold Methane Seeps in Floodplains of West Siberian Rivers

ABSTRACT A complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4 flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH4 h−1, while some seeps emitted up to 5.54 g CH4 h−1. The δ13C value of methane released from these seeps varied between −71.1 and −71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4 ml−1 day−1) were measured in mud samples. Fluorescence in situ hybridization detected 107 methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH4 sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.

Methane fluxes in the southeastern Baltic Sea

New data from surveys of gas-bearing mud areas in the Gdansk Deep (southeastern Baltic Sea) were collected during four research cruises in 2009–2011. These revealed the presence of seven large pockmarks apart from the three already known, and enabled significant improvement of the existing digital map of gassy mud distribution. Based on geochemical sediment analyses, calculated diffusive methane fluxes from the upper (0–5xa0cm) seabed layer into near-bottom waters were highest—3.3xa0mmol/(m2u2009day)—in pockmark mud, contrasting strongly with the minimum value of 0.004xa0mmol/(m2u2009day) observed in typical, background mud. However, fluxes of less than 0.1xa0mmol/(m2u2009day) were observed in all sediment types, including pockmarks. In a newer attempt to roughly estimate budgets at a more regional scale, diffusive methane venting amounts to 280u2009×u2009106u2009mmol/day for southeastern Baltic Sea muddy sediments. Elongated pockforms in the southern Gotland Deep, known since the end of the 1980s as pockmarks, had methane concentrations that were similar to those of gassy mud from the Gdansk Basin, and there was no geo-acoustic evidence of considerably increased gas levels.

Activity and Distribution of Thermophilic Prokaryotes in Hydrothermal Fluid, Sulfidic Structures, and Sheaths of Alvinellids (East Pacific Rise, 13°N)

ABSTRACT Processes of inorganic carbon assimilation, methanogenesis, sulfate reduction, and acetate oxidation to CO2 occurring in samples from the East Pacific Rise at 13°N were traced, using radioisotopically labeled substrates, at temperatures ranging from 65 to 100°C. Molecular hydrogen stimulated lithotrophic methanogenesis and sulfate reduction but inhibited inorganic carbon assimilation. Active mineralization of acetate was observed in an organic-rich Alvinella-associated system at 80°C. Members of the Thermococcales were the most numerous hyperthermophilic archaea in these samples, their density achieving 108 cells per cm3, while the numbers of cultured hydrogen-utilizing thermophilic lithotrophs were several orders of magnitude lower.