Activity and Diversity of Aerobic Methanotrophs in Thermal Springs of the Russian Far East

C H A P T E R

1 Activity and Diversity of Aerobic Methanotrophs in Thermal Springs of the Russian Far East Ekaterina N. Tikhonova, and Irina K. Kravchenko Laboratory of Microbial Survival, Federal State Institution, Federal Research Centre Fundamentals of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation

O U T L I N E 1.1 Introduction

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1.2 Terrestrial Thermal Springs of the Russian Far East: Overviev of Thermal Springs

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1.3 Origin and Geographical Distribution of Terrestrial Russian Far East Thermal Springs

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1.4 Composition of Hydrothermal Fluids

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1.5 Microbial Communities of Hydrothermal Springs

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1.6 Methane Cycling in Hot Springs Methane Formation

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1.7 Composition of Magmatic Gases

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1.8 Methanogenic Activity

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1.9 Activity and Diversity of Methanotrophic Communities in Thermal Springs 1.9.1 Methane Oxidation

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1.14 Methane Oxidation in Hot Springs of Far-East Russian Volcanic Belt: Kamchatka and Kuril Islands

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1.15 Intensity of Ch4 Oxidation Evaluated by Ratio-Tracer Analysis

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1.16 Quantification of Aerobic Methanotrophs in Thermal Springs: Number of Copies of pmoA, mxaF and 16s rRNA Genes

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1.17 Evaluation of Active Methanotrophs by Fish Technique

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1.18 Diversity of Methanotrophs in Thermal Springs Based on PCR-DGGE Analysis of pmoA Genes

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1.19 Isolation and Characterization of Methane-Oxidation Cultures

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1.20 Growth of Enrichments

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1.10 Methanotrophs: A Brief Introduction

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1.21 Study of the Phylogenetic Diversity of Enriched Methanotrophic Cultures

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1.11 Classification of Methanotrophs

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1.22 Conclusions

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1.12 Thermophilic Methanotrophs

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References

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1.13 Methanotrophic Communities of Terrestrial Geothermal Springs

Further Reading

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New and Future Developments in Microbial Biotechnology and Bioengineering https://doi.org/10.1016/B978-0-444-64191-5.00001-8

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© 2019 Elsevier B.V. All rights reserved.

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1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

1.1 INTRODUCTION Methane (CH4) is a potent greenhouse gas, with a present mixing ratio of 1.8 p.p.m.v. and a global warming potential 25 times greater than carbon dioxide (Nisbet et al., 2014). Methane has a much shorter atmospheric lifetime (12 years) compared with carbon dioxide (5–200 years); so, it may be possible to regulate methane atmospheric concentrations through a reduction in emissions (IPCC, 2007). Methane mitigation strategies not only restrict atmospheric CH4 accumulation and the associated climate impact, but also provide a valuable industrial fuel source and chemical feedstock. Methane is a strong greenhouse gas and the major biogenic source, which is the end product of the microbial degradation of organic matter in anoxic environments. Major sources of abiogenic methane are underground reservoirs in geothermal regions, where methane is released to the atmosphere through seeps, gas venting, and degassing of spring water (Etiope and Klusman, 2002; Aronson et al., 2013; Nazaries et al., 2013). Methanotrophs are a unique group of microorganisms that use methane as their sole source of energy (Hanson and Hanson, 1996). Aerobic methanotrophs are present in different environments where methane and O2 are available and consume 10%–90% of the methane produced in the deep anoxic layers of wetland environments before it reaches the atmosphere. Methanotrophs include species adapted to extremes in pH (Pol et al., 2007; Islam et al., 2008; Dedysh et al., 2000; Danilova et al., 2013 Sorokin et al., 2000), salinity (Trotsenko and Murrell, 2008; Singh et al., 2017a, b), and temperature (Bowman et al., 1997; Berestovskaya et al., 2002; Bodrossy et al., 1997, 1999; Dunfield et al., 2007). Most known species of proteobacterial methanotrophs are mesophiles, but recently studies of thermal springs in Hungary and Japan led to the isolation of moderately thermophilic gammaproteobacterial methanotrophs belonging to the genera Methylocaldum (Bodrossy et al., 1997) and Methylothermus (Bodrossy et al., 1999; Tsubota et al., 2005) with optimal growth temperatures of 45–60°C. Methylothermus strain HB is the most thermophilic methanotroph known with a reported upper growth limit of 72°C (Bodrossy et al., 1999); however, this strain has been lost. A related strain with an upper growth limit of 67°C has been validated as Methylothermus thermalis (Tsubota et al., 2005). Comparative analysis of a signature methane oxidation gene (pmoA) or 16S rRNA genes has identified proteobacterial methanotrophs in geothermal waters and sediments from hot springs at 55–70°C (Bodrossy et al., 1999), 13.4–76.4°C (Sharp et al., 2014), and 46.5–99°C (Kizilova et al., 2013). The Russian Far East is one of the most active in the world terrestrial geothermal region with volcanic areas covered by springs of varying temperature (Grasby et al., 2000). Among the various groups of hot springs the chapter looks into the following two: the first group is closely related to modern volcanic activities of the Pacific plate (Eastern Kamchatka, Uzon geothermal area) and second—volcanoes of Kuril Islands. Until now, very limited data were available for CH4 oxidation and consumption in Russian terrestrial hot springs. So, monitoring and process understanding of CH4 consumption in these ecosystems is required to estimate global greenhouse gases balance and the contribution to global warming. This chapter, firstly to our knowledge, synthesizes the environmental and climatic factors influencing methane consumption regarding activity and diversity of methanotrophic communities in geothermal springs of the Russian Far East. Relationships between biomarkers and environmental variables including pH and temperature were evaluated to determine the factors that control microbial diversity in these geothermal environments.

1.2 TERRESTRIAL THERMAL SPRINGS OF THE RUSSIAN FAR EAST: OVERVIEV OF THERMAL SPRINGS Thermal springs are natural geological phenomena that occur on all continents (Fig. 1.1). There is no accurate distinction in temperature for springs and thermal springs, but usually the definition of a thermal spring is based on the mean annual air temperature of the specific site (Pentecost et al., 2003). Thermal springs originate either from recent plutonic activity (volcanic origin) or from rainwater that percolates into the ground through permeable rocks or via pipes in less permeable rocks (meteoric origin) (Moreaux and Tanner, 2001). The temperature of meteoric origin water usually increases at a rate 2–3°C per 100 m (geothermal gradient) (Press and Siever, 1986). If hot, the groundwater meets a fracture zone or an impermeable barrier; it may reach the surface as a thermal spring. Thus, the temperature of the thermal spring reflects the depth of the water and the rate at which it reaches the surface (Grasby and Hutcheon, 2001). Hydrothermal fluids are hot aqueous solutions circulating in the Earth’s crust and participating in the processes of movement and deposition of minerals. They come to the surface in areas of active volcanism in places of cracks, at the junction of lithospheric plates, carrying dissolved chemical elements; they form hydrotherms on the land and in the

1.3 ORIGIN AND GEOGRAPHICAL DISTRIBUTION OF TERRESTRIAL RUSSIAN FAR EAST THERMAL SPRINGS

FIG. 1.1

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Map of the Earth with the distribution of the volcanes. From http://volcano.si.edu/.

shallow and deep ocean areas (Brock, 1986). In some cases, hot springs can be found far from the active areas of volcanism. The water in such hot springs penetrates deep enough to contact with the heated rocks due to the so-called “geothermal gradient.” However, the majority of hydrothermal springs are fed by water that is heated by the igneous intrusions in areas of active volcanism. In these regions, the greatest concentration and diversity of surface hydrotherms occurs. Hot springs and volcanic areas, such as Yellowstone Park in United States, Italy, New Zealand, Kamchatka, and the Caucasus, have a variable chemical composition of water and different temperatures, as ground water mixed in different proportions with volcanic gases and reacts differently with the host rocks through which they seep into the depths. The waters are sodium-chloride, acid sulfate-chloride, acid sulfate, sodium, calcium-bicarbonate, etc. (Koronovskyi and Yakushova, 1991). Thermal waters contain many radioactive substances, in particular radon. Thus, the chemical composition of the hydrothermal solution is determined by the nature of its pathway in the depths of the Earth’s crust and reflects the composition of the rocks, as well as the time of its stay in the deep layers. Often, hydrothermal solutions are more or less enriched with various gas components (Von Damm, 1995). As minerals are generally more soluble in hot water, thermal springs are enriched with trace elements. The pH also affects the solubility of minerals. Hence, the specific chemical composition of spring water depends largely on the composition of rainwater, its temperature and pH, and the geology of the aquifer and the rocks through which the water rises to the surface. There are different classifications of thermal springs. A number are based on the origin of springs, and some on physical properties such as flow rate and/or temperature. Others are classified according to geology, chemical composition, or a combination of these (Moreaux and Tanner, 2001). Each is an isolated ecotope with a unique set of characteristics (high temperatures, high concentrations of different elements, and extreme pH values). Taking into account that hydrothermal deposits were formed throughout the long-term development of the Earth’s crust from 2.5 billion years ago to the present day (Klales et al., 2012), it can be argued that each of them is a separate ecosystem.

1.3 ORIGIN AND GEOGRAPHICAL DISTRIBUTION OF TERRESTRIAL RUSSIAN FAR EAST THERMAL SPRINGS Hydrothermal formations in the Russian Far East are concentrated on the Kuril-Kamchatka island arc (Fig. 1.2). Kamchatka, which is about 370,000 km2, is one of the most active regions of volcanism because it is located in the transitional zone where the Eurasian, the North American, and the Pacific plates meet. As a result, Kamchatka has numerous hydrothermal systems, which constantly release geothermal gases and fluids out to the Earth surface. The Kamchatka Peninsula is one of the biggest areas of modern volcanic activity on the Earth and the location of more than one hundred volcanoes and numerous thermal fields with hot springs, geysers, mud pools, and other features

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1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

FIG. 1.2 Sketch map of the study region.

of postvolcanic activity. There are over 30 active volcanoes and numerous hot springs and most of them are located in central and eastern Kamchatka. Eastern Kamchatka may be subdivided in five areas: Geyser Valley, Uzon Caldera, Academii Nauk Caldera, Pauzhetka, and Kireunckaya (Karpov et al., 2000). Uzon Caldera and Geyser Valley systems located along a collapsed caldera are the best described hydrothermal systems in the area.

1.4 COMPOSITION OF HYDROTHERMAL FLUIDS Being created by hot mineralized gas-liquid solutions circulating in the Earth’s crust, hydrothermal deposits are of great importance for the extraction of many important minerals. This is especially so for nonferrous, rare, noble, and radioactive metals, the vast majority of copper, lead, zinc, antimony, molybdenum, mercury, silver, cadmium, and lithium, as well as a significant proportion of gold, cobalt, uranium, tin, and tungsten. The same genesis has the number of nonmetallic raw materials: chrysotile asbestos, magnesite, fluorite, barite, as well as rock crystal, calcite, phlogopite, graphite. The accumulation of minerals in this case occurs as a result of the deposition of mineral masses in the cavities of rocks and in connection with the replacement of the latter. Numerous studies of modern mineral sources give evidence of mineral formation from hydrotherms. Hot water (80–96°C) of the Uzon-Gay system in Kamchatka for 100 years made (in thousand tons): arsenic-26, antimony-5, mercury—2.5, zinc-2, lead, and copper 2.5 (Chudaev, 2013). The fumaroles of the Thousand Smoke Valley in Alaska annually produce over a million tons of hydrochloric acid and about 200 thousand tons of hydrofluoric acid. The hot waters of the deep borehole of southern California are represented by highly concentrated (36%) hydrothermal solution, with alkali chlorides, 2 g/t of silver, 15 g/t of copper, 100 g/t of lead, and 700 g/t of zinc (Chudaev, 2003). Aqueous fluids have been analyzed by a variety of methods and the concentration of salts was generally less than 10% by weight, but may range from more than 50 to practically 0%. The salts consist of major amounts of Na, K, Ca, Mg, Cl, and SO4, with lesser amounts of Li, Al, BO3, PO4, HSiO6, HCO3, and many other ions. Many individual ions may predominate, but Na and Cl are generally the most abundant. Free carbon dioxide, both as gas and liquid, is not uncommon and may be dominant. In high-temperature springs from Kamchatka and Kuril Islands, bicarbonate contents are not significant; chlorine and sulfate—ions are predominant (Chudaev, 2013). The geothermal system in Kamchatka has considerable mineral and hydrocarbon resources. Volcanic ore formation prevails in the high thermal activity regions of Kamchatka. For instance, sulfur and Hg-Sb-As-FeS are two major ores observed in the Uzon Caldera. It is estimated that the total deposition of elements in the area is about 1000 tons of sulfur, 7000 tons of arsenic, 350 tons of antimony, and 200 tons of mercury (Karpov, 1992; Karpov and Naboko, 1990). The formation of oil is also observed in the Uzon Caldera. Structural analyses indicate that oils are dominated by n-alkanes with C18 as the most abundant compound. Analyses of biomarker and carbon isotopic

1.5 MICROBIAL COMMUNITIES OF HYDROTHERMAL SPRINGS

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signatures suggest that oils are generated from multiple origins including recent plants and ancient biomass (Bazhenova et al., 1998). Volcanic activity releases geothermal fluids containing large amounts of gases generated through both biotic and abiotic processes. In Kamchatka, free N2 and CO2 prevail in the outflows but H2, CH4, and H2S also occur frequently (Dymkin et al., 1988). The gas composition appears to correspond to the specific water chemistry and surface water temperature. In addition, aromatic hydrocarbons and alkenes are present in fumaroles, which may be generated through thermal degradation of organic matter (Capaccioni et al., 1993). In the outflow geothermal gases, N2 and CO2 usually prevail, but H2, CH4, and H2S also occur frequently. Hot spring waters in Kamchatka may have multiple origins including meteoric and magmatic water. The temperature of these hot springs ranges from 20°C to greater than 90°C. Water chemistry also varies dramatically with a pH range from 3.1 to 9.8. The origin of the water can be determined based on the stable isotopes data of hydrogen and oxygen. Values of δD and δ18O that fall along the meteoric line having a slope of 8 are characteristic of meteoric water, whereas values that fall off the line may be modified by other processes, for example, evaporation or mixing (Clark and Fritz, 1997). The δD of the hot springs values fall in the range of
119% to
74% and the δ18O falls in the range of
16% to
2% (Karpov et al., 2000). These values suggest that waters in these hot springs are predominantly meteoric water in origin with some dominated by magmatic water, which typically have a window of δ18O 3% to 11% and δD
94% to
40%. Because magmatic water usually cooccurs with magmatic gas emissions, the isotope data also indicate that some waters having H2S contain magmatic components while waters dominated by N2, CH4, and CO2 are practically free of magmatic components (Cheshko, 1994). Spring waters in Kamchatka also vary in pH (3.1–9.8). The chemical composition of the water varies with the geographical origin and the maximal surface-temperature of the spring. For example, the Geyser Valley-Uzon Caldera systems are dominated by high-temperature outflow springs, which are enriched with sodium chloride, silicic acid, and boron (Karpov and Naboko, 1990). Based on the temperature of venting water and the dominant chemical composition, five hydrochemical types are distinguished: Na-Cl—hyper high temperature, Na-Cl-SO4-HCO3—high temperature, Na-Ca-SO4—medium temperature, Na-HCO3-Cl—medium temperature, and Na-Ca-HCO3-SO4—low temperature. Waters with high temperature and acidic to neutral pH generally contain more salts than water with lower temperature and acidic water (Karpov et al., 2000). Furthermore, NadCl waters are formed in the basement of volcanoes, whereas Na-Ca-Cl-HCO3 waters are formed in the peripheral part and each stage of transformation can be characterized by a distinct set of secondary minerals (Chudaev, 2013).

1.5 MICROBIAL COMMUNITIES OF HYDROTHERMAL SPRINGS The systematic study of Kamchatka thermophilic microbial communities was initiated by the academician Georgy Zavarzin at the beginning of 1980s, and it resulted in a series of ecological and isolation works (Bonch-Osmolovskaya, 2004). In the following decades, several Russian and international research teams continued the exploration of Kamchatka hot springs by radioisotopic and molecular approaches as well as cultivation (Bonch-Osmolovskaya et al., 1999; Whitaker et al., 2003; Prokofeva et al., 2006; Perevalova et al., 2008; O’Neill et al., 2008; Kochetkova et al., 2011; Wagner et al., 2013; Chernyh et al., 2015, etc.). Microbial communities of hydrotherms can be divided into two main types: with the dominance of phototrophic microorganisms and with the dominance of chemotrophic microorganisms. Chemotrophic communities often develop in the form of fouling. Phototrophic communities in hydrotherms, in the absence of eukaryotic predators, may have a significant biomass and form microbial mats—organomineral structures, different from bacterial fouling by their structure (stratification). The boundary between phototrophic and chemotrophic communities is apparently determined by the resistance of the photosynthetic apparatus to environmental factors, primarily to temperature (Brock, 1978). In springs with pH 5–10, the upper temperature limit of the phototrophic microbial mat distribution is located at 61–73°C, but in acidic hydrotherms with pH 1–5, the developed mats are found only at temperatures below 55°C and are formed from eukaryotic algae Cyanidium caldarium (Castenholz, 1969, 1984; Hiraishi et al., 1999). At higher temperatures, or in the absence of light, chemotrophic communities develop. Substrates for them are H2, CO, NH3, H2S, SO, and other compounds that reach the surface of the Earth’s crust as components of volcanic gases. High temperature is the main limiting factor, and the diversity of the microorganisms decreases with the temperature increase. The high temperature prevents oxygen from dissolving, and hydrogen sulfide presenting in many hydrothermal solutions significantly reduces the amount of residual O2. This creates favorable conditions for the development of microaerophilic and anaerobic bacteria and narrows the variety of aerobes.

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1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

In the past 40 years, active research of hydrothermal activity has been carried out, and attention is increasingly focused on thermophilic microbial communities. The discovery of microorganisms living in hot springs on land and in marine hydrotherms was the basis for the development of new theories of the origin of life (Baross and Hoffman, 1986; Paces, 1991; Shock, 1996; Martin et al., 2008; Holm and Baltschejfsky, 2011; Simoncini et al., 2011; Mulkidjanian et al., 2012). In these extreme habitats, microbiological and geochemical processes are closely intertwined; biocenoses of such ecotopes can serve as a model for the study of the direct relationship of life with the deep substance of the Earth (Reysenbach and Cady, 2001). Hydrotherms are the source of many basic compounds for the initial synthesis of organic molecules (Sleep et al., 2011; Mulkidjanian et al., 2012). In the early stages of the Earth’s development, thermal habitats were much more widespread (Smith, 1981), and the atmosphere contained much higher concentrations of H2 and CO2 (Kasting, 1993). So, it was hypothesized that the common ancestor of prokaryotes was the chemolitotrophic organism that lived in hot springs (Weiss et al., 2016). Modern approaches in microbiology combine culture-dependent and culture-independent technologies to understand the microbial diversity and ecological function in certain environments. The cultivation approach offers systematic studies regarding the physiology and phylogeny of individual species; thus, their specific roles in a certain environment can be assessed. However, currently less than 1% of the assumed total amount of microbial species can be cultivated. Culture-independent approaches, such as phylogenetic analysis based on small subunit (SSU) ribosomal RNA gene sequences, overcome this deficiency and are essential for understanding microbial diversity in natural settings. Unlike some well-studied hot springs such as those in the Yellowstone National Park, hot springs in Kamchatka have not been explored in any detail in terms of microbial diversity. Therefore, it is difficult to assess the microbial robustness of the region. Microbiologists have isolated plenty of novel microorganisms from various regions of the Kamchatka peninsula in the past 20 years. Many of them are anaerobic thermophiles and were collected from hot springs of Uzon Caldera–Geyser Valley systems. These microorganisms include not only autotrophic hydrogen-oxidizing bacteria, carbon monoxide-oxidizing bacteria, and iron-oxidizing or reducing bacteria, but also heterotrophic fermentative or sulfur-reducing bacteria and archaea, and sulfur-oxidizing archaea as well (BonchOsmolovskaya, 2011). Recent studies based on quantitative measurements of phospholipid fatty acids showed that cyanobacteria, greensulfur bacteria, and green non-sulfur bacteria may be dominant autotrophic species (Zhang et al., 2004), which could contribute significant amounts of carbon to the total community in the hot springs. This may be particularly important in pools having significant outflow and negligible terrestrial input from surface runoff. Anaerobic chemolithoautotrophs appear to be important in Kamchatka hot springs. These include methanogens, autotrophic sulfate-reducing bacteria, carbon monoxide-oxidizing bacteria, sulfur-reducing bacteria, and ironreducing bacteria (Bonch-Osmolovskaya et al., 1999). However, acetogenesis, a process that produces acetate from CO2, was detected in pools over a wide range of pH (3.5–8.5) and temperature (60–80°C). Maximal acetogenesis occurred at pH 8.5°C and 60°C with a rate of 20 μg C L
1 day
1 (Bonch-Osmolovskaya et al., 1999). Carboxydotrophs are organisms capable of oxidizing CO to CO2 and assimilating part of oxidized CO2. These microorganisms may contribute significantly to the carbon cycle because of the availability of CO in these environments. Anaerobic oxidization of CO is observed to couple with hydrogen production in hot spring water columns at pH 8.5 (Bonch-Osmolovskaya et al., 1999). Representative bacteria include Carboxydothermus hydrogenoformans with an optimal temperature of 70°C and Carboxydocella thermautotrophica with an optimal temperature of 68°C (Gerhardt et al., 1991; Svetlichny et al., 1991; Sokolova et al., 2002). The CO utilization rate of C. hydrogenoformans can reach up to 178 μmol C mL
1 day
1 (Svetlichny et al., 1991). Lithotrophic reduction of sulfur and sulfate is detected in acidic hot springs with temperature optima of 60°C and 80°C, which coincide with the rate maxima of sulfidogenesis at these temperatures. This indicates that the lithotrophic metabolism of sulfur or sulfate can be performed by both thermophilic and hyperthermophilic microorganisms. Moderately thermophilic sulfur reducers are represented by the genus Desulfurella, which is a group of obligate anaerobic bacteria that can grow lithoautotrophically by using H2, CO2, and S (Miroshnichenko et al., 1998). Isolates of archaeal sulfur-reducers are mainly heterotrophic or fermentative species. For example, Desulfurococcus amylolyticus and Thermoproteus uzoniensis belong to the phylum Crenarchaeota and both have an optimal growth temperature of about 90°C. D. amylolyticus grows heterotrophically on peptides, amino acids, starch, and glycogen using S as electron acceptor. T. uzoniensis is a facultative sulfur reducer and can ferment peptides (BonchOsmolovskaya et al., 1990). Thermococcus litoralis and Thermococcus stetteri, belonging to Euryarchaeota, grow on peptides and have optimal growth temperatures of 88°C and 75°C, respectively (Neuner et al., 1990; Miroshnichenko et al., 1989). Processes of sulfur cycling in the natural environment are examined using tracers and rate measurements. Rates of sulfate- and sulfur-reduction vary spatially. Temperature may play a major role in such variation. For instance, the rate

1.6 METHANE CYCLING IN HOT SPRINGS METHANE FORMATION

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of sulfate reduction increases from 0.16 to 6 μg S cm
2 h
1 when the water temperature drops from 73°C to 40°C in some microbial mats (Bonch-Osmolovskaya et al., 1999). Enumerations showed that thermophilic (60°C) sulfate-reducing bacteria are present in 10–105 cultivable cells per mL sediment in various hot springs of Uzon Caldera. Nevertheless, sulfur- and sulfate-reducing microorganisms are potentially important for biogeochemical cycling of sulfur species in hot springs and could be the major energy source for microorganisms in hot spring ecosystems. Recent studies in Yellowstone geothermal ecosystems based on 16S rRNA gene clone libraries reveal that H2 should be the primary fuel for hot spring microbial communities (Spear et al., 2005). It would be interesting to see whether this holds true for Kamchatka hot springs. However, quantitative approaches, such as fluorescent in situ hybridization (FISH), should be used to complement the clone library approach. In addition to sulfur-reducers, sulfur-oxidizers are another important group that influences the sulfur cycle. As an example, the archaeal species Sulfurococcus mirabilis represents a group of the hyperthermophilic, facultative lithotrophic sulfur oxidizers that oxidize sulfur to sulfate under acidic conditions (pH 1.0–5.8) and 50–86°C (Golovacheva et al., 1987). Lithotrophic Fe (III)-reducing bacteria occur under various pH and temperature conditions in the hot springs of Kamchatka. Thermoanaerobacter siderophilus is a facultative Fe (III)-reducing bacterium growing lithoautotrophically on H2 and CO2 with Fe (III) as the electron acceptor. T. siderophilus grows optimally at 69–71°C (Slobodkin et al., 1999). Lithotrophic iron-oxidizing bacteria have also been isolated from Kamchatka hot springs. For example, Leptospirillum thermoferroxidans is an obligate chemoautotrophic bacterium that oxidizes Fe (II) to Fe (III) with an optimal growth temperature of 56.2°C (Golovacheva et al., 1992). Next generation sequencing was applied to study biogeography in geothermal springs at a regional level across New Zealand (Power et al., 2018). Analysis of 46 physicochemical parameters, and metadata from 1019 geothermal spring samples demonstrated that pH was the main biogeochemical driver of the diversity and community complexity structures within geothermal springs with temperatures <70°C. Surprisingly, community composition was dominated by two genera (Venenivibrio and Acidithiobacillus) across physicochemical spectrums of 13.9–100.6°C and pH < 1–9.7. The unusual prokaryotic community was found in five hydrothermal features sourced from both meteoric water and seawater on volcanic Raoul Island, approximately 1000 km from New Zealand (Stewart et al., 2018). The dominant taxa were mesophilic to moderately thermophilic, phototrophic, and heterotrophic marine groups related to marine Planctomycetaceae. Microbial communities of the Uzon Caldera have been recently studied using a molecular approach by analyzing 16S rRNA gene clone libraries (Burgess et al., 2012) and pyrosequencing and analyzing 16S rRNA gene fragments (Gumerov et al., 2011; Mardanov et al., 2011; Rozanov et al., 2014; Chernyh et al., 2015; Merkel et al., 2017). Three of these works were devoted to the investigation of the same source, Zavarzin Pool, where photosynthetic communities are present providing accompanying organotrophs with energy substrates (Burgess et al., 2012; Gumerov et al., 2011; Rozanov et al., 2014). Thermal acidic soil was found to contain a majority of uncultured archaea but no assumptions about the energy and carbon cycling in this community were made (Mardanov et al., 2011). Only the bacterial component of the community was studied in Arkashin Shurf, and representatives of the genus Hydrogenobaculum were supposed to be the primary producers in this ecosystem, oxidizing arsenite to arsenate (Burgess et al., 2012). Another attempt to study the producing part of microbial community was made for Bourlyashchy, the hottest pool in the Uzon Caldera, where the composition of water and sediment microbial communities was compared (Chernyh et al., 2015).

1.6 METHANE CYCLING IN HOT SPRINGS METHANE FORMATION Methane is the second most important greenhouse gas after carbon dioxide in the Earth’s atmosphere. In addition to direct participation in the formation of climate, it affects the composition of the atmosphere indirectly by chemical reactions with ozone, which explains the undying interest in the study of the methane cycle. The formation of methane and its entry into the atmosphere occurs as a result of various processes. The origin of the methane is divided into three types (Schoell, 1988): • Thermogenic (organo-thermocatalytic) methane is formed at high temperatures (200–300°C) and pressures during cracking of buried organic matter, from coal and oil shale, from oil in deep-loaded deposits. It enters the atmosphere, mainly in the development of mineral deposits; • Microbial (biochemical) methane is of biological origin. Its formation in the biosphere is carried out under anaerobic conditions, both from carbon dioxide and hydrogen, and from low—molecular organic compounds, as a result of the activity of a specific group of microorganisms, methanogens. This process is found in the bottom sediments of swamps

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1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

and other bodies of water, in the digestive system of insects and animals (mainly ruminants). According to the results of radioisotope studies (14C), 70%–90% of methane in the atmosphere is of biogenic origin (Cicerone, Oreland, 1988); • Abiogenic (thermal, high temperature) is formed in catalytic reactions at high temperature (600–800°C) and pressure in the deep layers of the lithosphere (by the reaction of CO2 + H2). This methane enters the atmosphere as a part of volcanic gases and hydrothermal solutions.

1.7 COMPOSITION OF MAGMATIC GASES The participation of the terrestrial thermal springs in the methane cycle is not well understood. Only recently, magmatic juvenile gases associated with the Earth’s crust faults became known. Geothermal gases usually contain from 0.01% to 1% methane (Etiope and Klusman, 2002); however, in some geothermal systems, the proportion of methane can reach 11%, or up to 27% of gas emissions (Giggenbach, 1995). This source is underestimated in the global methane budget, although it accounts for up to 10% of total emissions (Etiope and Ciccioli, 2009). These values indicate that magmatic juvenile gases are the second natural source of methane after swamps with emissions up to 110 Tg of methane per year. The total contribution of emissions in geothermal areas is 40–60 Tg CH4 year
1 (Etiope, 2010). When considering hydrotherms as methane sources, researchers are primarily interested in the origin of this gas. The isotopic composition of methane (13C, 14C, and D content) can be used to judge the origin of methane and the time of its formation (Rust, 1981). 14C isotope is formed in the atmosphere due to cosmic radiation. The half-life of 14C is 5730 years, which is much greater than the life of most wildlife on the Earth. 14C reacts with oxygen with formation of 14СО2. Further, these molecules through the processes of photosynthesis along with molecules of the stable carbon 12СО2 are included into the living organic matter formation. During its life, a living organism accumulates a certain amount of radioactive carbon, but this accumulation stops after the death and burial of the body. Since that time, the amount of radioactive carbon has only been falling. Therefore, by measuring the proportion of radioactive carbon relative to stable carbon, it is possible to determine the date of life of the body. Methane coming from nonbiological sources, such as coal mining, is very old and does not contain 14C. However, it was found that 21  3% of atmospheric methane is of fossil origin. Thus, during the biochemical processing, methane is enriched with a light carbon isotope. With the aging of the source of biological material and its chemical transformation, the content of heavy carbon is gradually increased (Wahlen et al., 1989). By analyzing the data of the radioactive isotope 14C, it is possible to determine the proportion of biological and nonbiological methane. Biogenic methane is usually an indicator of δ13C-СН4 in the interval from
55% to
88%, and abiogenic from
25% to
55%. Table 1.1 data show that the composition of hydrothermal methane is dominated by light isotopes, indicating its abiogenic origin. This fact is consistent with the data regarding the structure of hydrotherms. However, due to the TABLE 1.1 Carbon-13 Isotopic Composition of the Major Sources of the Atmospheric Methane Source

δ13С (%)

Lakes


40

Rice paddies


62

Bogs


61

Termites


60

Herbivores


60

Natural gas


41

Geothermal springs


27

Dumps


51

Coal mining


41

Biomass burning


25

Methane hydrates


65

From Craig, H., Chou, C.C., Welhan, J.A., 1988. The isotopic composition of methane in polar ice core. Science 242, 1535–1539.

1.9 ACTIVITY AND DIVERSITY OF METHANOTROPHIC COMMUNITIES IN THERMAL SPRINGS

9

large number of hydrothermal outputs and the fragmentary nature of the data, this aspect requires further study. Perhaps, after the development of a unified theory and generalizations from the data obtained, it will be possible to extract new results (common underground systems, geological structure of layers, etc.).

1.8 METHANOGENIC ACTIVITY Methane-metabolizing archaea play an important role in the global carbon cycle because they are the key participants in the formation or oxidation of methane (Reeburgh, 2007; Thauer et al., 2008). The recent discoveries of methanogenic archaea in the phyla Bathyarchaeota (Evans et al., 2015) and Verstraetearchaeota (Vanwonterghem et al., 2016) show that the current phylogeny of methanogens or archaeal methanotrophs will be expanded. The methyl-coenzyme M reductase complex not only acts in methanogenesis, but also activates methane to methyl-coenzyme M in anaerobic methane oxidation (Knittel and Boetius, 2009; Orphan et al., 2001). A culture-independent metagenomic study revealed previously unrecognized methane-metabolizing Crenarchaeota in two hyperthermal hot springs above 80°C in Malaysia (Wang et al., 2018). In some hydrotherms, radioisotope studies revealed the process of CH4 biological formation (Bonch-Osmolovskaya and Karpov, 1987). In situ radioisotope experiments with H14CO3 show that the maximal rate of methanogenesis is about 0.2 μg C L
1 day
1 at pH 7.0 and 60°C (Bonch-Osmolovskaya et al., 1999). No methanogenesis was found in acidic springs. Analysis of thermal microbial communities demonstrated the presence of methanogens by amplification and sequencing of the mcrA gene, which is a key functional and phylogenetic marker of methanogenesis (Sayeh et al., 2010; Amenábar et al., 2013; Takeuchi et al., 2011; Kizilova et al., 2013). Using cultural and molecular-ecological approaches (Merkel et al., 2013), the study of the distribution and diversity of methanogenic microorganisms in the terrestrial hot springs of Kamchatka Peninsula (Russia) and San Miguel Island, Portugal, was carried out. It was shown that methanogens were present in most of the terrestrial hot springs, constituting a small part of the microbial population (less than 0.1% of the total number of prokaryotes). For the first time, the presence of methanogens of MCR-2c, Methanocellales, and Methanomethylovorans was shown for terrestrial hot springs. The variety of cultivated methanogens is restricted by the representatives of the genera Methanothermobacter, Methanosaeta, Methanomethylovoran, etc. Summing up, it can be noted that studies of hydrotherms as methane sources are fragmentary and often involve only a few or even one spring in a particular area; so, there is no uniform system of statistics. Methanogens were detected and the process of methanogenesis was recorded in some hydrotherms, but its intensity is small and the methane is mostly of abiogenic origin.

1.9 ACTIVITY AND DIVERSITY OF METHANOTROPHIC COMMUNITIES IN THERMAL SPRINGS 1.9.1 Methane Oxidation When studying microorganisms with important biogeochemical functions, it is important not only to detect them in the studied ecosystem, but also to evaluate their contribution to biogeochemical cycling. Among the methods of assessment of microbial activity in natural habitats, the radioisotopic method is the most sensitive for the determination of the intensities of various metabolic processes, such as sulfate reduction, CO-oxidation, dark assimilation of carbon dioxide, and the formation and consumption of methane (Pimenov and Bonch-Osmolovskaya, 2006). Radioisotope analysis of microbial oxidation of methane is widely used in the study of aquatic ecosystems. The biogeochemistry of methane oxidation was investigated in a number of mid-latitude, Antarctic lakes and marine ecosystems (Galchenko et al., 1989; Galchenko, 2001). Studies of the activity of methanotrophs inhabiting hydrothermal springs are very few. Methane oxidation activity up to 48.2 μg C-CH4 L
1 day
1 was revealed in samples from Alla, Kuchiger, Dry, and Sowing springs in Buryatia, Russia, with temperatures 48–55°С (Tsyrenzhapova et al., 2007). Subsequently, the enriched cultures of methanotrophs and one pure culture referring to the genus Methylocystis were obtained from these hydrotherms. The results of the radioisotope study of the potential rate of methane oxidation in thermal springs of the coastal strip of Baikal Lake, namely Sukhaya, Zmeiniy, and Goryachinsk, 36–51°C, pH 9.0–9.4, evaluated methane consumption, as 7.2–92.7 μg C-CH4 L
1 day
1. A significant part of labeled carbon (25%–86%) was converted into cell biomass and

10

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

extracellular metabolites. On further investigation, aerobic methanotrophs were detected in all samples and methane oxidation was correlated with methanotroph number (Zelenkina et al., 2009). Potential rates of methane oxidation were detected in 19 of 36 sediment samples taken from 11 Canadian hot springs with a temperature range of 22.4–50.0°C and ranged from 16.7 to 141 μmol CH4 g
1 day
1 (Sharp et al., 2014).

1.10 METHANOTROPHS: A BRIEF INTRODUCTION The first methanotroph was isolated by S€ ohngen at the beginning of the last century and named Bacillus methanicus (S€ ohngen, 1906). Since then, the most significant contribution was made in 1970, in which over 100 methane-utilizing bacteria were isolated, characterized, and compared (Whittenbury et al., 1970). Playing an important role in the global methane cycle, MOB are found in a broad range of natural environments, in nearly all samples taken from soil, swamps, rivers, oceans, ponds, and sewage sludge, reportedly representing up to 8% of the total “heterotrophic” population (Higgins et al., 1981). As methane is naturally produced mainly through the anaerobic decay of organic matter, methanotrophs are found primarily at oxic-anoxic interfaces. The majority of known methanotrophs are aerobic; however, methane oxidation is known to occur in anaerobic environments by coupling oxidation to sulfate (Valentine and Reeburgh, 2000) and nitrite reduction (Ettwig et al., 2010). As expected from their prevalence within the environment, methanotrophs are found in both mesophilic (Singh et al., 2018) and extreme environments. Strains have been isolated from temperatures as low as 4°C (Bowman et al., 1997) and as high as 72°C (Bodrossy et al., 1999), and it has been demonstrated that populations of methanotrophs in nature adapt to different temperatures (Hanson and Hanson, 1996). Two populations of methanotrophs have been identified that exist depending on environmental methane availability (Bender and Conrad, 1992). Low-affinity methanotrophs are able to use methane at high concentrations (>40 ppm), and are detected in environments with high methane content. High-affinity methanotrophs are able to oxidize ambient methane concentrations (2 ppm) and although their existence within soil samples has been verified using molecular techniques, isolation of such bacteria has not yet been possible. Analysis of nucleic acids (DNA and RNA), phospholipids, methane oxidation rates, and stable isotope probing (SIP) using 13C labeled methane confirmed the relatively low abundance of these high-affinity methanotrophs in soils. In contrast to the relatively high abundance of methanotroph populations present in the environment, these high-affinity methanotrophs account for less than 0.01% of total bacteria biomass in soils (Maxfield et al., 2009). The characteristic unique feature of methane-oxidizing bacteria is their possession of methane monooxygenase (MMO) enzymes (Singh and Singh, 2017) that catalyze the initial the oxidation of methane to methanol, followed by methanol oxidation to formaldehyde by methanol dehydrogenase (MDH), oxidation of formaldehyde to formate by formaldehyde dehydrogenase (FADH), and finally formate to carbon dioxide by formate dehydrogenase (FDH). Formaldehyde is assimilated into biomass by either the ribulose monophosphate (RuMP) pathway, or the serine pathway. Fig. 1.3 illustrates the metabolism of methane by methanotrophs.

CytCred O2

pMMO

H2O CytCox

RuMP pathway

CH4

CH3OH NADH O2 NADH

CO2

Serine pathway

FDH

sMMO

NAD+

XH2 HCOOH

CytCox

H2O NAD+

FADH

MDH X

Type I methanotrophs

Type II methanotrophs

CytCred HCHO

Carbon assimilation

FIG. 1.3 Pathways for methane oxidation in aerobic methanotrophic bacteria. From Hanson, R.S., Hanson, T.E., 1996. Methanotrophic bacteria. Microbiol. Rev. 60, 439–471.

11

1.11 CLASSIFICATION OF METHANOTROPHS

Two types of MMO have been found in methanotrophic bacteria, a soluble cytoplasmic form (sMMO) and a particulate membrane-bound form (pMMO) (Singh, 2016). All methanotrophs known to date, with the exception of members of the genera Methylocella (Dedysh et al., 2005) and Methyloferula (Vorobev et al., 2011), have the ability to produce pMMO; however, Type II methanotrophs are also able to produce sMMO. For Type II methanotrophs, the type of enzyme expressed within the cell primarily depends on the availability of copper. Under conditions of copper excess (>0.85 μmol g
1 dry weight of cells), pMMO is produced preferentially, while under conditions of limited copper availability, sMMO is generated (Stanley et al., 1983). The dependence on copper availability is attributed to its presence in the active site of the pMMO enzyme (Semrau et al., 1995).

1.11 CLASSIFICATION OF METHANOTROPHS Traditionally, all aerobic MOB belonged to the phylum Proteobacteria, and were classified into two major groups: Type I and Type II, based on differences in physiological and morphological traits, with Type X methanotrophs further differentiated from Type I (Hanson and Hanson, 1996). Recent characterization of new genera and species of methanotrophs demonstrated that this classification system was no longer useful to characterize all known species and has been updated. The comprehensive review of the current taxonomy of aerobic methanotrophs is presented by Knief (2015). Currently, methanotrophs are now known in Proteobacteria, Verrucomicrobia, and candidate phylum NC10. Proteobacteria methanotrophs are divided into two classes: Alphaproteobacteria and Gammaproteobacteria. Current classification further divides Gammaproteobacteria, of the order Methylococcales, into three families: Methylococcaceae, which is further separated into Type Ia, including a total of 13 genera, and Type Ib (genera Methylococcus, Methylocaldum, Methylogaea, and Methyloparacoccus); Methylothermaceae (genera: Methylothermus, Methylohalobius, and Methylomarinovum); and Crenotrichaceae, which includes a single genus (Crenothrix polyspora) that to date has not been isolated as a pure culture. The ability to oxidize methane and its close relationship with methanotrophs were shown for Clonothrix fusca by molecular biological, physiological, biochemical, and cytological methods (Vigliotta et al., 2007). These organisms are filamentous bacteria living in underground storage and drinking water sources. Despite their popularity for more than 140 years, these microorganisms could not be cultivated, because of which they remain poorly understood. Methanotrophs Type Ia of the family Methylococcaceae use the RuMP cycle for carbon assimilation (Bowman et al., 1995) and have intracytoplasmic membranes arranged as a uniform array of bundles of vesicular disks distributed across the cell (Fig. 1.4-1). Methanotrophs formally identified as Type X were renamed Type Ib and express low levels of the ribulose-1, 5-bisphosphate carboxylase enzyme (RuBisCo), in addition to the RuMP pathway. The methanotrophic Alphaproteobacteria have been divided into two families: Methylocystaceae, Type IIa (genera: Methylocystis, Methylosinus) and Beijerinckiaceae, Type IIb (genera: Methylocella, Methylocapsa, Methyloferula) methanotrophs. Type II methanotrophs of the family Methylocystacea utilize the serine pathway for formaldehyde assimilation, with the intracytoplasmic membrane arranged as stacks of vesicles in parallel to the cell membrane (Fig. 1.4-3). Methanotrophs FIG. 1.4 Structure of the intracytoplasmatic membrane system in the methanotrophic cells of Methylomonas—1; Methylobacter and Methylococcus—2; Methylosinus and Methylocystis— 3. ICM—intracytoplasmatic membranes; CW—cell wall; CM—cell membrane. From Galchenko, V.F., 2001. Methanotrophic bacteria. Moscow: Geos. p. 500.

12

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

of the family Beijerinckiaceae do not contain the intracytoplasmic membranes, while in Methyloferula they are found only on one side of the cell. Extremophilic methanotrophs belonging to the phylum Verrucomicrobia, of the genus Methylacidiphilium, are sometimes described as Type III. Three strains of thermoacidophilic methanotrophs belonging to Verrucomicrobia and combined in the genus Methyloacidiphilum: Methyloacidiphilum fumarolicum were obtained almost simultaneously from the mud volcano Solfata near Naples, southern Italy (Pol et al., 2007), Methyloacidiphilum kamchatkensis (Islam et al., 2008) from an acidic thermal spring near the Uzon Caldera, Russia, and Methyloacidiphilum infernorum (Dunfield et al., 2007) from the Hell’s Gate geothermal source, New Zealand. These organisms do not have typical methanotroph membrane systems; instead, cells contain polyhedral or tubular organelles, whose functions are not yet clear. Presumably, they contain a special form of MMO (Trotsenko and Khmelenina, 2008; Op den Camp et al., 2009), which is not detected by functional genes of known methanotrophs. As with the majority of methanotrophs, they possess pMMO but lack the familiar formaldehyde assimilation pathways, instead using the Calvin-Benson cycle for carbon assimilation (Dunfield et al., 2007). Able to grow across a wide range of temperatures, they are unique in comparison with all other known methanotrophs due to their extremely acidophilic phenotype (Sharp et al., 2014; van Teeseling et al., 2014). Since the late 1970s, data on the anaerobic oxidation of methane (AOM) in marine sediments and water columns have been available (Barnes and Goldberg, 1976; Reeburgh, 1976), and the process soon associated with a community of microorganisms comprising methanogenic archaea and sulfate-reducing bacteria (Hoehler et al., 1994; Hansen et al., 1998), when sulfate acted as the final electron acceptor instead of oxygen. Some researchers estimate the amount of anaerobically oxidized methane in marine sediments to be between 70 and 300 Tg/year (Valentine, 2002). According to studies carried out on polluted fresh water in the Netherlands, the AOM was directly related to microorganisms that carry out the process of denitrification (Raghoebarsing et al., 2006). Anaerobic methane oxidation, coupled with nitrite reduction, has been observed in bacteria of the candidate phylum NC10, and named Ca. Methylomirabilis oxyfera (Ettwig et al., 2010). In a number of works, the data where aerobic methanotrophs can be active in oxygen-free water layers are given. The participation of type I methanotrophs (Gammaproteobacteria), namely representatives of the genus Methylobacter, in the AOM is shown (Oswald et al., 2016; Tveit et al., 2013, 2014; Martinez-Cruz et al., 2017). A recent study showed that the presence of Fe and Mn ions stimulated the activity of the AOM, carried out by the Gammaproteobacterial MOB, either directly acting as an electron acceptor, or indirectly used as a trace metal (Oswald et al., 2016; Semrau et al., 2010). Thus, to date, information on the processes of AOM is quite contradictory. The consensus is that the implementation of anaerobic methane oxidation processes in marine and freshwater ecosystems is recognized, thanks to archaea and/or bacteria communities acting separately or as a consortium (Boetius et al., 2000; Raghoebarsing et al., 2006; Ettwig et al., 2010). The study of the above groups of microorganisms proves that the phenomenon of methanotrophy is represented in different bacterial taxa and is realized through several mechanisms involving different genes.

1.12 THERMOPHILIC METHANOTROPHS Most known species of proteobacterial methanotrophs are mesophiles and neutrophiles. The first moderately thermophilic known methanotroph was Methylococcus capsulatus Texas, which was isolated from wastewater in United States (Foster and Davis, 1966) and another strain of Methylococcus capsulatus Bath from a hot spring (Whittenbury et al., 1970). Following the representative kind, Methylococcus thermophilus was isolated after nearly 10 years of sludge waters and therapeutic muds studies (Malashenko et al., 1975). Studies of thermal springs in Hungary and Japan led to the isolation of moderately thermophilic methanotrophs belonging to the genera Methylocaldum (Md) (Bodrossy et al., 1997) and Methylothermus (Mt) (Bodrossy et al., 1999; Tsubota et al., 2005). The first of these currently contains three validated species (Md. szegediense, Md. gracile, Md. tepidum) (Bodrossy et al., 1997), and the second two species—Mt. thermalis (Tsubota et al., 2005) and Mt. subterraneus (Hirayama et al., 2011). These Gammaproteobacteria have optimal growth temperatures of 45–60°C. Methylothermus strain HB is the most thermophilic methanotroph known with a ported upper growth limit of 72°C (Bodrossy et al., 1999). However, this strain has been lost. A related strain with an upper growth limit of 67°C has been validated as M. thermalis (Tsubota et al., 2005). Recently, a thermotolerant methanotroph, Methylocystis strain Se48, was isolated from the Transbaikal hot springs, Russia, and had an upper growth limit of 53°C (Tsyrenzhapova et al., 2007). This organism is the first reported thermotolerant Alphaproteobacteria methanotroph, but has never been taxonomically validated. Isolates of Verrucomicrobia methanotrophs have an upper growth limit of 65°C but also require high acidity for growth (Dunfield et al., 2007; Pol et al., 2007; Islam et al., 2008).

1.13 METHANOTROPHIC COMMUNITIES OF TERRESTRIAL GEOTHERMAL SPRINGS

13

A survey of methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons found methanotrophic Verrucomicrobia in a temperature range of 22.5–81.6°C and a pH range of 1.8–5.0, suggesting this group requires acidic pH (Sharp et al., 2014). Two novel moderately thermophilic and acid-tolerant obligate methanotrophs were isolated from a tropical methane seep topsoil habitat in Bangladesh. Isolates grew at pH range 4.2–7.5 (optimal 5.5–6.0) and at a temperature range of 30–60°C (optimal 51–55°C). 16S rRNA gene phylogeny placed them in a well-separated branch forming a cluster together with the genus Methylocaldum as the closest relatives (93.1%–94.1% sequence similarity). These strains are the first isolated acid-tolerant moderately thermophilic methane oxidizers of the class Gammaproteobacteria and most likely represent a novel genus within the family Methylococcaceae (Islam et al., 2016). Thermophilic/thermotolerant methanotrophs living at high temperatures have a number of adaptations (Trotsenko and Khmelenina, 2008). Their fatty acid composition is very specific. It is dominated by saturated (C16:0), methylated fatty acids (C9-ome-16:0), and their cyclic derivatives (C17sus), which are involved in the stabilization of cell membrane structures and thus optimize their fluidity. Osmoprotectors are involved in the stabilization of proteins and the most studied is sucrose (see, e.g., Md. szegediense O-12), (Medvedkova et al., 2007). Representatives of the genus Methylocaldum form melanin, which is also referred to as thermal adaptation (Medvedkova et al., 2008), as toxic aromatic intermediates are formed at elevated temperatures, which are removed by polymerization to form melanin. High temperatures increase the level of oxygen radicals in the cell, which are converted to H2O2 or OH, causing oxidative stress and in order to protect against this stress cells produced peroxidase, superoxide dismutase, glutathione, and cytochrome compounds that utilize super-radicals in different ways.

1.13 METHANOTROPHIC COMMUNITIES OF TERRESTRIAL GEOTHERMAL SPRINGS There is still no clear evidence to support the association of methanotrophic species with certain habitats. Thus, mesophilic, thermotolerant, and thermophilic methanotrophs were isolated from the same ecosystems (Whittenbury et al., 1970). Contrarily, other researchers insist on the specific affinity of methanotrophs for specific ecosystems (Heyer, 1977). Since the composition of hydrothermal fluids includes CH4 along with CO, H2S, and CO2, it was suggested that thermophilic/thermotolerant methanotrophs are present in these habitats (Giggenbach, 1996). The solubility of gases in aqueous solutions decreases with increasing temperature, which is often considered the main limiting factor for the growth of methanotrophs at high temperatures. However, it is known that the solubility of methane in liquid media with low ionic strength decreases only to 33% when the temperature increases from 30°C to 60°C (Duan et al., 1992). This may explain the presence of methane oxidation in high-temperature biotopes. The composition of methanotrophic hydrotherm communities in Russian territory has been investigated occasionally (Tsyrenzhapova et al., 2007; Zelenkina et al., 2009). Some growth studies support the existence of moderately thermotolerant Alphaproteobacteria methanotrophs. A methanotroph affiliated with Methylocystis was previously grown from a thermal spring in the Transbaikalia region, Russia. Methylocystis strain Se48 had a reported optimal growth temperature of 37°C and a maximal growth temperature of 53°C (Tsyrenzhapova et al., 2007). Using fluorescence in situ hybridization (FISH), Zelenkina et al. (2009) reported that members of the genus Methylocystis constituted the majority (up to 99%) of the total alphaproteobacterial methanotrophs in enrichment cultures from the coastal thermal springs of Lake Baikal isolated at 45°C and 51°C. Information about methanotrophic organisms in thermal springs of the Russian Far East is poor. For a number of sources, the methane oxidation with maximum intensity at 37°С was discovered and Methylococcus, Methylomonas, Methylosinus, Methylocystis, and Methyloacidiphilum methanotrophs were found (Khmelenina et al., 2011). At the same time, the gas hydrothermal manifestations of the Far East region are characterized by a wide variety of temperatures and mineral composition, which suggests the existence of different activities and compositions of methanotrophic communities. Bacteria involved in methane oxidation were identified in Canadian warm geothermal sediments using DNA-SIP. Spring sediments incubated at 22°C, 30°C, or 37°C showed a predominance of OTUs with high similarity to the Gammaproteobacteria (Crenothrix, Methylosoma, Methylobacter, Methylocaldum, Methylococcus, Methylomicrobium, Methylomonas, Methylogaea, Methylomarinum, and Methylovulum spp.). Methanotrophic Alphaproteobacteria (Methylosinus, Methylocystis, Methyloferula, Methylocapsa, and Methylocella spp.) have been described in geothermal environments below 37°C from cultivation studies (Sharp et al., 2014). The methanotrophic communities present in the hightemperature incubations (45°C) were less diverse and moderate thermophilic gammaproteobacterial methanotrophs of the genera Methylococcus and Methylocaldum have been found.

14

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

1.14 METHANE OXIDATION IN HOT SPRINGS OF FAR-EAST RUSSIAN VOLCANIC BELT: KAMCHATKA AND KURIL ISLANDS Aerobic methane oxidation has been mostly studied in environments with moderate to low temperatures and the intensity of aerobic methane oxidation differs substantially between natural ecosystems. In contrast to these habitats, our knowledge of methane oxidation in habitats with elevated temperatures is scarce. The existence and activity of thermophilic methanotrophs have been questioned not only because of the instability of cell inner membrane structures, which are essential for methane metabolism at elevated temperatures (e.g., Bonch-Osmolovskaya, 2011), but also due to low methane solubility at high temperatures (e.g., Duan et al., 1992). To date, little research on the subject has been done; so, we have undertaken the study characterizing the methanotrophic bacteria inhabiting the hot springs in the Russian Far East, specifically the Kamchatka Peninsula and Kunashir Island. Kamchatka Peninsula and Kuril Islands are the part of the Pacific Ring of Fire (Figs. 1.1 and 1.2), a tectonic instability region where the Pacific Plate subducts underneath the Eurasian Plate at a rate of about 80 mm per year. Kamchatka with its length of more than 1500 km stretches from north to south, and along its longitudinal axis, the peninsula is framed by two mountain belts, a Sredinny (Central) and a shorter Vostochny (Eastern) mountain ridge. Kamchatka is characterized by a number of active as well as dormant volcanoes and the highest volcano Kluchevskaya sopka (4750 m) is located between mountain belts, dominating the whole region. Kamchatka has numerous hydrothermal systems, which constantly release geothermal gases and fluids out to the earth’s surface. In the outflow geothermal gases N2 and CO2 usually prevail, but H2, CH4, and H2S also occur frequently. Hot spring waters in Kamchatka may have multiple origins including meteoric and magmatic water. The temperature of these hot springs ranges from 20°C to greater than 90°C. Water chemistry also varies dramatically with pH ranging from 3.1 to 9.8. The Uzon Caldera in Kamchatka is a unique area of contemporary volcanism. Hydrothermal activity was detected within a narrow East-West oriented zone along the central line of the caldera. In the Uzon Caldera, there are four hydrothermal fields, namely Zapadnoye, Severnoe, Vostochnoe, and Oranzhevoe, as well as Lake Fumarolnoe. In these fields, there are numerous boiling gryphons, mud pools, small volcanoes, and hot platforms with steam and water vents and temperatures varying from 45°C to 98°C. About 160 groups of thermal springs with temperatures up to 98°C inhabited by different communities of thermophiles are presently known, and the majority of microbiological studies were carried out in the Vostochnoe thermal field. In July 2008, silt and cyanobacterial mats were sampled at 18 hot springs near Lake Fumarolnoe, Karbonatnoe Field, Kamchatka, and the composition of enriched methanotrophic communities was analyzed (Dvoryanchikova et al., 2011). In the course of our expedition to the Uzon Caldera in July 2010, samples of sedimentary material were collected from the vents of 36 hot springs with temperatures and pH ranging from 37°C to 86.6°C and from 2.6 to 6.8, respectively. The properties of the studied springs are listed in Table 1.2. The investigated thermal springs were located in different zones of the Uzon Caldera (Fig. 1.5); most of them were found in Vostochnoe field (18 springs), near Lake Fumarolnoe (6 springs), and near the Izvilistyi stream (8 springs). The Vostochnoe field of the Uzon Caldera has been studied in more detail. All the studied hot springs are characterized by low acidity (pH 5.3–6.8), high temperatures (48.7–86.6°C), and a mildly reducing environment (Eh from +24 to
107 mV). Near the Izvilistyi stream, six acidic springs (pH 2.6–3.8) with temperatures ranging from 40.6°C to 69.2°C were studied. The redox status of the spring may be described as transient with the unstable geochemical regime and variable contents of hydrogen sulfide and oxygen. The third group of hot springs near Lake Fumarol’noe was characterized by low acidity, temperatures ranging from 56.1°C to 65°C, and transient redox status. One of these hot springs, Ryzhik, is characterized by transient conditions (+43 mV) and lower temperature (39°C). Some springs in the Oranzhevoe and Severnoe fields are slightly acidic, of high temperature (65°C and 70°C), and with reducing conditions (
37 and
2 mV). Hot springs near Lake Khloridnoe have different temperatures (37°C and 64.1°C); however, they are slightly acidic and are characterized by transient redox status. Kuril Islands are a volcanic chain of islands stretching southwest from the tip of Kamchatka (Fig. 1.2). The islands themselves are summits of volcanoes that are a direct result of the subduction of the Pacific Plate under the Okhotsk Plate. The chain has around 100 volcanoes, some 40 of which are active, and many hot springs and fumaroles, and frequent seismic activity. Kunashir is the southernmost island and extends from the northeast to the southwest for approximately 122 km at a width from approximately 4 to 30 km. The island comprises the Golovnin, Mendeleev, Rurui, and Tyatya volcanoes occurring at the gas-hydrothermal stage. A limited number of studies on the Kunashir thermal springs exist to date and most of them have been focused on geological and geochemical features (Markhinin and Stratula, 1977). Our knowledge on microorganisms inhabiting the thermal springs of Kunashir Island is scarce and

1.14 METHANE OXIDATION IN HOT SPRINGS OF FAR-EAST RUSSIAN VOLCANIC BELT: KAMCHATKA AND KURIL ISLANDS

15

TABLE 1.2 Bacic Physicochemical Properties of the Studied Thermal Spring of the Uzon Caldera Sample no.

Spring

Coordinates

pH

T (°C)

Eh, мВ

Concentration of СН4 (μL/L)

VOSTOCHNOE FIELD 1

Treshchinnyi

N54 29. 938, E160 00. 930

5.6

86,6


90

10.58

2

Zavarzina

N54 29. 881, E160 00. 871

5.6

56.7


30

15.10

3

Tsyklop

N54 29. 918, E160 00. 701

6.3

59.3


41

6.26

4

Termofilnyi

N54 29. 912, E160 00. 691

5.9

73.2


107

19.63

5

Feniks

N54 29.956, E160 00. 665

6.8

63.0


8

11.70

6

Molochnyi

N54 29. 962, E160 00. 653

6.3

67.4

+14

7.48

7

Entselad

N54 29. 966, E160 00. 670

5.8

48.7


56

71.58

8

Fobos

N54 29. 978, E160 00. 684

5.9

50.3


60

40.48

9

Venera

N54 29. 982, E160 00. 693

6.1

59.6


8

28.14

10

Vertoletnyi

N54 30. 002, E160 00. 737

5.9

64.5


56

27.77

11

Deimos-1

N54 29. 969, E160 00. 779

6.5

49.6

+24

10.39

12

Zheltyi

N54 29. 928, E160 00. 786

5.2

66.8


64

16.34

31

Deimos-2

N54 29. 969, E160 00. 783

6.2

65.3

nd

nd

32

Yupiter

N54 29. 941, E160 00. 676

5.7

50.8


13

5.78

33

Mars

N54 29. 927, E160 00. 732

5.9

63.1


7

5.19

34

Zatsepina

nd

6.2

78.8


40

8.66

36

Saturn

nd

5.2

76.3

nd

24.49

37

Premer

nd

5.3

70.6


93

6.28

IZVILISTYI STREAM 13

Rodzher

N54 30. 047, E160 00. 459

5.0

69.7

+98

10.14

14

Geizeritovyi

N54 30. 046, E160 00. 456

5.1

85.7


42

24.37

15

Izvilistyi stream

N54 30. 019, E160 00. 448

2.6

40.6

+242

6.94

16

Mutnyi

N54 30. 041, E160 00. 450

3.8

69.1

+123

23.24

17

Kometa

N54 30. 128, E160 00. 444

2.8

51.1

+165

17.89

18

Sbornyi

N54 30. 076, E160 00. 435

2.7

54.2

+286

70.11

LAKE FUMAROLNOE 19

Kulturnyi

N54 30. 115, E159 59. 279

5.2

63.0

+280

26.48

20

Terra

N54 29. 887, E159 59. 410

6.2

65.0

+273

24.39

21

Stroma

N54 29. 854, E159 59. 477

5.3

56.1

+205

15.99

22

Kvadrat

N54 29. 862, E159 59. 485

6.3

64.5

+178

9.94

23

Glaz Drakona

N54 29. 885, E159 59. 468

6.2

59.8

н.о

10.32

24

Ryzhyk

N54 30. 005, E160 00. 118

6.6

39.0

+43

11.26

N54 30. 446, E160 00. 047

6.0

65.0


37

44.44

N54 30. 670, E160 00. 312

6.0

70.0


2

18.15

ORANZHEVOE FIELD 25

Atsyclik

SEVERNOE THERMAL FIELD 26

Lokon

Continued

16 TABLE 1.2 Sample no.

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

Bacic Physicochemical Properties of the Studied Thermal Spring of the Uzon Caldera—cont’d Spring

Coordinates

pH

T (°C)

Eh, мВ

Concentration of СН4 (μL/L)

THERMAL SITE “RAZVILKA” NEAR LAKE KHLORIDNOE 27

Tsytron

N54 30. 035, E160 00. 410

3.4

64.1

+60

48.03

28

Izvilistyi stream

N54 30. 022, E160 00. 411

3.1

46.7

+203

29.08

THERMAL FIELD NEAR LAKE KHLORIDNOE 29

Kholodnyi

N54 29. 944, E160 00. 554

6.2

37.0

+153

13.26

30

Tretiy

N54 29. 943, E160 00. 587

5.3

64.1

+150

47.35

nd ¼ No data.

(A)

(B)

FIG. 1.5 Sketch map of the study region on Kamchatka (A) and distribution of studied thermal springs in Uzon Caldera (B).

is currently limited to a few reports on algal flora (Nikulina and Kociolek, 2011) and extremely thermophilic, obligately anaerobic, carboxydotrophic eubacteria (Svetlichny et al., 1991). Samples were collected in July 2013 from Kurilsky nature reserve, located on the northern and southern tips of Kunashir Island. Our study was focused on six geothermal areas with various physical and chemical properties (Fig. 1.6; Table 1.3) and collected samples from a total of 30 thermal springs (Kizilova et al., 2013). Three investigated geothermal areas, namely Stolbovskie (ST), Tretyakovskie (T), and Goryachii Plyazh (GP), belong to the siliceous neutral or alkaline thermal waters with traces of bromine and sodium, carbon dioxide, chlorides, nitrates, and sulfates with low water mineralization (0.4–5 g L
1. The three other geothermal areas, Yuzno-Alekhinskie (YA), Neskuchenskie (NI), and Golovnina volcano (OK), belong to the sulfureted low-mineralized waters with low or semineutral pH and traces of bromine, aluminum, fluoride, iron, manganese, strontium, and phosphorus (Markhinin and Stratula, 1977). All springs, where samples were screened for methanotrophic presence, contained dissolved methane (3%–12%) in accord with previous measurements in the area (Markhinin and Stratula, 1977).

1.15 INTENSITY OF CH4 OXIDATION EVALUATED BY RATIO-TRACER ANALYSIS Among techniques estimating methane oxidation rates, gas chromatography and radioactive tracer technique with C-labeled methane are the most commonly used ones. Despite its complexity, the radioactive tracer technique has great potential for the analysis of methane oxidation in thermal springs due to extremely high sensitivity.

14

1.16 QUANTIFICATION OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS: NUMBER OF COPIES OF pmoA, mxaF AND 16S rRNA GENES

17

FIG. 1.6 Location of the study sites on Kunashir Island. The description of thermal area and springs is provided in Table 1.3. From Kizilova, A.K., Sukhacheva, M.V., Pimenov, N.V., Yurkov, A.M., Kravchenko, I.K., 2013. Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island, Russian Far East. Extremophiles. 18, 207–218.

It was shown that all studied samples from the Uzon Caldera contained dissolved methane (29.9–361.6 μL L
); so, the next step was to determine the intensity of microbial oxidation of CH4 using the radioisotope method. Methane oxidation was recorded in 8 of the 36 hydrothermal springs (Table 1.4). To assess the efficiency of methane use by microorganisms, the share of methane carbon not only completely oxidized to CO2, but also included into their biomass and exometabolites was taken into account. Highest rates of methane oxidation to CO2 (energy metabolism) were registered in sources Terra, Stroma, and Kvadrat (1338; 513; and 165 ng C-CH4 L
1 day
1, respectively). The highest values of the inclusion of the 14C label in the biomass (constructive metabolism) were found in Sroma, Terra, and Sborniy (141; 132; and 21 ng C-CH4 L
1 day
1, respectively) (Table 1.4). The total value of methane oxidation ranged from 26 to 1471 ng C-CH4 L
1 day
1. The highest intensity of methane oxidation was found in the Terra spring (65°C, pH 6.2), the lowest in the Izvilistiy stream (40.6°C, pH 2.6). Samples from 15 thermal springs from all six thermal sites were selected for measuring potential methane oxidation by means of the radioisotope tracer technique utilizing 14C-labeled methane on Kunashir Island. Potential methane oxidation rates in sediments of thermal springs of Kunashir Island were detected in 14 samples and varied between 0.04 and 104 μM CH4 L
1 day
1 (Table 1.5). Highest rates of methane oxidation were observed in Tretyakovskie thermal springs (T4 and T1) showing 80–104 μM CH4 L
1 day
1.Interestingly, the process of methane oxidation was detected in several springs with temperatures equal to or above 67°C (the highest growth temperature of M. thermalis). Specifically, we were able to estimate methane oxidation in springs ST1 (74°C), ST3 (78°C), ST6 (68.4°C), GP3 (99°C), GP4 (67°C), and YA4 (83°C). Potential methane oxidation rates in ST1 and ST3 spring sediments, incubated at 75°C, were nearly 3 and 1 μM CH4 L
1 day
1, respectively, and these springs also showed incorporation of labeled methane into biomass (Kizilova et al., 2013).

1.16 QUANTIFICATION OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS: NUMBER OF COPIES OF pmoA, mxaF AND 16S rRNA GENES The presence of bacterial 16S rRNA genes in hot springs of the Uzon Caldera was detected by real-time PCR using the DNA extracted from sediment samples as the template. In hot springs with neutral or slightly acidic pH, their number reached 108–109 copies mL
1 irrespective of the temperature. At the same time, in the samples taken from the springs located near the Izvilistyi stream (pH 2.6–2.8), it was significantly lower, not exceeding 105 copies mL
1 sediment (Table 1.6). In 10 out of the 30 studied springs, the number of copies of the pmoA gene, the most widely used molecular marker of methane oxidation, exceeded the detection level (3–6  103 copies mL
1) (Table 1.6). The highest and the lowest numbers of gene copies differed by three orders of magnitude; the highest values (2.8 and 1.1  107 pmoA copies mL
1) were

18

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

TABLE 1.3 Springs

Some Hydrological and Chemical Characteristics of the Investigated Thermal Springs on the Kunashir Island

Coordinates, elevation

pH

T (°C)

CH4

Mineralization (mg/L)

Water composition

2702

Neutral and slightly acid waters with carbon dioxide, nitrogen, sulfates, and sodium. Traces of boron and bromide. High content of silicic acid. Up to 12% dissolved CH4

Moderate mineralization

Nitrogen, chloride, sodium, manganese, and strontium. High content of silicic acid. Up to 3% dissolved CH4

Up to 4400

Silicon, sulfates, chlorides, and complex cationic composition (Na, К, Са, Mg, Al, Fe, Н)

2414

Slightly alkaline waters with chlorides and sodium. High content of silicic acid and phosphorus. Traces of strontium and metaboric acid

Low mineralization

Sulfates, hydrocarbonates with various cations

STOLBOVSKIE ST1

N44 00.424, E145 40.997, 28 m

7

74

+

ST2

N44 00.433, E145 40.985, 21 m

7

44

+

ST3

N44 00.417, E145 40.994, 23 m

6.03

77

+

ST4

N44 00.432, E145 40.990, 24 m

5.7

64.2

+

ST5

N44 00.420, E145 40.992, 25 m

5.9

76.6

+

ST6

N44 00.422, E145 40.990, 25 m

6.0

68.4

+

TRETYAKOVSKIE T1

N43 59.147, E145 39.244, 29 m

46.5

6.2

+

T2

N43 59.148, E145 39.248, 29 m

70

6.5

+

T4

N43 59.151, E145 39.255, 31 m

47

5.36

+

T5

N43 59.107, E145 39.349, 31 m

80

5.6

+

MENDELEEVA VOLCANO MCH40

N43 59.324, E145 43.686, 385 m

1.5–2.0

55

nd

M-CH4

N43 59.258, E145 43.597, 384 m

2.1–2.2

49.3–50

nd

YUZNO-ALEKHINSKIE P1

N43 54.227, E145 29.060

50-–53

7.2-–7.4

+

P3

N43 54.291, E145 29.162

61

8.5

+

P4

N43 54.291, E145 29.178

83

7.3

+

NESKUCHENSKIE NI1

N44 29.434, E146 06.635, 45 m

88

6.2

nd

NI2

nd

46

5.5

nd

NI3

nd

56

6

nd

NI4

nd

40

6

nd

NI5

nd

80

4–5

nd

NI6

N44 29.397, E146 06.577 47, m

55

4

nd

NI7

nd

58

4

nd

NI8

nd

48

4

nd

NI9

nd

60

4

nd

19

1.17 EVALUATION OF ACTIVE METHANOTROPHS BY FISH TECHNIQUE

TABLE 1.3 Some Hydrological and Chemical Characteristics of the Investigated Thermal Springs on the Kunashir Island—cont’d Springs

Coordinates, elevation

pH

T (°C)

CH4

Mineralization (mg/L)

Water composition

4655

nd

6648

Sulfureted (164 mg/L H2S) waters with iron (up to 200 mg/L) and silicic acid. Manganese (up to 1.7 mg/L), strontium (up to 1.8 mg/L), bromide (up to 3.1 mg/L), fluorine (up to 1.6 mg/L), and phosphorus (up to 5 mg/L)

GORYACHIIPLYAZH GP1

nd

65

6.3–7

GP2

N43 59.539, E145 47.933

66.5

6.3

GP3

N43 59.539, E145 47.933

99

4.5–5

GP4

nd

67

6

GOLOVNINA VOLCANO (KIPYASCHEE LAKE) OK1

N43 51.857, E145 30.084, 147 m

65

5.7

+

OK2

N43 51.857, E145 30.078, 141 m

62

6.5

+

OK3

52.8

5.58

+

OK8

46.5

2

nd

nd ¼ No data.

TABLE 1.4 Microbial Methane Oxidation in Thermal Springs of Uzon Caldera Evaluated by c-Radiotracer Experiments 14

СО2

14

Spring

CH4 transformation rate (ng C-CH4 L21 d21) C-biomass + dissolved 14С (% of total)

14

Potential methane oxidation rate (ng C-CH4 L21 day21)

Izvilistyi stream

21.2

4.9 (18.7)

26.1

Kometa

42.5

2.6 (5.8)

45.1

Sbornyi

64.7

20.6 (24.2)

85.3

135.8

10.5 (7.1)

146.3

1338.3

132.4 (9.0)

1470.7

Stroma

513.4

140.7 (21.5)

654.1

Kvadrat

164.7

6.8 (4.0)

171.5

78.3

9.9 (11.3)

88.2

Kulturnyi Terra

Glaz Drakona

detected in the Kulturnyi and Kvadrat springs, respectively. In other springs, these values were considerably lower, ranging from 2.8  106 to 4.0  104 pmoA copies mL
1 (Kizilova et al., 2012). The number of pmoA copies in sediments of the thermal springs of Kunashir Island varied between 104 and 106 copies per mL of sediment (Kizilova et al., 2013). This provides evidence for the presence of methanotrophs in the springs, including those characterized by temperatures exceeding 67°C, namely ST1, ST3, ST5, ST6, GP3, GP4, and YA4. The highest number of pmoA copies (106) was found in ST2-ST6, T4, and GP1. Numbers of mxaF gene copies generally exceeded the numbers of pmoA copies and ranged from 105–107 copies per mL of sediment. Total numbers of bacteria in sediments of thermal springs were estimated to be between 106 and 109 cells per mL. No correlation between the number of gene copies (pmoA, mxaF, and 16S rRNA) and hot spring properties (temperature, pH, and type of spring) was revealed.

1.17 EVALUATION OF ACTIVE METHANOTROPHS BY FISH TECHNIQUE The presence of methanotrophic Gammaproteobacteria in 8 springs in the Uzon Caldera was revealed by FISH using the probe set M84 + M705 (Table 1.6). Type II methanotrophs (Alphaproteobacteria) were not detected in any of the studied

20

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

TABLE 1.5 Potential Methane Oxidation Rates Evaluated by c-Radiotracer Experiments in the Sediments of Thermal Springs of the Kunashir Island 14

CH4 transformation rate (mL CH4 L21 day21)

Thermal spring

Group of springs

Incubation (°C)

14

14

Potential methane oxidation rate (μM CH4 L21 day21)

ST1

Stolbovskie

75

0.05

0.02

3.05

ST3

Stolbovskie

75

0.02

0

1.03

ST4

Stolbovskie

60

0.44

0.26

31.70

ST5

Stolbovskie

75

0.00

0

0.04

ST6

Stolbovskie

75

0.02

0

0.94

M-CH4

Mendeleeva

50

0.03

0

0.96

NI1

Neskuchisnkie

75

0.00

0

0

NI3

Neskuchisnkie

60

0.04

0

1.18

NI4

Neskuchisnkie

40

0.05

0

2.08

T1

Tretyakovskie

50

1.10

1.25

104.43

T4

Tretyakovskie

50

1.28

0.56

79.32

GP2

Goryachii Plyazh

75

0.00

0

0

GP3

Goryachii Plyazh

75

0.01

0

0.49

GP4

Goryachii Plyazh

75

0.01

0

0.57

OK2

Golovnina

60

0.01

0

0.51

YA1

Yuzno-Alekhinskie

50

0.02

0

0.85

YA4

Yuzno-Alekhinskie

75

0.01

0

0.3

СО2

C-biomass + dissolved 14С

TABLE 1.6 Quantitative Characteristics of the Microbial Community Obtained by Direct Microscopic Counts and Real-time PCR in the Uzon Caldera Thermal Springs Number of cells (mL21 sediment) Thermal springa

DAPI

Number of gene copies (mL21 sediment)

М-84 + М-705

EUB338 mix

Kulturyi

(2.40  0.22)  10

7

(9.70  0.39)  10 (40.4%)

Terra

(1.10  0.39)  107

Stroma

16S рРНК

pmoA

(1.50  0.46)  10 (15.5%)

8

(10.5  1.26)  10

(2.83  0.25)  107

(4.20  1.40)  106 (38.2%)

(5.0  1.9)  105 (11.9%)

(3.04  0.08)  107

(1.12  0.28)  105

(3.30  0.56)  107

(1.00  0.160)  107 (30.3%)

(1.90  0.61)  106 (19.0%)

(8.47  0.71)  108

(2.76.  0.25)  106

Kvadrat

(3.60  0.45)  107

(2.10  0.33)  107 (58.3%)

(2.00  0.49)  106 (9.5%)

(2.42  0.02)  108

(1.13  0.20)  107

Isvilistyi

(3.30  0.12)  107

(2.00  0.07)  107 (60.6%)

(7.00  0.34)  105 (3.5%)

(9.50  2.40)  105

(4.67  0.19)  104

Kometa

(3.30  0.34)  107

(1.40  0.16)  107 (42.4%)

(1.50  0.61)  106 (10.7%)

(2.00  1.13)  105

(2.17  0.08)  104

Sbornyi

(1.70  0.220)  107

(1.00  0.15)  107 (58.8%)

(3.50  1.10)  105 (35.0%)

(5.00  0.98)  104

(0.2  0.00)  104

a b c

For information see Table 1.2. % of the total number of microorganisms. % of the number of metabolically active eubacteria.

6

b

6

c

1.18 DIVERSITY OF METHANOTROPHS IN THERMAL SPRINGS BASED ON PCR-DGGE ANALYSIS OF pmoA GENES

21

springs (Kizilova et al., 2012). The numbers of methanotrophs varied within a wide range, from 1.3  104 cells mL
1 in the Glaz Drakona spring to 3.5  106 cells mL
1 sediment in the Sbornyi spring (Table 1.6). The shares of methanotrophs in the bacterial communities of these springs (probe EUB 338 mix) reached 0.57% and 35%, respectively.

1.18 DIVERSITY OF METHANOTROPHS IN THERMAL SPRINGS BASED ON PCR-DGGE ANALYSIS OF pmoA GENES For sediment samples collected in springs Izvilistyi (Izl5), Kometa (Km17), Sbornyi (Sbl8), Kulturnyi (Ku19), Terra (T20), Stroma (S21), Kvadrat (Kv22), and Glaz Drakona (DY23), the product of the pmoA gene fragment of the expected size was amplified, and the amplicon mixture was separated by DGGE (Kizilova et al., 2012). The number of specific bands for each sample ranged from 1 to 3, indicating the low diversity of methanotrophic bacteria. The only exception was the Stroma spring, for which eight well-defined separate bands were obtained. However, after reamplification and sequence analysis of these bands, all the sequences obtained were found to be identical (100% similarity of the deduced protein sequences) and exhibited high similarity with Methylothermus subterraneus sequences. Phylogenetic analysis revealed the presence of methanotrophs most closely related to Methylothermus HB (Fig. 1.7), but significantly different from it (less than 90% similarity), in four springs located near Lake Fumarolnoe (Kulturnyi, Terra, Stroma, and Kvadrat). In the samples from the Kulturnyi and Kvadrat springs, methanotrophs with high similarity to the HB strain were detected. Two methanotrophs were found in Terra spring and one of them was a quite distant relative of the HB strain and Methylothermus subterraneus isolated from a subsurface geothermal water stream in a Japanese gold mine (Hirayama et al., 2011) was the closest relative of the methanotroph detected in the Stroma spring (S21). We failed to reveal the methane-oxidizing Verrucomicrobia in hot springs with high acidity (Izvilistyi, Kometa, and Sbornyi) by use of the A189F—A682R primer set and a low annealing temperature reducing the primer specificity as recommended by Pol et al. (2007). At the same time, methane-oxidizing Gammaproteobacteria (sequences Izl5–23, Kml7–18, and SM8–21) with Methylomonas methanica as the closest cultivable relative of the isolated methanotrophs were 0.10

Methylomonas methanica MC09 (AEF98753) uncultured bacterium (ACS 72334) Km17-18 DY23-25 86 53 Iz15-23 T20-10 Sb18-21 Methylovulum miyakonense (AB501285) Methylosoma difficile (DQ119047) 74 56 68 Methylobacter tundripaludum (AJ414658) 100 Km17-22 Methylomicrobium japanense (AB253367) Methylosarcina lacus (AY007286) 93 100 Methylocaldum gracile (U89301) 100 Methylocaldum tepidum (U89304) Methylococcus capsulatus str. Bath (NC_002977) 77 Methylohalobius crimeensis (AJ581836) 90 Methylothermus thermalis (AY829010) 100 Methylothermus subterraneuss (AB536748) 63 97 S21-05 T20-11 54 78 thermophilic methanotroph HB (U89302) Kv22-14 100 100 Ku19-04 99 Methylocapsa acidiphila (AJ278727) 100 Methylosinus trichosporium (AJ868409) Methylocystis parvus (U31651) Crenothrix polyspora (DQ295904) Methylacidiphilum infernorum V4 (NC_010794) 61

FIG. 1.7 Phylogenetic tree constructed on the basis of the deduced amino acid sequences of the pmoA gene fragments from the Uzon springs sediments. The sequences determined in the present work are in bold. The GenBank accession numbers of the gene fragment sequences obtained are given in parentheses. Scale bar: 10 amino acid substitutions per 100 amino acid residues. The numerals show the significance of the branching order as determined by bootstrap analysis of 100 alternative trees (only bootstrap values above 50% are shown).

22

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

TABLE 1.7 Physicochemical Properties, Mineral Composition of the Water, and Methanotrophs Detected in the Springs Near Fumarolnoe Lake Ion content (mg/L) Temperature (°C)

NO2 3

Ca2

рН

Cl2

Mg2

Spring

SO22 4

+

+

Na+

K+

NH+4

Methanotrophs

Stroma

56

5.3

36.6

0

223

27

15

16.1

1.8

0

Methylothermus subterraneus

Glaz Drakona

60

6.2

249

0.9

51

5.7

14.8

385

28.3

2.2

Methylomonas sp.

Kulturnyi

63

5.2

35

0

28

1.2

7.4

4

0.9

0

Methylothermus HB

detected in these three hot springs. In Kometa spring, another methane-oxidizing microorganism (Km 17–22) exhibiting the highest similarity with Methylobacter tundripaludum was found (Fig. 1.7). Despite similar values of the hydrothermal parameters and numbers of microorganisms, the diversity of the pmoA gene demonstrated substantial differences. To explain the possible reasons, three hot springs, namely Stroma, Glaz Drakona, and Kulturnyi, were compared (Table 1.7). In two of them, Stroma and Kulturnyi, Methylothermus like bacteria were found to exhibit high similarity with different representatives of this genus. For instance, methanotrophic bacteria most closely related to Methylothermus HB isolated were detected in Kulturnyi spring with a high temperature and a low mineralization of water. At the same time, a methanotroph most closely related to Methylothermus subterraneus was detected in Stroma spring with high sulfate content. In the Glaz Drakona spring, only uncultured Methylomonas were detected. This spring differs from the other two by the extremely high level of mineralization, especially by the contents of chloride, sodium, and potassium ions, as well as by the presence of mineral nitrogen compounds (ammonium and nitrate). Analysis of pmoA genes was used to study methanotrophs diversity in Kunashir Island thermal springs (Kizilova et al., 2013). It was shown that methanotrophs were detectable in 19 out of 30 springs and belong to both the Alphaand Gammaproteobacteria (Fig. 1.8). DGGE analysis revealed that 10 out of 19 springs harbor one methanotroph, whereas 9 springs (ST1–ST6, NI2, GP3, and T1) yielded 2 or more. The highest richness was detected in six springs (ST1, ST2, ST3, ST5, ST6, and NI2). The majority of gammaproteobacterial amino acid sequences belong to the genus Methylothermus (Fig. 1.8). A Methylothermus-like group with sequences related to both M. thermalis (99% similarity) and Methylothermus subterraneus (100% similarity) was found in springs Stolbovskie (ST2–ST6), Goryachii Plyazh (GP1, GP3), Golovnina volcano (OK2), and Tretyakovskie (T1). Sequence identical to the PmoA gene of M. thermalis was detected in Stolbovskie springs (ST4). Interestingly, two sequences from the Goryachii Plyazh thermal spring (GP3) were identical to the thermophilic methanotroph HB (Bodrossy et al., 1999), which has the highest upper temperature growth limit among studied pure cultures of methanotrophs, that is, 72°C. Apart from sequences that clustered with the two species of Methylothermus with validly published names, PmoA sequence analyses revealed a smaller cluster comprising sequences obtained from Stolbovskie springs (ST1, ST3, and ST5), which are characterized by temperatures higher than 74°C. These sequences showed a lower degree of similarity (97%– 99%) to Methylothermus subterraneus and M. thermalis than other sequences in this cluster. Analysis of the ST2 probe also yielded a sequence that belongs to the family Methylococcaceae and is related (97% similarity) to an uncultured Methylobacter sp. discovered from a Chinese wetland soil (Yun et al., 2012). This spring has the lowest temperature among the springs of the Stolbovskie geothermal area, 44°C. Spring groups Stolbovskie (ST1–ST3 and ST6), Tretyakovskie (T1), Neskuchenskie (NI2, NI3), Yuzno-Alekhinskie (YA1, YA4), and Golovnina volcano (OK3) harbored Methylocystis-like methanotrophs (Fig. 1.8), clustering with the so-called type II methanotrophs Methylocystis hirsuta, Methylocystis sp. SC2, and a number of uncultured methane oxidizers, all detected in various aquifers and soils.

1.19 ISOLATION AND CHARACTERIZATION OF METHANE-OXIDATION CULTURES During the study of methane oxidation in Uzon Caldera thermal springs, 13 stable methane oxidation cultures were enriched: K14, Ku19 (Kulturyi spring), K7 and K8 (Glaz Drakona spring), K11, K12, S21 (Stroma), Iz15 (Izvilistyi), Km17 (Kometa), SB18 (Sbornyi), T20 (Terra), Kv22 (Kvadrat), and DY23 (Glaz Drakona). According to the molecular biological analyses data, each culture contained one methanotroph and the ratio of methanotrophs was 80%–97%.

1.20 GROWTH OF ENRICHMENTS

0.08

23

ST6_22 ST5_19 ST4_17 ST4_13 GP1_1 ST3_7 OK2 ST2_5 GP3_37 97 ST4_15 Methylothermus thermalis (AY829010) Methylothermus subterraneus (AB536748) ST6_24 T1_27 ST3_9 Gammaproteobacteria 100 ST1_1 ST5_20 71 ST3_11 97 GP3_35_36 Thermophilic methanotroph HB (U89302) ST2_6 100 100 uncultured Methylobacter sp. clone LD pmoA 9 (EU124864) Methylobacter psychrophilus (AY945762) 67 Methylocaldum gracile (U89301) Methylococcus capsulatus (AE017282) 81 74 T5 93 NI4 87 Uncultured methanotrophic bacterium clone SKB8 (FJ009656) 90 Uncultured bacterium clone 90-2000B-661r (HE617688) T4 61 GP4 92 Uncultured bacterium (JN591304) NI2_31 ST2_4 ST5_21 ST1_2 ST1_3 Methylocystis sp. SB2 (GU734137) NI3 YA4 Alphaproteobacteria ST6_23 Methylocystis hirsuta (DQ364434) NI2_32 NI2_33 100 OK3 100 T1_28 YA1 Methylosinus sporium (DQ119048) Methylacidiphilum infemorum V4 (CP000975)

FIG. 1.8 Phylogenetic tree of pmoA sequences determined for the thermal springs of Kunashir Island. The scale indicates the number of expected substitutions accumulated per site. GenBank accession numbers are given in parentheses. From Kizilova, A.K., Sukhacheva, M.V., Pimenov, N.V., Yurkov, A.M., Kravchenko, I.K., 2013. Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island, Russian Far East. Extremophiles. 18, 207–218.

1.20 GROWTH OF ENRICHMENTS High-enriched cultures of thermophilic methanotrophs with methane as a sole source of carbon and energy were obtained on mineral growth media by sequential passages. With cultivation conditions, the characteristics of hydrotherms were taken into account, from which cultures were obtained (temperature, pH, and mineralization). Throughout the entire period of cultivation, the presence of methanotrophs was confirmed by detection of the pmoA gene and in FISH with M-84 + M-705 probes (Type I methanotrophs) (Dvoryanchikova et al., 2011). Cultures Iz15, Km17, and Sb18 were grown at temperatures of 40°C, 50°C, and 55°C, respectively. All these cultures were characterized by low growth rate, relatively small proportion of methanotrophs, and a wide variety of accompanying organisms (6–8 components). No growth occurred on solid media. All other cultures were grown at 50–55°C in medium with different mineralization. K14 and Ku19 cultures developed a uniform suspension, without flakes when grown on liquid media, and K14 formed colonies on agar media. Culture S21 was characterized by a stable uniform growth of single cells in a liquid mineral medium during the exponential and linear growth phase and formed flakes at the stationary phase. After 1–2 weeks growth on the agar medium, round, convex, smooth, light, beige, opaque mucous colonies 0.4–0.8 mm in diameter were formed. All enrichments needed a mineral source of nitrogen for growth and the nifH gene was not detected.

24

1. ACTIVITY AND DIVERSITY OF AEROBIC METHANOTROPHS IN THERMAL SPRINGS OF THE RUSSIAN FAR EAST

FIG. 1.9 Electronic micrographs of whole cells (A, B; marker
0.5 μm), thin sections (E–F; marker
0.1 μm) of methanotrophs enriched from thermal springs of Uzon Caldera: (A, C) S21 culture, Stroma, (B, D) Ku19 culture Kulturnui spring, (E) T20 culture, Terra spring, (F) S21 culture Stroma spring.

The presence of polar flagellum in cells of culture S21 was revealed by electron microscopy (Fig. 1.9A); no flagella were found in other cultures. Transmission electron microscopy of thin sections showed a stacked arrangement of internal membranes characteristic of Type I methanotrophs (Fig. 1.9C). Cells, releasing a significant amount of mucus, are enclosed in covers, which are placed bacterial satellites (Fig. 1.8D). Through the use of a fixing solution of ruthenium red, the fibrillar structure of the mucous material is detected. In old cells, electron-dense inclusions in the form of granules of irregular shape are clearly visible (Fig. 1.9E and F).

1.21 STUDY OF THE PHYLOGENETIC DIVERSITY OF ENRICHED METHANOTROPHIC CULTURES The study of the phylogenetic diversity of methanotrophs was carried out by molecular cloning and PCR-DGGE analysis of 16 s rRNA and pmoA genes. The data for the analysis of K14 culture is presented in Table 1.8. It was found that the dominant organism (29 clones out of 50) was the methanotroph closest to the strain HB (Bodrossy et al., 1999).

25

REFERENCES

TABLE 1.8 Closest Relatives to Genbank Sequences of 16 s rRNA Clones of the K14 Methanotrophic Culture According to MegaBLAST comparison Clones number (% of library)

Nearest database neighbor in NCBI (accession number)

% DNA identity

29 (58)

Methylotermus HB (U89302)

98

10 (20)

Uncultured Chlorobi (EU631213)

95

5 (10)

Thermoactinomyces sacchari (AM161154)

98

1 (2)

Uncultured Acidobacteriaceae ZB_P10_C06 (GQ328567), Uzon, Zavarzin spring

83

1 (2)

Pseudomonas putida (AM161157)

98

2 (4)

Uncultured bacterium ZB_P14_B04 16S(GQ328675), Uzon, Zavarzin spring

97

1 (2)

Uncultured bacterium ZB_P13_F12 (GQ328655), Uzon, Zavarzin spring

85

1 (2)

Uncultured bacterium TP146 (EF205576), Tibet hot spring Uncultured Acidobacteria (AM749745), New Zealand hydrothermal soils

97 94

Minor components (1–5 clones) are represented by thermophilic organisms that exhibit a high degree of similarity with bacteria isolated from the thermal sources of Kamchatka, Yellowstone National Park, Tibet, and New Zealand geothermal soils. Another example of the use of molecular biological methods for the diagnosis of methanotrophs was the analysis of a fragment of the 16 s rRNA gene by PCR-DGE for the culture e S21. The DGGE-profile consists of three bands and a comparative sequence analysis showed that two of them are highly similar to Methylothermus (94%–98% similarity of nucleotide sequences). Analysis of the third band sequences showed a high degree of similarity (98%) with representatives of the genus Thermaerobacter.

1.22 CONCLUSIONS Thermal springs are extreme ecosystems of considerable interest, both for basic research and for potential practical applications, particularly as they relate to microbial organisms. Due to the relative constancy of the chemical composition and temperature, they are convenient model systems for studying the ecology of living organisms. Study of the extreme systems and extremophilic microorganisms is important from the point of view of determining the physicochemical boundaries of the functioning of living systems, including when considering the existence of life on planets other than Earth. Scientific interest in the extreme environments is also related to investigation of the adaptation mechanisms and microbial evolution. Finally, these habitats harbor rich bacterial diversity that could be the source of commercially important products. We have studied the diversity and activity of methane-oxidizing microorganisms in the hydrotherms of the Far-East Russian volcanic belt region. For the first time, a large number of hydrotherms with a wide temperature range from 20°C to 98°C and pH from 2 to 11 were investigated. Due to CH4-rich magmatic gases, the thermal springs of Kamchatka and Kunashir Island provide a sedimentary environment for the MOB. Methane oxidation activity was estimated up to 75°C, the highest growth temperature for known methanotrophs. Successful amplification of pmoA genes from spring sediments suggested a contribution of methanotrophs from both Alpha- as well as Gammaproteobacteria. The presence of many taxonomically unsolved pmoA sequences from these springs could be a sign of novel microbe richness in these less known environments and that the diversity of methanotrophs in hot cosystems is broader than previously suggested. Cultivation in mineral salts medium resulted in stable high-enriched methane-oxidation cultures with novel thermophilic methanotrophs. Further studies with cultivation followed by physiological analysis of these important microbes would be required to determine their precise functional roles within these communities.

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Further Reading Craig, H., Chou, C.C., Welhan, J.A., 1988. The isotopic composition of methane in polar ice core. Science 242, 1535–1539. Eshinimaev, B.T., Medvedkova, K.A., Khmelenina, V.N., Suzina, N.E., Osipov, G.A., Lysenko, A.M., Trotsenko, Y.A., 2004. New thermophilic methanotrophs of the genus Methylocaldum. Microbiology 73 (4), 448–456. J€ ackel, U., Thummes, K., K€ampfer, P., 2005. Thermophilic methane production and oxidation in compost. FEMS Microbiol. Ecol. 52, 175–184. Whittenbury, R., Krieg, N.R., 1984. Family Methylococcaceae. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology. In: vol. 1. The Williams & Wilkins Co., Baltimore, pp. 256–261.