View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Kyoto University Research Information Repository Detection of anaerobic carbon monoxide-oxidizing Title thermophiles in hydrothermal environments. Yoneda, Yasuko; Kano, Sanae I; Yoshida, Takashi; Ikeda, Eitaro; Fukuyama, Yuto; Omae, Kimiho; Kimura-Sakai, Author(s) Shigeko; Daifuku, Takashi; Watanabe, Tetsuhiro; Sako, Yoshihiko Citation FEMS microbiology ecology (2015), 91(9) Issue Date 2015-09 URL http://hdl.handle.net/2433/207705 This is a pre-copyedited, author-produced PDF of an article accepted for publication in 'FEMS microbiology ecology' following peer review. The version of record [Yasuko Yoneda, Sanae I. Kano, Takashi Yoshida, Eitaro Ikeda, Yuto Fukuyama, Kimiho Omae, Shigeko Kimura-Sakai, Takashi Daifuku, Tetsuhiro Watanabe, Yoshihiko Sako. Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal Right environments. Volume 91, Issue 9, fiv093, September 2015] is available online at: http://femsec.oxfordjournals.org/content/91/9/fiv093.; The full- text file will be made open to the public on 28 July 2016 in accordance with publisher's 'Terms and Conditions for Self- Archiving'.; This is not the published version. Please cite only the published version. この論文は出版社版でありません。 引用の際には出版社版をご確認ご利用ください。 Type Journal Article Textversion author Kyoto University 1 Title: Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal 2 environments 3 4 Authors: Yasuko Yoneda1,§, Sanae I. Kano1, Takashi Yoshida1, Eitaro Ikeda1, Yuto 5 Fukuyama1, Kimiho Omae1, Shigeko Kimura-Sakai1,¶, Takashi Daifuku1, Tetsuhiro 6 Watanabe 2 and Yoshihiko Sako1,*. 7 8 Authors Affiliations: 9 1Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, 10 Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. 11 2Laboratory of Soil Science, Graduate School of Agriculture, Kyoto University, 12 Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. 13 §Current affiliation: Bioproduction Research Institute, National Institute of Advanced 14 Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 15 305-8566, Japan. 16 ¶Current affiliation: School of Environmental Science, The University of Shiga 17 Prefecture, 2500 Hassaka-cho, Hikone city, Shiga 522-8533, Japan. 18 19 Running title: Anaerobic CO-oxidizing thermophiles in hydrothermal environments 20 21 *Corresponding Author: Yoshihiko Sako 22 Phone: +81 75 753 6217 23 Fax: +81 75 753 6226 24 E-mail: [email protected] 25 26 Abstract 27 Carboxydotrophic anaerobic thermophiles have been isolated from various 28 hydrothermal environments and are considered to be important carbon monoxide (CO) 29 scavengers or primary producers. However, the ecological factors that influence the 30 distribution, abundance and CO-oxidizing activities of these bacteria are poorly 31 understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus 32 spp. in a hot spring sample and found that they constituted up to 10% of the total 33 bacterial cells. In this study, we investigated environmental features, potential microbial 34 CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot 35 springs to determine environmental factors that affect CO-oxidizers and to see whether 36 Carboxydothermus spp. are common in those environments. We detected potential 37 microbial CO-oxidation activities in samples that showed relatively high values of total 38 organic carbon (TOC), total nitrogen (TN), oxidation–reduction potential (ORP) and 39 soil-water content. The abundance of Carboxydothermus spp. did not correlate with the 40 presence of potential microbial CO-oxidation activities; however, Carboxydothermus 41 spp. were detected in a wide range of environments, suggesting that these bacteria are 42 widely distributed in spite of the relatively low population size. This study implies 43 thermophilic CO-oxidizers occur in a wide range of environments and oxidize CO in 44 somewhat oxidative environments rich in organic matter. Graphical Abstract Figure 45 46 47 One-sentence summary 48 Carbon-monoxide-oxidizing thermophiles are widely distributed in a range of 49 environments, and high potential microbial CO-oxidation activity is associated with the 50 levels of organic matter, oxidation–reduction potential and soil-water content of 51 environments. 52 53 54 Keywords 55 carboxydotroph, carbon monoxide, carbon monoxide dehydrogenase, thermophile, hot 56 spring 57 58 Introduction 59 It is believed that approximately 2,500–2,600 Tg of carbon monoxide (CO) is produced 60 annually and emitted to environments via the combustion of fossil fuels, oxidation or 61 photochemical degradation of organic compounds, volcanic gas, and metabolism in 62 animals, plants and microbes (King & Weber, 2007). CO concentrations in 63 environments usually occur in trace amounts; e.g., 2–15 nM in seawater (Moran & 64 Miller, 2007), 20–33 nM in hot springs (Kochetkova et al., 2011) and approximately 92 65 ppb global mean mole in the atmosphere (WMO WDCGG, 2014). There is generally 66 equilibrium between CO production and consumption; approximately 90% of 67 atmospheric CO is consumed by reaction with hydroxyl radicals, and CO-oxidizing 68 microbes in soil are considered to be a CO sink accounting for 10% of the total CO 69 consumption (King, 2007). 70 Studies on microbiological CO consumption have been conducted under 71 aerobic conditions using soil, freshwater and seawater (Conrad & Seiler, 1980; Conrad 72 & Seiler, 1982). Aerobic CO-oxidizers utilize molybdenum-containing carbon 73 monoxide dehydrogenases (Mo-CODHs), which can be detected widely in 74 environments such as soils (plant root), sediments, volcanic deposits and the ocean floor 75 (King, 2003; King, 2007; Dunfield & King, 2004; Martin-Cuadrado et al., 2009). In 76 anaerobes, CO oxidation is catalysed by nickel-containing CO dehydrogenases 77 (Ni-CODHs) (Ragsdale, 2004). Ni-CODHs can be divided into the following two major 78 groups: the monofunctional CODH group and the bifunctional CODH/acetyl CoA 79 synthase (ACS) group. Bifunctional CODH/ACSs comprise CODH subdomains and 80 ACS subdomains and are known to be key enzymes in the Wood–Ljungdahl pathway 81 (reductive acetyl CoA pathway) (Ragsdale, 2004). The CODH subdomains reduce CO2 82 to CO, after which the ACS subdomains incorporate CO as a carboxyl group of 83 acetyl-CoA. Monofunctional CODHs catalyse oxidation of CO to CO2, thereby deriving 84 low potential electrons whose energy can be converted to the transmembrane potential 85 and ATP when they are transferred to some oxidants (Techtmann et al., 2009). In a 86 previous study, monofunctional and bifunctional CODH genes were predicted from 87 their genomic context. Monofunctional Ni-CODH genes are widely found in the 88 prokaryotic genomes of hydrogenogens, methanogens, sulphate reducers, acetogens and 89 gut microbes in animals (Techtmann et al., 2012). CODH genes are found in diverse 90 microbes in various environments; however, only a few studies have reported anaerobic 91 microbial CO oxidation in environmental samples from soil, wetland and hot springs 92 (Conrad & Seiler, 1980; Rich & King, 1999; King, 2007; Kochetkova et al., 2011), and 93 the environmental and physiological significance of anaerobic CO-oxidizers is less 94 understood. 95 The thermophilic bacterial genus Carboxydothermus is one of the most 96 studied CO-oxidizing anaerobes. Five species of the genus Carboxydothermus have 97 been isolated from hot springs and described (Yoneda et al., 2012; Sokolova et al., 98 2013). Four of the species produce hydrogen via CO-oxidization under 100% CO 99 atmospheric conditions. Because CO is a toxic gas and hydrogen is an important energy 100 source for many microbes in an anoxic environment, CO-oxidizers in the environment 101 are assumed to be important ‘CO scavengers’ or primary producers (Sokolova et al., 102 2009; Techtmann et al., 2009). 103 The genome of C. hydrogenoformans has four genes encoding 104 monofunctional CODH (cooS-I, cooS-II, cooS-IV and cooS-V), which have different 105 predicted functions and sequences (Wu et al., 2005). Previously, the quantitative 106 detection of Caboxydothermus spp. by targeting the CODH-II gene (cooS-II) using 107 real-time PCR showed that these microorganisms can constitute up to approximately 108 10% of the total bacterial cells in acidic hot spring sediment where Carboxydothermus 109 pertinax was previously isolated (Yoneda et al., 2013a). Considering that the optimal 110 growth pH of Carboxydothermus spp. is moderately acidic or neutral, the abundance of 111 these bacteria is intriguingly high. This led us to further question whether 112 Carboxydothermus spp. are common in other hot springs, where the environment is 113 more suitable for the growth of this genus, and are responsible for CO-oxidation in situ. 114 The physiological group of carboxydotrophic hydrogenogens has been isolated from 115 various environments, such as hot springs, deep-sea hydrothermal vents, lake sediments 116 and bioreactors (Sokolova & Lebedinsky, 2013). Unfortunately, the range of 117 environments that offer a niche for these microorganisms is still unknown. The aim of 118 the current study was to determine the answer to the above question. In the current study, 119 we measured potential microbial CO-oxidation activities under anoxic conditions and 120 the abundance of Carboxydothermus spp. in various hot spring samples. 121