Exploration and enrichment of methane-oxidizing bacteria derived from a rice paddy field emitting highly concentrated methane Shohei Yasuda, 1 Risako Toyoda, 1 Shelesh Agrawal, 2 Toshikazu Suenaga, 3 Shohei Riya, 1 Tomoyuki Hori, 4 Susanne Lackner, 2 Masaaki Hosomi, 1 and Akihiko Terada 1 , 3, * Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan, 1 Department of Civil and Environmental Engineering Science, Institute IWAR, Chair of Wastewater Engineering, Technische Universität Darmstadt, Franziska-Braun-Straße 7, 64287 Darmstadt, Germany, 2 Global Innovation Research Institute, TokyoUniversity of Agriculture and Technology, 3-8-1 Harumi-cho, Fuchu, Tokyo 185-8538, Japan, 3 and Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology,16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan 4 Received 18 January 2020; accepted 20 April 2020 Available online xxx Methane-oxidizing bacteria (MOB) possess the metabolic potential to assimilate the highly potent greenhouse gas, CH 4 , and can also synthesize valuable products. Depending on their distinct and fastidious metabolic pathways, MOB are mainly divided into Type I and Type II; the latter are known as producers of polyhydroxyalkanoate (PHA). Despite the metabolic potential of MOB to synthesize PHA, the ecophysiology of MOB, especially under high CH 4 flux conditions, is yet to be understood. Therefore, in this study, a rice paddy soil receiving a high CH 4 flux from underground was used as an inoculum to enrich MOB using fed-batch operation, then the enriched Type II MOB were characterized. The transi- tions in the microbial community composition and CH 4 oxidation rates were monitored by 16S rRNA gene amplicon sequencing and degree of CH 4 consumption. With increasing incubation time, the initially dominant Methylomonas sp., affiliated with Type I MOB, was gradually replaced with Methylocystis sp., Type II MOB, resulting in a maximum CH 4 oxidation rate of 1.40 g-CH 4 /g-biomass/day. The quantification of functional genes encoding methane monooxygenase, pmoA and PHA synthase, phaC, by quantitative PCR revealed concomitant increases in accordance with the Type II MOB enrichment. These increases in the functional genes underscore the significance of Type II MOB to mitigate greenhouse gas emission and produce PHA. Ó 2020, The Society for Biotechnology, Japan. All rights reserved. [Keywords: Methane-oxidizing bacteria; Polyhydroxyalkanoate; Culture; 16S rRNA gene amplicon sequencing; Microbial community transition; quantitative PCR; pmoA; phaC] Methane (CH 4 ) is the second most abundant gas contributing to global warming with a global warming potential about 25 times that of carbon dioxide (1). Most CH 4 is microbiologically produced via consecutive anaerobic reactions of organic matter present in natural environments including paddy fields. In contrast, more than 90% of the CH 4 produced in anaerobic environments of paddy fields is oxidized by methane-oxidizing bacteria (MOB) under aer- obic conditions (2). MOB, capable of assimilating CH 4 , facilitate the mitigation of excessive CH 4 emission as a sink and, therefore, contribute to the prevention of global warming. MOB, phylogenetically diverse methanotrophs, are mainly divided into Type I and Type II, affiliated with Gamma- proteobacteria and Alpha-proteobacteria, respectively (3,4). Type I MOB cover 16 genera so far, including Methylocaldum, Methyl- ococcus, and Methylomonas (5e7), while Type II include Methylocella, Methylocapsa, Methylocystis, and Methylosinus (6,8,9). Type I and Type II MOB inhabit many natural environments, e.g., soils, wetlands, lakes, and oceans (10e14). Interestingly, MOB can not only assimilate CH 4 but also display the metabolic potential to synthesize valuable products (15). Polyhydroxyalkanoate (PHA) is one such valuable product syn- thesized especially by Type II MOB (16). Despite the relatively higher cost of PHA synthesis when compared with a normal resin manufactured from crude oil, replacing oil with naturally abun- dant CH 4 can reportedly reduce the environmental burden and raw material cost, contributing to, at maximum, 30% reduction in the production cost (17). Despite the intriguing metabolic potential of MOB, their phylog- enies and physiologies under a myriad of environmental conditions are yet to be holistically investigated. Among these conditions, high CH 4 concentrations determine the structure of MOB communities, particularly favoring Type II MOB (3). Given the ecological niche to preferentially grow Type II MOB, environments emitting a high concentration of CH 4 (ca. 90% v/v) (18, 19) are likely hotspots, and thus suitable for their enrichment. Specifically, a site close to a nat- ural gas plant received a higher CH 4 concentration and flux than landfill sites that have been deemed a well-known CH 4 source (20). MOB capable of efficiently degrading tetrachloroethylene via com- etabolism with CH 4 oxidation were obtained from the site. However, * Corresponding author at: Department of Chemical Engineering, Tokyo Univer- sity of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan. Tel.: þ81 42 388 7069; fax: þ81 42 388 7731. E-mail addresses: s170883x@st.go.tuat.ac.jp (S. Yasuda), rsk65@st.go.tuat.ac.jp (R. Toyoda), S.Agrawal@iwar.tu-darmstadt.de (S. Agrawal), suenagat@ go.tuat.ac.jp (T. Suenaga), sriya@cc.tuat.ac.jp (S. Riya), hori-tomo@aist.go.jp (T. Hori), S.Lackner@iwar.tu-darmstadt.de (S. Lackner), hosomi@cc.tuat.ac.jp (M. Hosomi), akte@cc.tuat.ac.jp (A. Terada). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx 1389-1723/$ e see front matter Ó 2020, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2020.04.006 Please cite this article as: Yasuda, S et al., Exploration and enrichment of methane-oxidizing bacteria derived from a rice paddy field emitting highlyconcentrated methane, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.04.006