title: Reversing methanogenesis to capture methane for liquid biofuel precursors.
abstract: BACKGROUND
  Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones.
  
  
  RESULTS
  Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using (13)C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing.
  
  
  CONCLUSIONS
  We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. valwc.soo@gmail.com.
authors.forename: Valerie W C
authors.initials: VW
authors.lastname: Soo
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. mum278@psu.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McAnulty
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. sript.art@gmail.com.
authors.forename: Arti
authors.initials: A
authors.lastname: Tripathi
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. zhufy6@hotmail.com.
authors.forename: Fayin
authors.initials: F
authors.lastname: Zhu
authors.affiliation: Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, 16802-4400, USA. luz26@psu.edu.|Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China. luz26@psu.edu.
authors.forename: Limin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802-4400, USA. euc15@psu.edu.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Hatzakis
authors.affiliation: The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802-4400, USA. pbs13@psu.edu.
authors.forename: Philip B
authors.initials: PB
authors.lastname: Smith
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland, 0632, New Zealand. saumya.agrawal@gmail.com.
authors.forename: Saumya
authors.initials: S
authors.lastname: Agrawal
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. hnbokaee@psu.edu.
authors.forename: Hadi
authors.initials: H
authors.lastname: Nazem-Bokaee
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. sxg375@psu.edu.
authors.forename: Saratram
authors.initials: S
authors.lastname: Gopalakrishnan
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. salis@psu.edu.
authors.forename: Howard M
authors.initials: HM
authors.lastname: Salis
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802-4400, USA. jgf3@psu.edu.
authors.forename: James G
authors.initials: JG
authors.lastname: Ferry
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. costas@engr.psu.edu.
authors.forename: Costas D
authors.initials: CD
authors.lastname: Maranas
authors.affiliation: Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, 16802-4400, USA. adp117@psu.edu.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Patterson
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802-4400, USA. tuw14@psu.edu.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802-4400, USA. tuw14@psu.edu.
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D056804:Biofuels; D008697:Methane / Q000378:metabolism*; D017020:Methanosarcina / Q000201:enzymology / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism
chemical_list: D056804:Biofuels; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D008697:Methane