Lawrence Berkeley National Laboratory Recent Work Title Formation of Zerovalent Iron in Iron-Reducing Cultures of Methanosarcina barkeri. Permalink https://escholarship.org/uc/item/1b183465 Journal Environmental science & technology, 54(12) ISSN 0013-936X Authors Shang, Haitao Daye, Mirna Sivan, Orit et al. Publication Date 2020-06-01 DOI 10.1021/acs.est.0c01595 Peer reviewed eScholarship.org Powered by the California Digital Library University of California 1 Formation of zero-valent iron in iron-reducing cultures of 2 Methanosarcina barkeri 3 4 Haitao Shang 1*, Mirna Daye 1, Orit Sivan 2, Caue S. Borlina 1, Nobumichi Tamura , 5 Benjamin P. Wei$$ 1 and Tanja Bo$a& 1 6 7 1Department of )arth, *tmospheric and "lanetary Science, Ma$$achusett$ Institute of Technology, 8 Cambridge, M*, US* 9 2Department of -eological and )nvironmental Science$, Ben -urion ,niversity of the Negev, Beer 10 Sheva, +$rael 11 *dvance% Light Source, La/rence Berkeley National Laboratory, Berkeley, CA, US* 12 13 *Email0 ht$1mit.edu 14 Abstract 15 Methanogenic archaea have been $ho/n to reduce iron from ferric 23e4+++56 to ferrous 23e4++56 $tate, 16 but mineral$ that form during iron reduction by di7erent methanogens remain to be characteri8ed. 17 Here, we show that 8ero-valent iron 4:;+5 mineral$, ferrite 2<93e(0)] and austenite 2>93e(0)], appear 18 in the ?-ray di7raction $pectra minute$ after the addition of ferrihydrite to the culture$ of the 19 methanogenic archaeon Methanosarcina barkeri 4M. barkeri). M. barkeri cell$ and redox-active, 20 non-enzymatic $oluble organic compounds in organic-rich $pent culture $upernatant$ can promote 21 the formation of :;+A the latter compounds al$o likely $tabili8e :;I. Methanogenic microbe$ that 22 inhabit organic9 and 3e(III)-rich anaerobic environment$ may $imilarly reduce oxidi8e% iron to 23 3e4++5 and :;+, /ith implications for the pre$ervation of paleomagnetic $ignal$ during $ediment 24 diagene$i$ and potential applications in the protection of iron metal$ against corrosion and in the 25 green synthe$i$ of Z;I. 26 Abstract Art 27 28 29 1 Introduction 30 Microorgani$m$ mediate numerous redox transformations of iron in natural environment$ and 31 couple the biogeochemical cycle$ of iron, carbon, oxygen, $ulfur, nitrogen and other element$ 21B 32 C]. In $oil$ and $ediment$, microbe$ can utili8e ferric iron 23e4+++56 a$ the electron acceptor for 33 di$$imilatory iron reduction [1,C,D]. The reduction of Fe(III) produce$ ferrous iron [Fe(II)] that can 34 be incorporate% into di7erent 3e(II)-containing mineral$ $uch a$ magnetite 2E,F], vivianite 2G,H6 35 and siderite 210,11]. The formation of $peciIc mineral pha$e$ i$ thought to depend on pH, electron 1 1 36 donors, pCO2 and other environmental factors [11]. 37 Methanosarcina barkeri 4M. barkeri), a coccoi% methanogen, can grow on methanol, acetate 38 and carbon dioxideJhydrogen 212,13]. The growth physiology of M. barkeri depends on the redox 39 potential of the ambient environment0 thi$ microbe i$ able to $urvive high redox-potential 40 conditions by generating it$ own low potential environment 214]. #hen M. barkeri produce$ 41 methane (CHC5 /ith hydrogen ga$ (H25 a$ the electron donor, it use$ $everal electron carriers /ith 0' 0' 42 low redox-potential$ such a$ ferredoxin (E K -500 m;), coenzyme FC20 (E K -360 m;), coenzyme 0' 0' 43 B 4E K -140 m;5 and methanophena8ine 4E K -165 m;5 2156. The$e enable M. barkeri to reduce 0' 44 a range of oxidi8e% compounds including ferrihydrite 23eOOH(am5 L 3e(II), E K -50m; 21666 45 into product$ /ith rather low reduction potential$. M. barkeri /a$ $ho/n to reduce amorphous 46 217B196 and cry$talline 220,216 3e4+++5 to 3e(II). Some of the$e $tudie$ al$o reporte% the formation 47 of iron mineral$ $uch a$ magnetite 2196 and vivianite 220]. However, only mineral pha$e$ that 48 contain 3e4+++5 and/or 3e4++5 have been reported, although other mineral$, $uch a$ ilmenite, /ere 49 hypothe$i8e% a$ well [17]. 50 The low redox-potential environment$ /here M. barkeri grow$ and persi$t$ may $upport the 51 formation of other iron pha$e$ /ith low reduction potential, such a$ 8ero-valent iron 4:;I) [Fe4+++5 − 0' 52 M e L 3e(0), E K 9 F m; 222]]. :;+ i$ unstable in most $urface environment$ because it i$ 53 ea$ily oxidi8e% to 3e4++5 or 3e4+++5 by both abiotic 2236 and biological 224B276 reactions in a 54 proce$$ known a$ iron corrosion. The reverse proce$$ B that i$, the reduction of oxidi8e% iron to 55 :;+ B ha$ al$o been observe% in $ome abiotic reactions 228B31]. One example i$ the formation of 56 3e4=5 in a/aruite, a nickel and iron-containing alloy, in $erpentini8ing environment$ 231]. Several 57 $tudie$ have al$o reporte% the reduction of 3e4+++5 in aqueous tea9lea( extract$ to :;+ 232B C]. 58 However, to the be$t of our knowledge, only one $tudy reporte% the pre$ence of $mall ?-ray 59 di7raction pea&$ of 3e4=5 in microbial enrichment culture$ of Geobacter sulfurreducens and 60 Shewanella denitrificans that gre/ on ochre pigment [ D]. 61 Here, /e explore the biominerali8ation of iron in low-potential environment$ that $upport 62 microbial methanogene$i$. Thi$ i$ done by characteri8ing mineral$ that form in iron-reducing 63 culture$ of M. barkeri and exploring mechani$m$ that produce and $tabili8e the$e mineral$. Our 64 re$ult$ demonstrate the formation of titanomagnetite (or magnetite5 and :;+ in active M. barkeri 65 culture$ and $pent culture $upernatant$. The ability of M. barkeri to reduce oxidi8e% iron to it$ 66 metallic $tate may influence the cycling of nutrient$ and toxins in the environment$, /ith potential 67 applications in the protection against iron corrosion and in the green synthe$i$ of Z;I. 68 69 2 Materials and Methods 70 2.1 Cell Incubation 71 M. barkeri (DSM G==5 /a$ obtaine% from Deut$che Sammlung von Mikroorgani$men und 72 :ellkulturen (DSM:, Braunschweig, -ermany). *ll $erum bottle$ (160 m.5 /ere autoclave% at 73 120 °C for = mins. Media /ere prepare% according to modiIe% medium recipe (Oregon 74 Collection of Methanogens Medium for Methanogens 21765 4Supporting Information, Table S1). 75 The medium containe% either a low organic content (1.0 g/. of D=/D= /tQ yea$t extract and 76 ca$itone, D+3CO5 or a high organic content (4.0 g/. of D=/D= /tQ yea$t extract and ca$itone, 77 D+3CO). Organic-free medium /a$ prepare% according to the $ame recipe, but /ithout yea$t 78 extract and ca$itone. The media /ere titrate% /ith a $aturate% NaHCO $olution to 'H 6.8. *ll 79 media /ere prepare% anaerobically, Ilter $terili8e% and adde% into the autoclave% $erum bottle$. 2 2 80 M. barkeri cannot grow in the pre$ence of O2, $o the vacuum-vorte@ technique 2 E6 /a$ use% to 81 generate anaerobic conditions in the serum bottle$. Ga$ mixture of H2/CO2 4G=Q/20%) wa$ adde% 82 into $erum bottle$ a$ the hea%$pace atmosphere. )ach 160 m. $erum bottle containe% D= m. of 83 liqui% and 110 m. of hea%$pace ga$. The Inal pre$$ure of hea%$pace atmosphere /a$ 100 &"a. 84 )ither Ti4+++59citrate (2.56 mM Inal concentration) or .-cysteine (0.5 mM Inal concentration) 85 /ere use% a$ reducing agent$. SulIde /a$ not use% a$ a reducing agent to avoi% reactions /ith 86 iron $pecie$ and the formation of $ulIde mineral$. "reliminary experiment$ u$e% di7erent Inal 87 concentrations of Ti4+++59citrate (0.85 mM, 2.56 mM and 7.67 mM5 and found that 2.56 mM 88 Ti(III)-citrate /a$ optimal for M. barkeri to grow and produce CHC. *ll culture$ and control$ 89 /ere incubate% at 37 °C. 90 91 2.2 Experimental Design 92 The experimental de$ign i$ summari8e% in Table 1. Initially, we explored the biominerali8ation in 93 M. barkeri culture$ in the pre$ence of ferrihydrite a$ a function of: (1) the content of organic 94 additive$ in medium, (2) the timing of ferrihydrite addition, and (3) the composition of hea%$pace 95 ga$ in $erum bottle$. The M. barkeri inocula for all experiment$ /ere grown in the medium /ith 96 1 g/. organic additive$, the$e culture$ /ere inoculate% at 1:10 v/v into media that containe% 97 either 1 g/. or C g/. organic additive$ for gro/th and/or iron-reduction experiment$. Most 98 experiment$ de$cribe% in /hat follow$ u$e% 1 g/. of the$e additive$ (yea$t extract and ca$itone), 99 a fourfol% reduction relative to C g/. in the original recipe 217]. "oorly crystalline ferrihydrite 100 /a$ prepare% by titrating 3eCl /ith 10N NaOH to pH F to a Inal concentration of 7.5 mM of 101 ferrihydrite. Before the addition of ferrihydrite, the hea%$pace$ of triplicate M. barkeri culture$ 102 and triplicate $terile control$ /ere Oushe% by N2/CO2 4G=Q/20%) for 1 hour. To te$t i( the 103 hea%$pace ga$ composition influence$ the precipitation of 3e(0), the hea%$pace$ of additional 104 $erum bottle$ /ith triplicate M. barkeri culture$ or $terile media /ere not Ou$he% to remove H2 105 and CHC. *queous 3eCl (7.5 mM Inal concentration) /a$ adde% to the M. barkeri culture$ to 106 te$t the influence of di7erent iron source$ on the production of Fe(0). 107 "recipitate$ forme% in M. barkeri culture$ and $terile control$ /ere $ample% = mins, 28 108 days and 42 days after the addition of ferrihydrite.