AEM Accepts, published online ahead of print on 16 May 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.00895-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Direct Interspecies Electron Transfer Between Geobacter
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metallireducens and Methanosarcina barkeri
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Amelia-Elena Rotarua, *,, Pravin Malla Shresthaa, , Fanghua Liua, b, Beatrice
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Markovaitea, Shanshan Chena, c, Kelly Nevina, Derek Lovleya
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Department of Microbiology, University of Massachusetts Amhersta; Yantai
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Institute of Coastal Zone Researchb; School of Environmental Science and
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Engineering, Sun Yat-sen University, Guangzhou 510275, Chinac
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Running Head: DIET by Methanosarcina barkeri
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*Address correspondence to Amelia-Elena Rotaru: [email protected].
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University of Southern Denmark, Denmark;
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Present address: A. -E. Rotaru, Nordic Center for Earth Evolution,
Present address: P.M. Shrestha, Department of Plant and Microbial Biology, University of California Berkeley, United States.
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ABSTRACT
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Direct interspecies electron transfer (DIET) is potentially an effective form of
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syntrophy in methanogenic communities, but little is known about the diversity of
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methanogens capable of DIET. The ability of Methanosarcina barkeri, to participate
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in DIET was evaluated in co-culture with Geobacter metallireducens. Co-cultures
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formed aggregates that shared electrons via DIET during the stoichiometric
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conversion of ethanol to methane. Co-cultures could not be initiated with a pilin-
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deficient G. metallireducens, suggesting that long-range electron transfer along pili
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was important for DIET. Amendments of granular activated carbon permitted the
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pilin-deficient G. metallireducens to share electrons with M. barkeri, demonstrating
30
that this conductive material could substitute for pili in promoting DIET. When M.
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barkeri was grown in co-culture with the H2-producing Pelobacter carbinolicus,
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incapable of DIET, M. barkeri utilized H2 as an electron donor, but metabolized little
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of the acetate that P. carbinolicus produced. This suggested that H2, but not electrons
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derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri co-
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cultures did not aggregate, demonstrating that, unlike DIET, close physical contact
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was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen
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found to accept electrons via DIET and the first methanogen known to be capable of
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using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M.
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barkeri is genetically tractable, making it a model organism for elucidating
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mechanisms by which methanogens make biological electrical connections with other
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cells.
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INTRODUCTION
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43
Effective interspecies electron transfer is key to the efficient functioning of
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methanogenic communities (1-4). Promoting interspecies electron transfer to
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methanogens enhances the anaerobic digestion of wastes and appropriate models of
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the pathways for interspecies electron transfer are necessary in order to predictively
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model the response of methanogenic communities to environmental change.
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The best-known strategy for interspecies electron transfer is H2 interspecies
49
transfer (HIT), in which electron-donating microorganisms reduce protons to H2 and
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methanogens oxidize the H2 with the reduction of carbon dioxide to methane (1). In
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some instances, formate substitutes for H2 as the electron carrier (2-6). The
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abundance of H2/formate-consuming methanogens in anaerobic soils (7, 8) and
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sediments (9, 10) as well as some anaerobic digesters (11) suggests that HIT plays an
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important role in methane production in those environments. HIT has been
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documented in studies with defined co-cultures of H2-donating microorganisms and
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H2-consuming methanogens, and the physiology and biochemistry of both H2
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production and H2 consumption are relatively well understood (1, 12, 13).
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Direct interspecies electron transfer (DIET) is an alternative to HIT.
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Methanosaeta haurindacaea, which is representative of the Methanosaeta species that
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are responsible for a substantial portion of the global methane production (14),
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directly accepted electrons from Geobacter metallireducens for the reduction of
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carbon dioxide to methane in defined co-cultures (15). DIET in these co-cultures
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required that G. metallireducens produce pili (15), that have a metallic-like
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conductivity (16, 17), but further details of the interspecies electrical connections are
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as yet unknown. Multiple lines of evidence suggested that Methanosaeta species were
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also participating in DIET in anaerobic digesters treating brewery wastes (15, 18).
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Geobacter species were abundant in the digester granules, which possessed a
3
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metallic-like conductivity (18) similar to the metallic-like conductivity of Geobacter
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pili (16-18). Granule conductivity in different digesters was directly correlated with
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the abundance of Geobacter species (19). The potential importance of conductive pili
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in electron transfer to Methanosaeta species is consistent with the clear importance of
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conductive pili in DIET in co-cultures of G. metallireducens and G. sulfurreducens,
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cooperating to oxidize ethanol with the reduction of fumarate (20-23). The electrical
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connections that M. harundinacea employs for DIET are unknown and elucidating
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them may be technically difficult because a system for genetic manipulation of this
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organism has not yet been identified.
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Another genus of acetoclastic methanogens, Methanosarcina, is often
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abundant in methanogenic soils and sediments (24-26), coal mines (27, 28), landfills
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(29), and anaerobic digesters (30, 31). Several studies suggested that Methanosarcina
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species could accept electrons from non-biological extracellular surfaces (32, 33). For
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example, M. barkeri attached onto electrically conductive granular activated carbon
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(GAC), which served as a mediator for electron transfer between M. barkeri and G.
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metallireducens (33). Conductive iron minerals were proposed to function as
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mediators between Geobacter and Methanosarcina species in a similar manner, based
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on the abundance of these genera in mineral-amended enrichment cultures (32). Here
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we provide evidence that M. barkeri does not require a conductive mediator for DIET
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because it is capable of forging direct biological electrical connections to receive
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electrons from G. metallireducens.
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MATERIALS AND METHODS
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Strains and conditions for cultivation
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Methanosarcina barkeri (DSM 800) and Pelobacter carbinolicus (DSM 2380)
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were purchased from the DSMZ culture collection. Geobacter metallireducens
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(ATCC 53774) as well as gene-deletion strains in which either the gene for PilA, the
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structural pilin protein (Gmet 1399) (34), or gene Gmet 1868, encoding a putative
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outer-surface c-type cytochrome (35), was deleted were obtained from our laboratory
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culture collection. Strict anaerobic culturing procedures with either pressure tubes or
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serum bottles sealed with thick butyl rubber stoppers were used throughout.
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M. barkeri was grown on 30 mM acetate in a modification of DSM medium 120 medium, which was determined to also support good growth of G.
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metallireducens. The DSM 120 modifications were as follows: 0.5 mM sulfide, 1 mM
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cysteine, 0.002 g/L CaCl2 × 2H2O, 1 g/L NaCl, no yeast extract, no casitone, no
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resazurin, and 2 g/L NaHCO3.
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G. metallireducens wild type and mutant strains were routinely cultured on a
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ferric citrate medium amended with 10 mM acetate (34). However, prior to initiating
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co-cultures with M. barkeri, the G. metallireducens strains were adapted for at least
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three transfers (5-10% inoculum) in ferric citrate medium in which the acetate was
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replaced with 20 mM ethanol.
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P. carbinolicus was routinely grown on 10 mM acetoin, as previously described (21). The co-culture inocula were obtained from cultures that were in late
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exponential or early stationary phase, as determined by monitoring methane (M.
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barkeri), Fe(II) production (G. metallireducens) or OD600nm (P. carbinolicus).
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The G. metallireducens/M. barkeri co-cultures were initiated with a 0.5 ml
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inoculum of the ethanol-adapted G. metallireducens and a 0.5 ml inoculum of an
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acetate-grown M. barkeri culture added to 9 ml of the modified DSM 120 medium
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described above, but with the acetate replaced with 20 mM ethanol as the electron
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donor. CO2/HCO3- was the only potential electron acceptor. After the co-culture was
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established it was routinely transferred into 45 ml of medium with a 10 % inoculum.
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Co-culture growth was also tested in the absence of sulfide and cysteine, but for long-
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term incubations both sulfide and cysteine are required as sulfur sources for the M.
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barkeri (36-38) and were therefore added consistently to the co-culture medium.
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When noted, granular activated carbon (GAC) was added as previously described (33).
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Similar to GAC-free co-cultures, 9 ml medium with GAC was inoculated with 0.5 ml
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of a G. metallireducens strain and 0.5 ml M. barkeri.
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M. barkeri grew in the same media as P. carbinolicus, but P. carbinolicus
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could not grow in DSM 120 media, or the modified version of it. Therefore, the P.
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carbinolicus - M. barkeri co-cultures were initiated with a 5 % inoculum of acetoin-
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grown P. carbinolicus and a 5 % inoculum of acetate-grown M. barkeri in the
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previously described P. carbinolicus medium (21). After establishing the co-culture,
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P. carbinolicus – M. barkeri were routinely transferred together using 10% inocula.
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Analytics
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Headspace methane (0.5 mL) was sampled at regular intervals using strict
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anaerobic sampling techniques (39), injected on a Shimadzu 8A GC gas
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chromatograph equipped with a 80/100 Hayasep Q column heated at 110°C. The
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injector port and FID detector were both set at 200°C (15). To determine the
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concentration of organic acids, and ethanol 200 µL of culture medium were sampled,
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diluted, and placed in tightly sealed vials specially designed for HPLC analysis (fatty
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acids) and GC analysis (ethanol). Organic acid samples were analyzed immediately,
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or stored at 4°C for maximum of one week. HPLC analysis were performed as
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previously described (40) by separating the organic acids on an Aminex NPX-87H
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column using 8 mM H2SO4 as eluent. Organic acids were detected at 210 nm by an
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UV detector. Under these conditions the following organic acids could be monitored:
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acetate, formate, succinate, fumarate, lactate, propionate, and butyrate. The detection
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limit for all was ca. 5 µM. In all our samples only acetate was detected. Gas
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chromatography analyses of ethanol were performed on a Perkin Elmer Clarus600 GC
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equipped with a headspace automatic sampler and an FID detector. Separation of
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ethanol was obtained on an Elite 5 (Perkin Elmer) column (30 m length, 0.25 mm ID)
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using He as carrier gas and the following separation parameters: 50ºC for 1 minute, a
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ramp of 12ºC per minute to reach 200ºC, and 1.5 minutes at 200ºC. The injector and
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detector temperatures were set at 200ºC and 300ºC, respectively. The chromatography
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data from GC and HPLC analyses were analyzed with an integrated TotalChrom data
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analysis system.
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The increase in aggregate biomass over time was determined as previously
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described (41) by harvesting (15 minutes, 3600 g) 50 ml of three M. barkeri – G.
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metallireducens co-cultures at different growth stages – during the initial growth
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phase (day 5) and in stationary phase (day 28). Aggregates were washed in isotonic
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buffer and freeze-dried for 48 hours on a Labconco lyophilizer at -50ºC. The weighted
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dry biomass was solubilized in 0.5% SDS by steaming. The total protein in the
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steamed biomass was determined using the PierceTM BCA Protein assay kit, following
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manufacturers’ instructions. Bovine serum albumin was used as protein standard.
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Microscopy
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To evaluate cell-to-cell contact, we checked unfixed cells immediately after
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removing them from a culture tube and visualized the cultures with phase contrast
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microscopy on a Leica Axioplann microscope. The presence of the methanogens was
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verified by fluorescence at 420 nm and their specific coccus shape. To better visualize
7
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how cells were distributed within the aggregates, we performed fluorescence in situ
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hybridization. Cells were sampled in mid-log, fixed in premixed glutaraldehyde
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(0.5%) and paraformaldehyde (1%), rinsed in 50 mM PIPES buffer, dehydrated (30
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min at 4°C, in ethanol series 30%, 50%, 70%, 80%, and 100%), and pre-embedded in
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a 50:50 mix of ethanol (100%) and butyl-methyl-metacrylate (BMM). Ultimately
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aggregates were embedded in BMM resin and polymerized on a Spectorlinker
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crosslink XL-1000 at room temperature over night. Sections of the aggregates were
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obtained by microtome and stained with Cy3-probes (red) targeting Methanosarcina
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species (MS1414) 5’-CTCACCCATACCTCACTCGGG-3’ (42) and Cy5-probes
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(green) specific for the Geobacter cluster (Geo3ABC, 5’-
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CCGCAACACCTRGTWCTCATC-3’) (43) or Cy5 (green) labeled probes specific
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for P. carbinolicus (PCARB1, 5’-GCCTATTCGACCACGATA-3’) (43) depending
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on the co-culture. The labeled co-cultures were visualized using a confocal laser
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microscope as previously described (21).
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RESULTS AND DISCUSSION
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Growth of M. barkeri via DIET with G. metallireducens
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Methanosarcina barkeri was co-cultured with Geobacter metallireducens in a
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medium with ethanol as the sole electron donor in order to determine whether it was
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capable of DIET. Multiple lines of evidence have indicated that G. metallireducens is
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unable to release the electrons derived from the oxidation of ethanol to acetate as
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either H2 or formate, but can directly transfer these electrons to microorganisms such
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as G. sulfurreducens or Methanosaeta harundinacea which are capable of DIET (15,
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20-22). After a lag of ca. 39 days, co-cultures of G. metallireducens and M. barkeri
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started metabolizing ethanol to methane (Fig. 1a). Neither G. metallireducens nor M.
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barkeri metabolized ethanol in pure culture (Fig. 1b). With continued transfer of the
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co-culture, the initial long lag in ethanol metabolism was eliminated and ethanol
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began to be metabolized to methane in less than 7 days (Fig. 1c).
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Acetate only transiently accumulated in the co-cultures (Fig. 1a and 1c). The
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total ethanol consumed (0.95 ± 0.01 mmol; mean ± standard deviation, n=4) resulted
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in 1.59 ± 0.04 mmol methane, consistent with the expected production of 1.5 moles of
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methane for each mole of ethanol consumed according to:
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2 C2H6O + H2O 3 CH4 + CO2. This stoichiometric conversion of ethanol to methane requires that M. barkeri
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not only metabolize the acetate that G. metallireducens produces from ethanol: 2
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C2H6O + 2 H2O 2 C2H4O2 + 8 H+ + 8 e- via the reaction 2 C2H4O2 2 CH4 + 2
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CO2, but also use the electrons released from ethanol metabolism to reduce carbon
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dioxide to methane: 8H+ + 8e- + CO2 CH4 + 2 H2O. Thus, the high electron
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recovery of electrons available from ethanol in methane, and the fact that G.
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metallireducens is incapable of producing H2 during ethanol metabolism (23),
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strongly suggested that M. barkeri was accepting electrons for carbon dioxide
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reduction via DIET.
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The fact that the co-culture could be continually transferred (10 % inoculum)
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indicated that a small fraction of the carbon and electrons derived from ethanol must
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also be consumed for biomass production. Biomass was examined by sacrificing
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triplicate co-cultures for protein analysis at day 5, when methane started increasing
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and at day 28 when methane production was complete. Protein increased from 233 ±
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62 µg ml-1 (mean ± standard deviation) to 861 ± 115 µg ml-1, verifying co-culture
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growth.
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Cells involved in DIET need physical connections. G. metallireducens and M.
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barkeri co-cultures formed aggregates of ca. 100-200 µm diameter (Fig. 2 a) in which
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both Methanosarcina and Geobacter were intertwined (Fig. 2b).
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Electrical Connections via Outer Surface Proteins and Granular Activated
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Carbon
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Previous studies have demonstrated that deleting pilA, which encodes the structural
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protein of the conductive pili of Geobacter species, eliminates the ability of G.
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metallireducens to participate in DIET (15, 22). Co-cultures initiated with a pilA-
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deficient strain (34) of G. metallireducens failed to metabolize ethanol or produce
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methane (Fig. 3a). Furthermore, co-cultures initiated with a strain of G.
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metallireducens in which a gene (Gmet 1868) for an outer-surface cytochrome
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required for Fe(III) oxide reduction was deleted (35) also failed to metabolize ethanol
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to methane (Fig. 3b).
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However, co-cultures initiated with either the pilA-deficient or Gmet 1868-
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deficient strains of G. metallireducens did effectively produce methane in the
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presence of granular activated carbon (GAC) (Fig. 3c). This is consistent with
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previous studies which demonstrated that GAC, which is electrically conductive,
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served as electrical conduit for DIET between G. metallireducens and G.
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sulfurreducens, permitting the two species to share electrons even when the co-culture
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was initiated with strains of G. sulfurreducens that could not produce pili or the pilin-
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associated c-type cytochrome OmcS (33). As previously noted (33), GAC
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amendments also stimulated the conversion of ethanol to methane in co-cultures
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initiated with wild-type G. metallireducens and M. barkeri (Fig. 3c), substantially
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reducing the 39 day long lag phase observed in the absence of GAC (Fig. 1a).
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Methane production of the co-cultures initiated with the pilA-deficient or Gmet 1868-
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deficient strains of G. metallireducens were slower than the wild-type co-cultures
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amended with GAC, but even in the absence of pili or the outer-surface c-type
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cytochrome methane production rates in the presence of GAC required less than a 6
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day lag period to initiate ethanol metabolism (Fig. 3b). Previous studies demonstrated
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that in co-cultures of G. metallireducens with either M. barkeri or G. sulfurreducens
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both syntrophic partners attached to the GAC surface, but the individual cells were
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too far separated for biological electrical connections between the species (33). These
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results suggest that GAC can substitute for pili and/or outer-surface cytochromes as
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the electrical conduit between the electron-donating G. metallireducens and the
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electron-accepting M. barkeri.
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Growth of M. barkeri via HIT with P. carbionolicus
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Like G. metallireducens, P. carbinolicus can grow on ethanol in the presence
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of a syntrophic partner, but it relies on HIT rather than DIET (21, 44). P. carbinolicus
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and M. barkeri grew in co-culture in medium with ethanol as the sole electron donor,
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but unlike G. metallireducens-M. barkeri co-cultures, there was a steady
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accumulation of acetate (Fig. 4a). This was probably due to the fact that acetate
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metabolism in M. barkeri is repressed when H2 is available (45-47).
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P. carbinolicus metabolizes ethanol to acetate and H2 according to: 2 C2H6O + 2
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H2O 2 C2H4O2 + 4 H2 (21). The metabolism of ethanol to acetate and H2 is only
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exergonic if M. barkeri can oxidize H2 with the reduction of carbon dioxide as
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follows: 4 H2 + CO2 CH4 + H2O.
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The stoichiometry of ethanol consumed (0.91 ± 0.07 mmol, mean ± standard
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deviation, n=4), methane produced (0.41 ± 0.07 mmol) and acetate accumulated (0.66
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± 0.07 mmol) were consistent with methane being produced almost exclusively from
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H2 oxidation with the reduction of carbon dioxide, assuming that some of the acetate
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produced was diverted to biomass production. A similar partial oxidation of ethanol
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with the accumulation of acetate was observed in the ethanol-metabolizing co-cultures
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of P. carbinolicus - G. sulfurreducens, which were dependent on HIT (21), whereas
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acetate did not accumulate in G. metallireducens-G. sulfurreducens co-cultures that
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relied on DIET (21).
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In contrast to the G. metallireducens - M. barkeri co-cultures (Fig 2a, b) the P.
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carbinolicus - M. barkeri co-cultures did not form multispecies aggregates (Fig. 4b, c).
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In a similar manner, P. carbinolicus - G. sulfurreducens co-cultures did not aggregate
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(21), whereas G. metallireducens-G. sulfurreducens did (20). These results are
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consistent with the need for direct cell-to-cell contact for DIET, but not for HIT.
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IMPLICATIONS
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The results demonstrate that M. barkeri is able to participate in DIET.
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It is only the second methanogen found to have this capability. Methanosarcina
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species are important contributors to methane production in some anaerobic digesters
278
(30) as well as soils and sediments (7-10). Thus the potential for DIET should be
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considered when attempting to promote the growth of Methanosarcina in digesters or
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when modeling their activity in soils and sediments.
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In previous pure culture studies, Methanosaeta haurindacea, which is
282
incapable of utilizing H2 as an electron donor, was found to be capable of DIET, but
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the two known H2-utilizing methanogens tested, Methanospirillum hungatei and
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Methanobacterium formicicum, were not (15). Thus, M. barkeri is more versatile
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than other methanogens that have been evaluated for DIET, with the capacity to either
286
accept electrons in the form of H2 from syntropic partners like Desulfovibrio (47)
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and Pelobacter (this study), or to participate in DIET.
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In the studies on DIET with M. harundinacea acetate was available as an
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additional substrate for methane production (15) as it was in the M. barkeri co-
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cultures reported here. However, studies with co-cultures of G. metallireducens and a
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strain of G. sulfurreducens unable to oxidize acetate demonstrated that electrons
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derived from DIET could support fumarate reduction and growth of G. sulfurreducens
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in the absence of acetate as an additional electron donor (23). Similar studies should
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be feasible with a M. barkeri mutant unable to metabolize acetate (48) to determine
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whether carbon dioxide reduction to methane with electrons derived from DIET
296
provides sufficient energy to support cell maintenance and growth. Other than the
297
obvious importance of DIET in some anaerobic digesters treating brewery wastes (15,
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18, 19) the prevalence of DIET in other methanogenic communities is unknown. The
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fact that at least two major genera of methanogens have evolved the capacity for
300
DIET suggests that there are conditions in some soils and sediments in which DIET
301
confers a selective advantage.
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The ability of GAC to promote methanogenic DIET has important
303
implications for the design of anaerobic digesters. Previous studies that have reported
304
stimulation in methane production when carbon cloth was introduced into digesters
305
have attributed this response to the cloth retaining methanogens (49-51). However,
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the possibility that these conductive materials might promote DIET should also be
307
considered.
308
Although the importance of electrically conductive pili and outer-surface
309
cytochromes in extracellular electron exchange, including DIET, is becoming
310
increasingly apparent for Geobacter species (52, 53) the potential extracellular
311
electron contacts that might permit methanogens to accept electrons via DIET are less
312
clear. Methanosaeta and Methanosarcina are the only genera of methanogens with
13
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membrane-bound cytochromes (54, 55), which could potentially play a role in
314
extracellular electron exchange. The availability of tools for genetic manipulation of
315
M. barkeri (56) suggests that it may be the ideal candidate for functional analysis of
316
DIET mechanisms in methanogens.
317
ACKNOWLEDGEMENTS
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We would like to thank Dr. Pier-Luc Tremblay and Jessica Smith for
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providing G. metallireducens deletion-mutant strains. We want to acknowledge the
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laboratory assistance of Dan Carlo Flores, Trevor Woodard and Joy Ward. The work
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was supported by the Office of Science, US Department of Energy, award number:
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DE-SC0004485
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FIGURE LEGENDS
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Fig. 1. Syntrophic growth of M. barkeri with G. metallireducens. (a)
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Metabolism of ethanol when the co-cultures were initiated with a 5 % (vol:vol)
326
inoculum of each species. (b) Lack of ethanol metabolism with single species. (c)
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Decreased lag after 25 transfers (10% inoculum) of the co-cultures. All cultures were
328
grown in triplicate (n=3). A difference in the Y-scale can be noted between a, b and c.
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This is because of differences in total incubation volume: 10 ml to initiate the co-
330
cultures (a, b), and 50 ml for later incubations (c).
331
Fig. 2. Co-culture composition. (a) Phase contrast micrograph of an
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aggregate of M. barkeri (cocci) and G. metallireducens (rods). (b) Scanning laser
333
confocal microscope image of co-culture aggregate after in situ hybridization, which
334
targeted Methanosarcina cells with a red probe (Cy3) and G. metallireducens with a
14
335
green probe (Cy5). Images are representative of triplicate samples taken during the
336
mid-exponential growth phase of the co-cultures.
337
Fig. 3. Mutant studies. (a) Lack of ethanol metabolism and methane
338
production in a co-culture initiated with a pilA-deficient G. metallireducens (b)
339
Methane production in co-cultures initiated with wild-type G. metallireducens or a
340
strain deficient in the gene Gmet 1868, which encodes a predicted extracellular
341
cytochrome. (c) GAC stimulation of methane production in co-cultures initiated with
342
wild-type G. metallireducens and enabling methane production in co-cultures initiated
343
with either the PilA- or cytochrome (Gmet-1868)-deficient strains of G.
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metallireducens. All incubations were set up with 5% (vol:vol) inoculum of each co-
345
culture partner.
346
Fig. 4. Syntrophic growth in co-cultures of P. carbinolicus and M. barkeri.
347
(a) Syntrophic growth in the third transfer of the co-culture. (b) Phase contrast image
348
illustrating lack of aggregation of P. carbinolicus (rods) and M. barkeri (cocci). (c)
349
Scanning laser confocal microscope image of co-culture after in situ hybridization,
350
which targeted Methanosarcina cells with a red probe (Cy3) and P. carbinolicus with
351
a green probe (Cy5). Images are representative of triplicate samples taken during the
352
mid-exponential growth phase of the co-cultures.
353
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23
1
Direct Interspecies Electron Transfer Between Geobacter
2
metallireducens and Methanosarcina barkeri
3
Amelia-Elena Rotarua, *,, Pravin Malla Shresthaa, , Fanghua Liua, b, Beatrice
4
Markovaitea, Shanshan Chena, c, Kelly Nevina, Derek Lovleya
5 6
Department of Microbiology, University of Massachusetts Amhersta; Yantai
7
Institute of Coastal Zone Researchb; School of Environmental Science and
8
Engineering, Sun Yat-sen University, Guangzhou 510275, Chinac
9 10 11
Running Head: DIET by Methanosarcina barkeri
12 13
*Address correspondence to Amelia-Elena Rotaru: [email protected].
14
15
University of Southern Denmark, Denmark;
16 17
Present address: A. -E. Rotaru, Nordic Center for Earth Evolution,
Present address: P.M. Shrestha, Department of Plant and Microbial Biology, University of California Berkeley, United States.
18 19
1
20
ABSTRACT
21
Direct interspecies electron transfer (DIET) is potentially an effective form of
22
syntrophy in methanogenic communities, but little is known about the diversity of
23
methanogens capable of DIET. The ability of Methanosarcina barkeri, to participate
24
in DIET was evaluated in co-culture with Geobacter metallireducens. Co-cultures
25
formed aggregates that shared electrons via DIET during the stoichiometric
26
conversion of ethanol to methane. Co-cultures could not be initiated with a pilin-
27
deficient G. metallireducens, suggesting that long-range electron transfer along pili
28
was important for DIET. Amendments of granular activated carbon permitted the
29
pilin-deficient G. metallireducens to share electrons with M. barkeri, demonstrating
30
that this conductive material could substitute for pili in promoting DIET. When M.
31
barkeri was grown in co-culture with the H2-producing Pelobacter carbinolicus,
32
incapable of DIET, M. barkeri utilized H2 as an electron donor, but metabolized little
33
of the acetate that P. carbinolicus produced. This suggested that H2, but not electrons
34
derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri co-
35
cultures did not aggregate, demonstrating that, unlike DIET, close physical contact
36
was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen
37
found to accept electrons via DIET and the first methanogen known to be capable of
38
using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M.
39
barkeri is genetically tractable, making it a model organism for elucidating
40
mechanisms by which methanogens make biological electrical connections with other
41
cells.
42
INTRODUCTION
2
43
Effective interspecies electron transfer is key to the efficient functioning of
44
methanogenic communities (1-4). Promoting interspecies electron transfer to
45
methanogens enhances the anaerobic digestion of wastes and appropriate models of
46
the pathways for interspecies electron transfer are necessary in order to predictively
47
model the response of methanogenic communities to environmental change.
48
The best-known strategy for interspecies electron transfer is H2 interspecies
49
transfer (HIT), in which electron-donating microorganisms reduce protons to H2 and
50
methanogens oxidize the H2 with the reduction of carbon dioxide to methane (1). In
51
some instances, formate substitutes for H2 as the electron carrier (2-6). The
52
abundance of H2/formate-consuming methanogens in anaerobic soils (7, 8) and
53
sediments (9, 10) as well as some anaerobic digesters (11) suggests that HIT plays an
54
important role in methane production in those environments. HIT has been
55
documented in studies with defined co-cultures of H2-donating microorganisms and
56
H2-consuming methanogens, and the physiology and biochemistry of both H2
57
production and H2 consumption are relatively well understood (1, 12, 13).
58
Direct interspecies electron transfer (DIET) is an alternative to HIT.
59
Methanosaeta haurindacaea, which is representative of the Methanosaeta species that
60
are responsible for a substantial portion of the global methane production (14),
61
directly accepted electrons from Geobacter metallireducens for the reduction of
62
carbon dioxide to methane in defined co-cultures (15). DIET in these co-cultures
63
required that G. metallireducens produce pili (15), that have a metallic-like
64
conductivity (16, 17), but further details of the interspecies electrical connections are
65
as yet unknown. Multiple lines of evidence suggested that Methanosaeta species were
66
also participating in DIET in anaerobic digesters treating brewery wastes (15, 18).
67
Geobacter species were abundant in the digester granules, which possessed a
3
68
metallic-like conductivity (18) similar to the metallic-like conductivity of Geobacter
69
pili (16-18). Granule conductivity in different digesters was directly correlated with
70
the abundance of Geobacter species (19). The potential importance of conductive pili
71
in electron transfer to Methanosaeta species is consistent with the clear importance of
72
conductive pili in DIET in co-cultures of G. metallireducens and G. sulfurreducens,
73
cooperating to oxidize ethanol with the reduction of fumarate (20-23). The electrical
74
connections that M. harundinacea employs for DIET are unknown and elucidating
75
them may be technically difficult because a system for genetic manipulation of this
76
organism has not yet been identified.
77
Another genus of acetoclastic methanogens, Methanosarcina, is often
78
abundant in methanogenic soils and sediments (24-26), coal mines (27, 28), landfills
79
(29), and anaerobic digesters (30, 31). Several studies suggested that Methanosarcina
80
species could accept electrons from non-biological extracellular surfaces (32, 33). For
81
example, M. barkeri attached onto electrically conductive granular activated carbon
82
(GAC), which served as a mediator for electron transfer between M. barkeri and G.
83
metallireducens (33). Conductive iron minerals were proposed to function as
84
mediators between Geobacter and Methanosarcina species in a similar manner, based
85
on the abundance of these genera in mineral-amended enrichment cultures (32). Here
86
we provide evidence that M. barkeri does not require a conductive mediator for DIET
87
because it is capable of forging direct biological electrical connections to receive
88
electrons from G. metallireducens.
89
MATERIALS AND METHODS
90
Strains and conditions for cultivation
4
91
Methanosarcina barkeri (DSM 800) and Pelobacter carbinolicus (DSM 2380)
92
were purchased from the DSMZ culture collection. Geobacter metallireducens
93
(ATCC 53774) as well as gene-deletion strains in which either the gene for PilA, the
94
structural pilin protein (Gmet 1399) (34), or gene Gmet 1868, encoding a putative
95
outer-surface c-type cytochrome (35), was deleted were obtained from our laboratory
96
culture collection. Strict anaerobic culturing procedures with either pressure tubes or
97
serum bottles sealed with thick butyl rubber stoppers were used throughout.
98 99
M. barkeri was grown on 30 mM acetate in a modification of DSM medium 120 medium, which was determined to also support good growth of G.
100
metallireducens. The DSM 120 modifications were as follows: 0.5 mM sulfide, 1 mM
101
cysteine, 0.002 g/L CaCl2 × 2H2O, 1 g/L NaCl, no yeast extract, no casitone, no
102
resazurin, and 2 g/L NaHCO3.
103
G. metallireducens wild type and mutant strains were routinely cultured on a
104
ferric citrate medium amended with 10 mM acetate (34). However, prior to initiating
105
co-cultures with M. barkeri, the G. metallireducens strains were adapted for at least
106
three transfers (5-10% inoculum) in ferric citrate medium in which the acetate was
107
replaced with 20 mM ethanol.
108 109 110
P. carbinolicus was routinely grown on 10 mM acetoin, as previously described (21). The co-culture inocula were obtained from cultures that were in late
111
exponential or early stationary phase, as determined by monitoring methane (M.
112
barkeri), Fe(II) production (G. metallireducens) or OD600nm (P. carbinolicus).
113
The G. metallireducens/M. barkeri co-cultures were initiated with a 0.5 ml
114
inoculum of the ethanol-adapted G. metallireducens and a 0.5 ml inoculum of an
115
acetate-grown M. barkeri culture added to 9 ml of the modified DSM 120 medium
5
116
described above, but with the acetate replaced with 20 mM ethanol as the electron
117
donor. CO2/HCO3- was the only potential electron acceptor. After the co-culture was
118
established it was routinely transferred into 45 ml of medium with a 10 % inoculum.
119
Co-culture growth was also tested in the absence of sulfide and cysteine, but for long-
120
term incubations both sulfide and cysteine are required as sulfur sources for the M.
121
barkeri (36-38) and were therefore added consistently to the co-culture medium.
122
When noted, granular activated carbon (GAC) was added as previously described (33).
123
Similar to GAC-free co-cultures, 9 ml medium with GAC was inoculated with 0.5 ml
124
of a G. metallireducens strain and 0.5 ml M. barkeri.
125
M. barkeri grew in the same media as P. carbinolicus, but P. carbinolicus
126
could not grow in DSM 120 media, or the modified version of it. Therefore, the P.
127
carbinolicus - M. barkeri co-cultures were initiated with a 5 % inoculum of acetoin-
128
grown P. carbinolicus and a 5 % inoculum of acetate-grown M. barkeri in the
129
previously described P. carbinolicus medium (21). After establishing the co-culture,
130
P. carbinolicus – M. barkeri were routinely transferred together using 10% inocula.
131
Analytics
132
Headspace methane (0.5 mL) was sampled at regular intervals using strict
133
anaerobic sampling techniques (39), injected on a Shimadzu 8A GC gas
134
chromatograph equipped with a 80/100 Hayasep Q column heated at 110°C. The
135
injector port and FID detector were both set at 200°C (15). To determine the
136
concentration of organic acids, and ethanol 200 µL of culture medium were sampled,
137
diluted, and placed in tightly sealed vials specially designed for HPLC analysis (fatty
138
acids) and GC analysis (ethanol). Organic acid samples were analyzed immediately,
139
or stored at 4°C for maximum of one week. HPLC analysis were performed as
140
previously described (40) by separating the organic acids on an Aminex NPX-87H
6
141
column using 8 mM H2SO4 as eluent. Organic acids were detected at 210 nm by an
142
UV detector. Under these conditions the following organic acids could be monitored:
143
acetate, formate, succinate, fumarate, lactate, propionate, and butyrate. The detection
144
limit for all was ca. 5 µM. In all our samples only acetate was detected. Gas
145
chromatography analyses of ethanol were performed on a Perkin Elmer Clarus600 GC
146
equipped with a headspace automatic sampler and an FID detector. Separation of
147
ethanol was obtained on an Elite 5 (Perkin Elmer) column (30 m length, 0.25 mm ID)
148
using He as carrier gas and the following separation parameters: 50ºC for 1 minute, a
149
ramp of 12ºC per minute to reach 200ºC, and 1.5 minutes at 200ºC. The injector and
150
detector temperatures were set at 200ºC and 300ºC, respectively. The chromatography
151
data from GC and HPLC analyses were analyzed with an integrated TotalChrom data
152
analysis system.
153
The increase in aggregate biomass over time was determined as previously
154
described (41) by harvesting (15 minutes, 3600 g) 50 ml of three M. barkeri – G.
155
metallireducens co-cultures at different growth stages – during the initial growth
156
phase (day 5) and in stationary phase (day 28). Aggregates were washed in isotonic
157
buffer and freeze-dried for 48 hours on a Labconco lyophilizer at -50ºC. The weighted
158
dry biomass was solubilized in 0.5% SDS by steaming. The total protein in the
159
steamed biomass was determined using the PierceTM BCA Protein assay kit, following
160
manufacturers’ instructions. Bovine serum albumin was used as protein standard.
161
Microscopy
162
To evaluate cell-to-cell contact, we checked unfixed cells immediately after
163
removing them from a culture tube and visualized the cultures with phase contrast
164
microscopy on a Leica Axioplann microscope. The presence of the methanogens was
165
verified by fluorescence at 420 nm and their specific coccus shape. To better visualize
7
166
how cells were distributed within the aggregates, we performed fluorescence in situ
167
hybridization. Cells were sampled in mid-log, fixed in premixed glutaraldehyde
168
(0.5%) and paraformaldehyde (1%), rinsed in 50 mM PIPES buffer, dehydrated (30
169
min at 4°C, in ethanol series 30%, 50%, 70%, 80%, and 100%), and pre-embedded in
170
a 50:50 mix of ethanol (100%) and butyl-methyl-metacrylate (BMM). Ultimately
171
aggregates were embedded in BMM resin and polymerized on a Spectorlinker
172
crosslink XL-1000 at room temperature over night. Sections of the aggregates were
173
obtained by microtome and stained with Cy3-probes (red) targeting Methanosarcina
174
species (MS1414) 5’-CTCACCCATACCTCACTCGGG-3’ (42) and Cy5-probes
175
(green) specific for the Geobacter cluster (Geo3ABC, 5’-
176
CCGCAACACCTRGTWCTCATC-3’) (43) or Cy5 (green) labeled probes specific
177
for P. carbinolicus (PCARB1, 5’-GCCTATTCGACCACGATA-3’) (43) depending
178
on the co-culture. The labeled co-cultures were visualized using a confocal laser
179
microscope as previously described (21).
180
RESULTS AND DISCUSSION
181
Growth of M. barkeri via DIET with G. metallireducens
182
Methanosarcina barkeri was co-cultured with Geobacter metallireducens in a
183
medium with ethanol as the sole electron donor in order to determine whether it was
184
capable of DIET. Multiple lines of evidence have indicated that G. metallireducens is
185
unable to release the electrons derived from the oxidation of ethanol to acetate as
186
either H2 or formate, but can directly transfer these electrons to microorganisms such
187
as G. sulfurreducens or Methanosaeta harundinacea which are capable of DIET (15,
188
20-22). After a lag of ca. 39 days, co-cultures of G. metallireducens and M. barkeri
189
started metabolizing ethanol to methane (Fig. 1a). Neither G. metallireducens nor M.
8
190
barkeri metabolized ethanol in pure culture (Fig. 1b). With continued transfer of the
191
co-culture, the initial long lag in ethanol metabolism was eliminated and ethanol
192
began to be metabolized to methane in less than 7 days (Fig. 1c).
193
Acetate only transiently accumulated in the co-cultures (Fig. 1a and 1c). The
194
total ethanol consumed (0.95 ± 0.01 mmol; mean ± standard deviation, n=4) resulted
195
in 1.59 ± 0.04 mmol methane, consistent with the expected production of 1.5 moles of
196
methane for each mole of ethanol consumed according to:
197 198
2 C2H6O + H2O 3 CH4 + CO2. This stoichiometric conversion of ethanol to methane requires that M. barkeri
199
not only metabolize the acetate that G. metallireducens produces from ethanol: 2
200
C2H6O + 2 H2O 2 C2H4O2 + 8 H+ + 8 e- via the reaction 2 C2H4O2 2 CH4 + 2
201
CO2, but also use the electrons released from ethanol metabolism to reduce carbon
202
dioxide to methane: 8H+ + 8e- + CO2 CH4 + 2 H2O. Thus, the high electron
203
recovery of electrons available from ethanol in methane, and the fact that G.
204
metallireducens is incapable of producing H2 during ethanol metabolism (23),
205
strongly suggested that M. barkeri was accepting electrons for carbon dioxide
206
reduction via DIET.
207
The fact that the co-culture could be continually transferred (10 % inoculum)
208
indicated that a small fraction of the carbon and electrons derived from ethanol must
209
also be consumed for biomass production. Biomass was examined by sacrificing
210
triplicate co-cultures for protein analysis at day 5, when methane started increasing
211
and at day 28 when methane production was complete. Protein increased from 233 ±
212
62 µg ml-1 (mean ± standard deviation) to 861 ± 115 µg ml-1, verifying co-culture
213
growth.
9
214
Cells involved in DIET need physical connections. G. metallireducens and M.
215
barkeri co-cultures formed aggregates of ca. 100-200 µm diameter (Fig. 2 a) in which
216
both Methanosarcina and Geobacter were intertwined (Fig. 2b).
217
Electrical Connections via Outer Surface Proteins and Granular Activated
218
Carbon
219
Previous studies have demonstrated that deleting pilA, which encodes the structural
220
protein of the conductive pili of Geobacter species, eliminates the ability of G.
221
metallireducens to participate in DIET (15, 22). Co-cultures initiated with a pilA-
222
deficient strain (34) of G. metallireducens failed to metabolize ethanol or produce
223
methane (Fig. 3a). Furthermore, co-cultures initiated with a strain of G.
224
metallireducens in which a gene (Gmet 1868) for an outer-surface cytochrome
225
required for Fe(III) oxide reduction was deleted (35) also failed to metabolize ethanol
226
to methane (Fig. 3b).
227
However, co-cultures initiated with either the pilA-deficient or Gmet 1868-
228
deficient strains of G. metallireducens did effectively produce methane in the
229
presence of granular activated carbon (GAC) (Fig. 3c). This is consistent with
230
previous studies which demonstrated that GAC, which is electrically conductive,
231
served as electrical conduit for DIET between G. metallireducens and G.
232
sulfurreducens, permitting the two species to share electrons even when the co-culture
233
was initiated with strains of G. sulfurreducens that could not produce pili or the pilin-
234
associated c-type cytochrome OmcS (33). As previously noted (33), GAC
235
amendments also stimulated the conversion of ethanol to methane in co-cultures
236
initiated with wild-type G. metallireducens and M. barkeri (Fig. 3c), substantially
237
reducing the 39 day long lag phase observed in the absence of GAC (Fig. 1a).
238
Methane production of the co-cultures initiated with the pilA-deficient or Gmet 1868-
10
239
deficient strains of G. metallireducens were slower than the wild-type co-cultures
240
amended with GAC, but even in the absence of pili or the outer-surface c-type
241
cytochrome methane production rates in the presence of GAC required less than a 6
242
day lag period to initiate ethanol metabolism (Fig. 3b). Previous studies demonstrated
243
that in co-cultures of G. metallireducens with either M. barkeri or G. sulfurreducens
244
both syntrophic partners attached to the GAC surface, but the individual cells were
245
too far separated for biological electrical connections between the species (33). These
246
results suggest that GAC can substitute for pili and/or outer-surface cytochromes as
247
the electrical conduit between the electron-donating G. metallireducens and the
248
electron-accepting M. barkeri.
249
Growth of M. barkeri via HIT with P. carbionolicus
250
Like G. metallireducens, P. carbinolicus can grow on ethanol in the presence
251
of a syntrophic partner, but it relies on HIT rather than DIET (21, 44). P. carbinolicus
252
and M. barkeri grew in co-culture in medium with ethanol as the sole electron donor,
253
but unlike G. metallireducens-M. barkeri co-cultures, there was a steady
254
accumulation of acetate (Fig. 4a). This was probably due to the fact that acetate
255
metabolism in M. barkeri is repressed when H2 is available (45-47).
256
P. carbinolicus metabolizes ethanol to acetate and H2 according to: 2 C2H6O + 2
257
H2O 2 C2H4O2 + 4 H2 (21). The metabolism of ethanol to acetate and H2 is only
258
exergonic if M. barkeri can oxidize H2 with the reduction of carbon dioxide as
259
follows: 4 H2 + CO2 CH4 + H2O.
260
The stoichiometry of ethanol consumed (0.91 ± 0.07 mmol, mean ± standard
261
deviation, n=4), methane produced (0.41 ± 0.07 mmol) and acetate accumulated (0.66
262
± 0.07 mmol) were consistent with methane being produced almost exclusively from
263
H2 oxidation with the reduction of carbon dioxide, assuming that some of the acetate
11
264
produced was diverted to biomass production. A similar partial oxidation of ethanol
265
with the accumulation of acetate was observed in the ethanol-metabolizing co-cultures
266
of P. carbinolicus - G. sulfurreducens, which were dependent on HIT (21), whereas
267
acetate did not accumulate in G. metallireducens-G. sulfurreducens co-cultures that
268
relied on DIET (21).
269
In contrast to the G. metallireducens - M. barkeri co-cultures (Fig 2a, b) the P.
270
carbinolicus - M. barkeri co-cultures did not form multispecies aggregates (Fig. 4b, c).
271
In a similar manner, P. carbinolicus - G. sulfurreducens co-cultures did not aggregate
272
(21), whereas G. metallireducens-G. sulfurreducens did (20). These results are
273
consistent with the need for direct cell-to-cell contact for DIET, but not for HIT.
274
IMPLICATIONS
275
The results demonstrate that M. barkeri is able to participate in DIET.
276
It is only the second methanogen found to have this capability. Methanosarcina
277
species are important contributors to methane production in some anaerobic digesters
278
(30) as well as soils and sediments (7-10). Thus the potential for DIET should be
279
considered when attempting to promote the growth of Methanosarcina in digesters or
280
when modeling their activity in soils and sediments.
281
In previous pure culture studies, Methanosaeta haurindacea, which is
282
incapable of utilizing H2 as an electron donor, was found to be capable of DIET, but
283
the two known H2-utilizing methanogens tested, Methanospirillum hungatei and
284
Methanobacterium formicicum, were not (15). Thus, M. barkeri is more versatile
285
than other methanogens that have been evaluated for DIET, with the capacity to either
286
accept electrons in the form of H2 from syntropic partners like Desulfovibrio (47)
287
and Pelobacter (this study), or to participate in DIET.
12
288
In the studies on DIET with M. harundinacea acetate was available as an
289
additional substrate for methane production (15) as it was in the M. barkeri co-
290
cultures reported here. However, studies with co-cultures of G. metallireducens and a
291
strain of G. sulfurreducens unable to oxidize acetate demonstrated that electrons
292
derived from DIET could support fumarate reduction and growth of G. sulfurreducens
293
in the absence of acetate as an additional electron donor (23). Similar studies should
294
be feasible with a M. barkeri mutant unable to metabolize acetate (48) to determine
295
whether carbon dioxide reduction to methane with electrons derived from DIET
296
provides sufficient energy to support cell maintenance and growth. Other than the
297
obvious importance of DIET in some anaerobic digesters treating brewery wastes (15,
298
18, 19) the prevalence of DIET in other methanogenic communities is unknown. The
299
fact that at least two major genera of methanogens have evolved the capacity for
300
DIET suggests that there are conditions in some soils and sediments in which DIET
301
confers a selective advantage.
302
The ability of GAC to promote methanogenic DIET has important
303
implications for the design of anaerobic digesters. Previous studies that have reported
304
stimulation in methane production when carbon cloth was introduced into digesters
305
have attributed this response to the cloth retaining methanogens (49-51). However,
306
the possibility that these conductive materials might promote DIET should also be
307
considered.
308
Although the importance of electrically conductive pili and outer-surface
309
cytochromes in extracellular electron exchange, including DIET, is becoming
310
increasingly apparent for Geobacter species (52, 53) the potential extracellular
311
electron contacts that might permit methanogens to accept electrons via DIET are less
312
clear. Methanosaeta and Methanosarcina are the only genera of methanogens with
13
313
membrane-bound cytochromes (54, 55), which could potentially play a role in
314
extracellular electron exchange. The availability of tools for genetic manipulation of
315
M. barkeri (56) suggests that it may be the ideal candidate for functional analysis of
316
DIET mechanisms in methanogens.
317
ACKNOWLEDGEMENTS
318
We would like to thank Dr. Pier-Luc Tremblay and Jessica Smith for
319
providing G. metallireducens deletion-mutant strains. We want to acknowledge the
320
laboratory assistance of Dan Carlo Flores, Trevor Woodard and Joy Ward. The work
321
was supported by the Office of Science, US Department of Energy, award number:
322
DE-SC0004485
323
FIGURE LEGENDS
324
Fig. 1. Syntrophic growth of M. barkeri with G. metallireducens. (a)
325
Metabolism of ethanol when the co-cultures were initiated with a 5 % (vol:vol)
326
inoculum of each species. (b) Lack of ethanol metabolism with single species. (c)
327
Decreased lag after 25 transfers (10% inoculum) of the co-cultures. All cultures were
328
grown in triplicate (n=3). A difference in the Y-scale can be noted between a, b and c.
329
This is because of differences in total incubation volume: 10 ml to initiate the co-
330
cultures (a, b), and 50 ml for later incubations (c).
331
Fig. 2. Co-culture composition. (a) Phase contrast micrograph of an
332
aggregate of M. barkeri (cocci) and G. metallireducens (rods). (b) Scanning laser
333
confocal microscope image of co-culture aggregate after in situ hybridization, which
334
targeted Methanosarcina cells with a red probe (Cy3) and G. metallireducens with a
14
335
green probe (Cy5). Images are representative of triplicate samples taken during the
336
mid-exponential growth phase of the co-cultures.
337
Fig. 3. Mutant studies. (a) Lack of ethanol metabolism and methane
338
production in a co-culture initiated with a pilA-deficient G. metallireducens (b)
339
Methane production in co-cultures initiated with wild-type G. metallireducens or a
340
strain deficient in the gene Gmet 1868, which encodes a predicted extracellular
341
cytochrome. (c) GAC stimulation of methane production in co-cultures initiated with
342
wild-type G. metallireducens and enabling methane production in co-cultures initiated
343
with either the PilA- or cytochrome (Gmet-1868)-deficient strains of G.
344
metallireducens. All incubations were set up with 5% (vol:vol) inoculum of each co-
345
culture partner.
346
Fig. 4. Syntrophic growth in co-cultures of P. carbinolicus and M. barkeri.
347
(a) Syntrophic growth in the third transfer of the co-culture. (b) Phase contrast image
348
illustrating lack of aggregation of P. carbinolicus (rods) and M. barkeri (cocci). (c)
349
Scanning laser confocal microscope image of co-culture after in situ hybridization,
350
which targeted Methanosarcina cells with a red probe (Cy3) and P. carbinolicus with
351
a green probe (Cy5). Images are representative of triplicate samples taken during the
352
mid-exponential growth phase of the co-cultures.
353
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