Extracellular Electron Uptake in Methanosarcinales Is Independent of Multiheme

bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 Extracellular electron uptake in Methanosarcinales is independent of multiheme

2 c-type cytochromes

3 Mon Oo Yee1 & Amelia-Elena Rotaru1

4 1Nordcee, Department of Biology, University of Southern Denmark, Odense, Denmark

6 Correspondence: [email protected]

8 Keywords:

9 multiheme c-type cytochromes, direct interspecies electron transfer, Methanosarcina, Methanothrix,

10 Methanosarcina mazei, electromethanogenesis, Rhodoferax ferrireducens, Geobacter hydrogenophilus,

11 Geobacter metallireducens

19 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

20 Abstract

21 The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of

22 direct interspecies electron transfer (DIET) in man-made and natural aquatic environments. We previously

23 reported that not all Geobacter are capable of DIET with Methanosarcina. Here we tested 15 new artificial

24 co-culture combinations with methanogens and electrogenic bacteria, including an electrogen outside of the

25 Geobacter clade – Rhodoferax ferrireducens.

26 Consistently, highly effective electrogenic bacteria (G. metallireducens, G. hydrogenophilus and R.

27 ferrireducens) formed successful associations with Methanosarcinales. Highly effective electrogens could

28 not sustain the growth of H2-utilizing methanogens of the genera Methanococcus, Methanobacterium,

29 Methanospirillum, Methanolacinia or Methanoculleus.

30 Methanosarcinales, including strict non-hydrogenotrophic methanogens of the genus Methanothrix (Mtx.

31 harundinacea and Mtx. shoeghenii) and Methanosarcina horonobensis, conserved their ability to interact

32 with electrogens. Methanosarcinales were classified as the only methanogens containing c-type

33 cytochromes, unlike strict hydrogenotrophic methanogens. It was then hypothesized that multiheme c-type

34 cytochromes give Methanosarcinales their ability to retrieve extracellular electrons. However, multiheme c-

35 type cytochromes are neither unique to this group of methanogens nor universal. Only two of the seven

36 Methanosarcinales tested had multiheme c-type cytochromes (MCH). In one of these two species - M. mazei

37 a deletion mutant for its MCH was readily available. Here we tested if the absence of this MHC impacts

38 extracellular electron uptake. Deletion of the MHC in M.mazei did not impact the ability of this methanogens

39 to retrieve extracellular electrons from G. metallireducens or a poised cathode. Since Methanosarcina did

40 not require multiheme c-type cytochromes for direct electron uptake we proposed an alternative strategy for

41 extracellular electron uptake. bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

42 Introduction

43 Direct interspecies electron transfer (DIET) was discovered in an artificial co-culture of an ethanol-oxidizing

44 Geobacter metallireducens with a fumarate-reducing Geobacter sulfurreducens where the possibility of

1,2 45 hydrogen gas (H2) or formate transfer was invalidated through genetic studies . Gene deletions rendering

1,2 46 H2 and formate transfer impossible resulted in active co-cultures , whereas deletion of genes for

47 extracellular electron transfer proteins (EET) such as pili, and multiheme c-type cytochromes made the

48 interspecies interaction impossible 1,3. Remarkably, a deletion mutant lacking an extracellular multiheme c-

49 type cytochrome (OmcS) could be rescued by the addition of extracellular conductive particles, whereas a

50 pili knock-out strain could not be rescued by conductive particles 4. Additionally, previous studies have

51 shown that G. metallireducens is also highly effective as an anode-respiring bacteria (ARB) generating some

52 of the highest current densities of all Geobacter tested 5. During DIET with G. sulfurreducens, G.

53 metallireducens requires pili and certain multiheme c-type cytochromes 3, which were also required during

54 anode respiration 6,7.

55 G. metallireducens is a strict respiratory microorganism unable to ferment its substrates to produce H2 for

8,9 56 interspecies H2-transfer . Consequently, G. metallireducens could not provide reducing equivalents for

57 strict hydrogenotrophic methanogens (Methanospirilum hungatei and Methanobacterium formicicum)10,11.

58 However, G. metallireducens did interact syntrophically with Methanosarcinales (Methanosarcina. barkeri

59 800, Methanosarcina horonobensis, Methanothrix harundinacea)10–12 of which the last two are unable to

13,14 60 consume H2 . When incubated with Methanosarcinales, G. metallireducens upregulated EET-proteins,

61 and if some of these EET proteins were deleted, co-cultures became inviable demonstrating the direct

62 electron transfer nature of the interaction 10,12.

63 For co-culture incubations, G. metallireducens has been typically provided with an ethanol as electron donor,

64 but without a soluble electron acceptor. In the absence of a soluble electron acceptor, G. metallireducens

65 releases electrons extracellularly (reaction 1) and uses the methanogen as its extracellular terminal electron

66 acceptor (reaction 2 & 3). If electrons released by G. metallireducens cannot reach a terminal electron bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

67 acceptor, ethanol oxidation would stop, because the electron transport chain would become ineffective, and

68 NADH produced during ethanol oxidation could not get re-oxidized.

69 Reaction 1. Ethanol oxidation with electrons release by G. metallireducens:

+ - 70 2CH3CH2OH + 2H2O → CH3COOH + 2CO2 + [8H + 8e ]

71 Reaction 2. Electron uptake coupled with CO2 reductive methanogenesis by Methanosarcinales:

+ - 72 CO2 + [8H + 8e ] → CH4 + 2H2O

73 Reaction 3. Acetoclastic methanogenesis: CH3COOH → CH4 + CO2

74 Reaction 4. Total DIET reaction by a syntrophic association between Geobacter and Methanosarcinales:

75 2CH3CH2OH → 3CH4 + CO2

76 There are indications for direct interspecies electron transfer occurs in methane producing environments such

77 as anaerobic digesters 15, rice paddy soils 16, and aquatic sediments 17,18 , as well as in methane consuming

78 environments such as hydrothermal vents 19,20. In these environments, DIET is typically inferred either

79 because conductive materials stimulate the syntrophic metabolism but also by the co-presence of DNA

80 and/or RNA of phylotypes related to DIET-microorganisms. DIET-pairing of Geobacter with methanogens

81 was only described in two Geobacter species (G. metallireducens, G. hydrogenophilus) and three

82 Methanosarcinales (Ms. barkeri 800, Ms. horonobensis, Mtx. harundinacea) 5,10–12. On the other hand, a

83 series of six Geobacter species were unable to interact syntrophically with Ms. barkeri 800. All these

84 Geobacter were modest anode respiring bacteria and did not produce high current densities at the anode 5.

85 No species outside of the Geobacter-clade have been shown to do DIET with methanogens.

86 In this study, we expand the list of syntrophic-DIET pairs and investigated whether other electrogenic

87 bacteria but Geobacter could interact syntrophically with methanogens. We determined whether DIET was

88 spread among other methanogens or was a specific trait of Methanosarcinales because of their high c-type

89 cytochrome content 21. Multiheme c-type cytochromes (MHC) were previously implicated in EET in bacteria bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

90 22 and an MHC of Methanosarcina acetivorans was required for AQDS respiration 23. Here we asked

91 whereas the OMCs of a Methanosarcina is required for DIET and electron uptake from electrodes.

92 Materials and methods

93 Cultures were purchased from the German culture collection (DSMZ) and grown on the media advised by

94 the collection until pre-adaption to co-cultivation media. The following strains of methanogens were tested:

95 Methanococcus maripaludis JJ (DSM 2067), Methanococcus voltae PS (DSM 1537), Methanoculleus

96 marisnigri JR1 (DSM 1498), Methanolacinia petrolearia (DSM 11571), Methanosarcina mazei Gö1 (DSM

97 3647), Methanospirillum hungatei JF1 (DSM 864), Methanosaeta harundinacea 8Ac (DSM 17206), three

98 strains of Methanosarcina barkeri MS (DSM 800), Fusaro (DSM 804), and 227 (DSM 1538). We used the

99 following strains of electrogenic bacteria: Rhodoferax ferrireducens (DSM 15236) and Geobacter

100 metallireducens GS-15 (DSM 7210) available in our own freezers stock collection at the University of

101 Massachusetts, or purchased from DSMZ, for experiments run at the University of Southern Denmark. A

102 mutant strain of Methanosarcina mazei lacking the multi-heme cytochrome c family protein MM_0633

103 (named M. mazei ∆0633) was kindly provided by Prof. Uwe Deppenmeier and Prof. Cornelia Welte. This

104 mutant strain has been characterized in detail in Christian Krätzer’s Ph.D. thesis 24.

105 Prior to co-cultivation, all Methanosarcina cultures were pre-grown on syntrophic DSMZ120c media lacking

106 ethanol 11,12 but containing their respective substrates (20-30mM acetate sometimes supplemented with 20

107 mM methanol). Methanothrix cultures were pre-grown on syntrophic media lacking ethanol 10 but containing

108 their respective substrate 20-80 mM acetate. Mc. maripaludis did not grow with its own substrate H2CO2

109 when provided with a low salt syntrophic freshwater-media (0.36g/L NaCl), whereas all the other strict

110 hydrogenotrophs (Mc. voltae, Mcl. marisnigri, Mlc. petrolearia, Msp. hungatei) were pre-grown on this

111 media with 1 bar overpressure of sterile H2: CO2 (80:20) gas. Mc. maripaludis had to be adapted to lower salt

112 on H2: CO2 supplemented syntrophic media. We started with the above media but adjusted the salt

113 concentration to their optima NaCl (18g/L). Afterwards, we successively transferred to 15g/L NaCl, then

114 10g/L and 5g/L. Cultures that grew at 15g/L NaCl were then transferred into 10g/L, when adapted these

115 were transferred into 5g/L NaCl media. Below 5g/L NaCl we did not observe effective growth of the bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

116 methanogen in this media. G. metallireducens, G. hydrogenophilus and Rhodoferax ferrireducens were

117 maintained on the same freshwater media mentioned above with 55 mM ferric citrate as electron acceptor.

118 The first two were maintained with 10-20 mM ethanol as electron donor 11,12 whereas the later was with

119 5mM glucose. Pelobacter carbinolicus which was used as the H2-donating strain was cultivated under

120 fermentative conditions on 10 mM acetoin 2. Co-cultures with Methanosarcina species were established as

121 previously described on syntrophic/modified media 120c (without added electron acceptors) with 10-20 mM

10–12 122 ethanol and 5 mM glucose (for R. ferrireducens). Co-cultures with strict H2-utilizing methanogens were

123 established as above, on a freshwater media with 0.36 g/L NaCl 10 for all strains exclusive of M. maripaludis

124 which received 5g/L salt. Co-cultures with hydrogenotrophs were set up without electron acceptors, but with

125 one of the following electron donors: 20 mM ethanol as electron donor for incubations with G.

126 metallireducens, G. hydrogenophilus and P. carbinolicus and 5 mM glucose for incubations with R.

127 ferrireducens.

128 Cultures and co-cultures were incubated at 37oC and granular activated carbon (GAC) was added at 25 g/L.

129 Samples were withdrawn anaerobically with N2: CO2 flushed hypodermic needles to verify the levels of

130 methane, hydrogen, acetate, ethanol, and glucose. Determination of methane (CH4), hydrogen gas (H2),

131 ethanol and volatile fatty acids (e.g. acetate, formate) was carried out as described before 11,12. Glucose was

132 determined at the end of the incubation, as previously described 25.

133 Incubations using a cathode as sole electron donor, were carried out as previously described 11, with the

134 following modifications: a resistor was used (250 ); and to ensure low carry over substrates, cells were

135 harvested in an anaerobic chamber and washed in fresh media prior to inoculation.

136 Results and discussion

137 Previously, two out of seven Geobacter species paired syntrophically with M. barkeri. These two Geobacter

138 were G. metallireducens and G. hydrogenophilus which exhibited the highest current densities on the anode

5 9 139 . Unlike G. metallireducens, G. hydrogenophilus has been shown to produce some H2 during respiration ,

140 but was not previously tested with hydrogen utilizing methanogens. Here we tested whether G. bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

141 hydrogenophilus can pair syntrophically with a strict hydrogenotrophic methanogen (Msp. hungatei) versus a

142 non-hydrogenotrophic methanogen (Mtx. harundinacea). Methane production from ethanol was used as a

143 proxy for the efficiency of the interaction. Over the course of 117 days, the co-culture with Methanothrix

144 accumulated 9-times more methane than a co-culture with Methanospirillum (Fig. 1, p=0.03). Control tests

145 with these two methanogens alone showed they could not sustain methane-production from ethanol (Fig. 1).

146 Although G. hydrogenophilus produces some H2 it was not an efficient H2 donor, unlike the H2-donating

147 syntroph - P. carbinolicus (Table 1).

148 With only two out of seven Geobacter showing aptitude for DIET with Methanosarcinales, we

149 decided to look outside the Geobacter clade. We tested the possibility for DIET or H2-transfer with the

150 effective anode respiring Betaproteobacteria - Rhodoferax ferrireducens25. Interestingly, Rhodoferax was

151 predicted to outcompete Geobacter in a subsurface environment with low substrate flux and relatively high

152 ammonia26, where a non-hydrogenotrophic Methanosarcina co-exists 27. The co-existence of Rhodoferax,

153 Geobacter and Methanosarcina was also noted in coastal Baltic Sea sediments 28. It is possible

154 Methanosarcina species could receive DIET-electrons from Rhodoferax as well as Geobacter. Here we

155 examined whether R. ferrireducens could establish interspecies electron transfer in co-cultures with 2 DIET

156 methanogens (Mtx. harundinacea, Ms. barkeri) or with 2 strict hydrogenotrophic methanogens (Msp.

157 hungatei and Mbt. formicicum). We expected that this efficient anode-respiring bacterium 25 would prefer

158 DIET syntrophic partners to H2-utilizing partners. R. ferrireducens cannot utilize ethanol, therefore these co-

159 cultures were provided with glucose (5 mM) as sole electron donor 29. On glucose, Rhodoferax acts a

160 respiratory organism and could not oxidize this substrate alone 30. In these co-cultures, methane was used as

161 a proxy for syntrophic metabolism. In order to estimate electron recovery from glucose, volatile fatty acid

162 accumulation was determined during stationary phase. All co-cultures consumed the 5 mM glucose added (<

163 4µM detected after 270 days). Product recoveries varied significantly in Rhodoferax co-cultures with DIET-

164 methanogens versus co-cultures with hydrogenotrophic methanogens (Fig. 2 and 2-inset). By comparing

165 methane production in co-cultures with Rhodoferax, it was evident that M. harundinacea was the most

166 effective at accumulating methane followed by Ms. barkeri and then strict hydrogenotrophs (Fig. 2). bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

167 Rhodoferax in co-culture with Methanothrix had the highest total electron recovery (45%) with all electrons

168 being recovered as methane, and none as acetate (Fig. 2-inset). The electrons not accounted for in products,

169 are likely assimilated into biomass, typical of methanogenic metabolisms 31. The Rhodoferax co-cultured

170 with Methanosarcina was 3-fold less effective at recovering electrons into products (14%; Fig. 2-inset), yet

171 the majority of the electrons were recovered as methane (12%) and only traces as acetate (2%). Both co-

172 cultures of Rhodoferax with strict hydrogenotrophs resulted in low electron recoveries of 22-23% mostly as

173 acetate (18%). These results show that Rhodoferax favors interactions with DIET-methanogens rather than

174 strict hydrogenotrophic methanogens. However, we do not know how Rhodoferax releases electrons to DIET

175 methanogenic partners, although hints about its EET metabolism have been projected from genome

176 screening 30,32. The genome of R. ferrireducens contains 45 putative c-type cytochromes 30 and the entire

177 Mtr-pathway suggesting R. ferrireducens may be doing EET similar to Shewanalla 32,33. It remains to be

178 tested whether this pathway is also used for DIET syntrophy with methanogens.

179 Strict hydrogenotrophic methanogens were incapable of DIET with G. metallireducens

180 To examine the ability for direct interspecies electron uptake in new strains of methanogens, G.

181 metallireducens was used as the default DIET-partner for co-culture experiments. We selected 7

182 methanogenic strains as representatives of two groups: hydrogenotrophic (H2-consuming) and non-

183 hydrogenotrophic methanogens.

184 We evaluated DIET between G. metallireducens and 4 strict hydrogenotrophic species: Methanoculleus

185 marisnigri, Methanolacinia petrolearia, Methanococcus voltae, and Methanococcus maripaludis. We

186 selected two members of the Methanomicrobiaceae family (Mlc. petrolearia, Mcl. marisnigri) whose

187 transcripts were abundant (13%) in rice paddies dominated by transcripts of Geobacter 34, hinting at the

188 possibility of an interaction between the two. We incubated G. metallireducens with Mlc. petrolearia for ca.

189 1 month, and with Mcl. marisnigri for ca. 5 months. The incubation time for Mcl. marisnigri was extended

190 because of preliminary observations showing a relatively slow growth in the co-culture media, even when

191 provided with their typical growth substrates. When co-cultured together with G. metallireducens neither

192 Methanolacinia nor Methanoculleus produced methane (Table 1). bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

193 Furthermore, we investigated two strains of Methanococcus for their ability to interact by DIET with G.

194 metallireducens. Methanococcus species are found in aquatic sediments from low to high salinity

195 environments 35 and have been associated with corrosion of metallic structures 36,37. Moreover,

196 Methanococcus cohabits with Geobacter in environments like for example a water flooded oil reservoir 38,

197 where they may interact cooperatively. The property of Methanococcus species to effectively retrieve

198 electrons from metallic iron39 or electrodes 40 either directly 40 or by making use of extracellular enzymes 41–

199 44 made them attractive candidates for testing DIET interactions with G. metallireducens. Growth of

200 Methanococcus is restricted by the low salt concentration required by Geobacter 45. Therefore, we had to

201 preadapt two Methanococcus species and G. metallireducens to grown in media with 5 g/L salt by successive

202 transfers at increasing salt concentrations. When all strains were effectively adapted, we co-inoculated each

203 methanogen with Geobacter and provided ethanol. Under these conditions, neither Methanococcus species

204 produced methane (Table 1).

205 DIET evaluation by co-cultivation with G. metallireducens has now been carried out for 6 strict

206 hydrogenotrophs from three major orders: Methanomicrobiales (Msp. hungatei, Mlc. petrolearia, Mcl.

207 marisnigri), Methanococcales (Mc. maripaludis, Mc. voltae) and Methanobacteriales (Mb. formicicum).

208 A clear pattern emerged regarding the inability of strict hydrogenotrophic methanogens to pair with G.

209 metallireducens (Table 1).

210 All seven Methanosarcinales tested did form ethanol-metabolizing consortia with G. metallireducens

211 Until now, Methanosarcinales is the only order of methanogens evaluated positive for DIET. Three strains

212 of this order have been reported to do DIET, of which 2 were strict non-hydrogenotrophic methanogens

213 (Methanothrix harundinacea and Methanosarcina horonobensis) whereas a third could utilize H2

10–12 214 (Methanosarcina barkeri 800) , yet it’s threshold for H2-uptake is 10-fold higher that of strict

46,47 215 hydrogenotrophs making it less effective at H2-utilization. This was evident since Ms. barkeri coupled

216 with P. carbinolicus (H2-donor) required 16 days to get to the same amount of methane whereas a strict

217 hydrogenotrophs required only 4 days (Table 1). bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

218 Previously we showed two Methanosarcina species could grow by DIET with G. metallireducens but only

219 one could grow on a cathode at -400mV (vs. SHE)11. Therefore, we investigated whether DIET may be

220 featured only by some Methanosarcina. To see if there are differences at the strain level, 2 new strains of M.

221 barkeri (M. barkeri 227 and Ms. barkeri 804) were evaluated for DIET with G. metallireducens.

222 Additionally, we tested M. mazei and a strict non-hydrogenotrophic methanogen, Mtx. concilii. Co-cultures

223 were provided with ethanol and were anticipated to reach mid exponential after circa two months, according

224 to preliminary tests and previous reports 10–12. After 75 days, all co-cultures of Geobacter and

225 Methanosarcinales oxidized ethanol and produced methane (Table 1). The respiratory metabolism of

226 Geobacter (reaction 1) resulted in extracellular transfer of electrons and transient formation of acetate (Fig.

227 3). The products of ethanol oxidation were then converted into methane (reaction 2 &3; Fig. 3b). In the

228 absence of Geobacter, none of the methanogens converted ethanol to methane (Fig. 3a).

229 Earlier, DIET has been reported to be accelerated by conductive particles such as granular activated carbon

230 (GAC)12,48 and not by non-conductive materials such as cotton cloth49. Conductive materials can promote the

231 respiratory metabolism of Geobacter until the material reaches charge-saturation50,51. Afterwards, only the

232 presence of a methanogen retrieving electrons would keep the process from coming to a halt 11. We subjected

233 co-cultures of G. metallireducens and three strains of Methanosarcina (M. mazei; M. mazei 663; M. barkeri

234 227) to 25g/L GAC to verify if we can stimulate their growth. All co-cultures reduced their lag-phases and

235 reached mid-exponential much quicker, while at a minimum tripling their methanogenesis rates (Fig. 3,

236 Table 1).

237 We have now expanded the list of methanogens capable of DIET to five species of Methanosarcinales plus

238 two additional strains of M. barkeri strain 227 and 804, besides the type strain 800. It appears that the genetic

239 makeup for DIET is conserved among Methanosarcinales, although the responsible genes have not been

240 identified.

241 The putative pentaheme c-type cytochrome of M. mazei was not required for DIET or cathodic EET bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

242 Many studies have shown that multi-heme c-type cytochromes (MHCc) are important for extracellular

52 1,3,12,19,53 243 electron transfer including EET during DIET . However, MHCc – cytochromes are neither

244 ubiquitous nor restricted to DIET-methanogens (Table 2). Of the hydrogenotrophic methanogens, Msp.

245 hugateii and Mcl. marisnigri contained potential multiheme cytochrome proteins (Table 2a), apparently

246 localized in the cytoplasm (Table 2b). Of the DIET-methanogens, 5 out of 7 species did not contain MHCs;

247 all of the Methanosarcina barkeri strains and both Methanothrix species. Ms. horonobensis and M. mazei

248 were the only 2 species with MHC-cytochromes apparently localized on the membrane or secreted (Table

249 2b). M. mazei’s predicted MHC cytochrome was detected in the surface/membrane-bound fraction by

250 biochemical testing and predicted to be secreted extracellularly through leaderless secretion 54 where it is

251 suggested to join a membrane-bound complex containing flavoproteins and iron-sulfur flavoproteins 55.

252 Our hypothesis was that if M. mazei required this MHC-cytochrome for extracellular electron transfer, cells

253 without it would be unable to interact with a DIET syntroph or with a poised electrode. This approach was

254 previously used to determine Geobacter’s necessity for cell surface MHC-cytochromes during EET to

255 electrodes 56, iron-oxide minerals 22 and DIET-partners 1,3,12.

256 To test this hypothesis, we used a knock-out mutant of M. mazei (∆0633) in which the gene (MM_0633)

257 encoding for the putative multiheme c-type cytochrome was deleted 57. The deletion mutant showed no

258 phenotypic variability to the wild type when growing on its typical substrates (methanol and acetate) 57. To

259 determine whether this MHCc was required to receive DIET electrons from Geobacter, M. mazei ∆0633 was

260 incubated with G. metallireducens in syntrophic media with ethanol (Fig. 3). Methane production and

261 ethanol oxidation progressed similar to wild type control incubations (Fig. 3) demonstrating that this MHC is

262 not required for DIET.

263 Recently, it was reported that M. mazei was not electroactive and incapable to retrieve electrons from a

264 poised cathode at -700 mV (vs. SHE) 58. However, these experiments were carried out for only 3 day, which

265 is too short compared to any other tests for electromethanogenesis in reactors with pure cultures 11,59. bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

266 Here we tested the wild type M. mazei and the MCH deletion mutant of M. mazei (∆0633) to see whether the

267 absence of its one and only MCH cytochrome impacts EET from a cathode. Both M. mazei strains with and

268 without the cytochrome were incubated with a cathode poised at a voltage of -400 mV (vs. SHE),

11 269 unfavorable for the H2-evolution reaction . Control experiments were run alongside, without applying a

270 voltage at the cathode, to verify whether methanogenesis can be induced by carry-over substrates. Only in

271 experiments with a poised cathode, methane production proceeded as effectively for ∆0633 and wild type M.

272 mazei (Fig. 4), showing that the MHC-cytochrome is not required for electron uptake from a cathode.

273 Previously, we observed that M. horonobensis which contains the highest number of MHCs among DIET-

274 methanogens was also unable to use a cathode as electron donor 11. Combined, these results disprove our

275 hypothesis that Methanosarcina species require a multiheme c-type cytochrome for extracellular electron

276 uptake.

277 For Methanosarcinales involved in EET/DIET, the first barrier for electrons to enter a cell is the cell

278 envelope. Thus, for DIET to take place, the cell surface of Methanosarcinales is anticipated to harbor charge

279 transferring molecules. DIET methanogens exhibit very different cell envelopes than strict hydrogenotrphic

280 methanogens. Methanospirillum is one exception being coated by a protein sheath comparable to that of

281 Methanothrix (Fig. 5). However, the protein sheath of Methanothrix contains 2-5 times more metal ions (Zn,

282 Cu, Fe, Ni) than that of Methanospirillum 60. Unlike Methanothrix, Methanosarcina are coated by

283 methanochondroitin sulfate, a type of exopolysacharide that resembles chondroitin sulfate in eukaryotes 61

284 where it confers conduction via axonal length 62. It is typical of exopolysaccharides to absorb metals 63 or

285 even trap redox cofactors and c-type cytochromes 64, thus the embedded redox centers within the surface

286 matrix may confer very different electric properties. These differences in surface biology may provide DIET-

287 methanogens with a specific niche where they could outcompete H2-utilizers (e.g. mineral rich

288 environments).

289 Conclusion

290 The incidence of Geobacter in methanogenic environments is often used as signature for direct interspecies

291 electron transfer, although only two out of seven Geobacter species which are highly electroactive were also bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

292 able to establish successful partnerships with Methanosarcinales, namely G metallireducens or G.

293 hydorgenophilus. Additionally, we tested whether DIET expands to another effective electrogens outside the

294 Geobacter-cluster. We observed that Rhodoferax ferrireducens promoted methanogenesis with Methanothrix

295 and Methanosarcina. On the other hand, neither of the electroctive strains (two Geobacter and Rhodoferax)

296 were able to grow in co-culture with strict H2-utilizing methanogens.

297 All Methanosarcinales tested formed metabolically active DIET consortia with G. metallireducens. In these

298 Methanosarcinales, electron uptake from an extracellular donor was assumed to require multiheme c-type

299 cytochromes. Here we highlight that multiheme c-type cytochromes are not ubiquitous nor restricted to

300 DIET-methanogens and show that the deletion of the sole MHC from M. mazei did not impact their ability to

301 retrieve extracellular electrons from a DIET-partner or from an electrode. This validates that multiheme c-

302 type cytochromes are not required for extracellular electron uptake in Methanosarcina.

303 Acknowledgements

304 This work was a contribution to a grant from the Innovationsfonden Denmark (4106-00017). We would like

305 to thank Lasso Ørum Smidt and Trevor Woodard for lab assistance. We would like to thank Prof. Derek

306 Lovley for reading and advising on the manuscript.

307 Statement of author contribution

308 AER and MOY designed the experiments, AER carried out some of the co-culture incubations including

309 analytical experiments at the University of Massachusetts, MOY carried out additional co-culture

310 incubations and analytical experiments at the University of Southern Denmark, did the bioinformatic

311 analyses and tested M. mazei strains in co-cultures and in a bioelectrochemical set-up. Both authors wrote the

312 paper.

313

314

315 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

316 Figures

317 Fig. 1. G. hydrogenophilus in co-culture with a non-hydrogenotrophic methanogen (Mtx. harundinacea)

318 versus a strict hydrogenotrophic methanogen (Msp. hungatei). Methane production in co-cultures (closed

319 symbols; n=2) provided with ethanol. Methane production in parallel control experiments with pure cultures

320 provided with ethanol (empty symbols; n=1). A small increase in methane production by Methanothrix,

321 could be traced back to acetate transfer with the inoculum. A 2nd transfer with ethanol showed absolutely no

322 methane production from ethanol. Whiskers represent standard deviations of the replicate incubations, and if

323 invisible they are smaller than the symbol.

Fig. 1

3 Co-cultures with G. hydrogenophilus

G. hydrogenophilus + Mtx. harundinacea Control Mtx. harundinacea G. hydrogenophilus + Msp. hungatei Control Msp. hungatei

)

(

e M 1

0 0 10 20 30 40 50 60 70 80 90 100 110 120 324 Time (days)

325 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

326 Fig. 2. Co-cultures of four species of methanogens with Rhodoferax ferrireducens. Methane profiles in co-

327 cultures provided with 5mM glucose as sole electron donor. (inset) Estimated electron recoveries into

328 products (acetate and methane). Incubations were carried out in triplicate (n=3). Whiskers represent standard

329 deviations of the replicate incubations, and if invisible, they are smaller than the symbol.

Fig. 2

Co-cultures with Rhodoferax ferrireducens 7 Mtx. harundinacea

Ms. barkeri 800 a

Msp. hungatei e

a i

6 u

e c

Mbt. formicicum i

n r

r k

a h

b .

5 t

x . p

t s s b

)

M M M M

m (

4 y

e a

v 40

h o

t c

e e

r 30 q

M 3

e e 20

M Acetate m 10 2 Methane 0

0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (days) 330

331 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

332 Fig. 3. Incubations with Methanosarcina mazei provided with ethanol as sole electron donor in the presence

333 (filled symbols) of absence (empty symbols) of conductive GAC-particles. a) Control incubations with pure

334 cultures of M. mazei; b) M. mazei wild type in co-culture with G. metallireducens; and c) M. mazei633 k.o.

335 MHC-cytochrome in co-culture with G. metallireducens. Whiskers represent standard deviations of the

336 triplicate incubations (n=3) and if invisible, they are smaller than the symbol.

Fig. 3

M. mazei M. mazei + G. metallireducens 12 a 12 b

10 10

) )

M 8

(

r t 6 t

n 6

C C 4 4

2 2

0 0 0 10 20 30 40 50 60 70 80 90 100 110 0 10 20 30 40 50 60 70 80 90 100 110 Time (days) Time (days)

M. mazei 633 + G. metallireducens LEGEND 12 c Methane (co-culture + GAC) Methane (co-culture, no ads) Ethanol (co-culture + GAC) Ethanol (co-culture, no ads) 10 Acetate (co-culture + GAC) Acetate (co-culture, no ads)

Hydrogen (co-culture + GAC) Hydrogen (co-culture + GAC) )

M 8

(

a r

t 6

o C 4

0 0 10 20 30 40 50 60 70 80 90 100 110 337 Time (days)

338 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

339 Fig. 4. M. mazei strains (wild type and MCH-cytochrome k.o.) incubated with cathodes as sole electron

340 donor. The cathode was either poised at -400mV versus SHE (closed symbols), or not poised (empty

341 symbols).

Fig. 4

45 M. mazei strains : Wild type @ -400mV 633 k.o. @ -400mV 40 Wild type control 633 k.o. control 35

) 30

(

n 25

h t

e 20 M

0 0 5 10 15 20 25 30 35 40 45 50 342 Time (days)

343 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

344 Fig. 5. Representative cell envelope structures of selected genera of methanogens. MC; methanochondroitin,

345 SL; S-layer, CM; cell membrane, PS; protein sheath, GP; glycosylated protein, PM; pseudomurein.

Fig. 5

SL Methanosarcina sp.

SL Methanospirillum sp. Methanothrix sp. CM

GP Methanococcus sp. SL Methanoculleus sp.

CM Methanolacinia sp.

PM Methanobacterium sp. CM 346

347 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

348 Table legends

349 Table 1. Overview of co-culture tests with 14 species of methanogens and various electroactive bacteria. In

350 this study we tested 7 new strains of methanogens. Tested substrates were 5 mM glucose for tests with

351 Rhodoferax; 20 mM ethanol for the rest of the co-cultures; except for tests with M. mazei strains, M.

352 horonobensis and M. barkeri 227, which got 10 mM ethanol.

)

(

)

(

S R Co-cultures with Geobacter metallireducens

s G. metallireducens Ms. barkeri 800 Yes/No 19.5 ± 1.4 89 3 Stationary Ethanol (20) Rotaru 2014a e

r G. metallireducens Ms. barkeri 804 Yes/No 25.9 ± 1.5 121 3 Eponential Ethanol (20) This study u

t G. metallireducens Ms. barkeri 227 Yes/No 9.1 ± 1.2 95 2 Exponential Ethanol (10) This study

l G. metallireducens Ms. mazeii Yes/No 7.7 ± 0.7 149 3 Exponential Ethanol (10) This study

u c

- G. metallireducens Ms. mazeii 663 k.o. Yes/No 4.6 ± 1.0 91 3 Exponential Ethanol (10) This study

o c G. metallireducens Ms. horonobensis No/No 13.1 ± 0.8 110 3 Exponential Ethanol (10) Yee 2019

T Strict non- E

I G. metallireducens Mtx. harundinacea No/No 14.2 ± 1.1 167 4 Exponential Ethanol (20) Rotaru 2014b

D hydrogenotrophs G. metallireducens Mtx. soehngenii No/No 1.8 ± 1.0 82 2 Exponential Ethanol (20) This study G. metallireducens Mcc. voltae Yes/Yes 0.1 ± 0.0 37 4 Inactive Ethanol (20) This study G. metallireducens Mcc. maripaludis Yes/No -0.1 ± 0.0 37 4 Inactive Ethanol (20) This study Strict G. metallireducens Mcl. marisnigri Yes/Yes 0.1 ± 0.0 135 10 Inactive Ethanol (20) This study hydrogenotrophic G. metallireducens Ml. petrolearius Yes/No 0.0 ± 0.0 35 4 Inactive Ethanol (20) This study G. metallireducens Mbt. formicicum Yes/Yes 0.1 ± 0.1 152 3 Inactive Ethanol (20) Rotaru 2014b methanogens G. metallireducens Msp. hungatei Yes/Yes 0.0 ± 0.1 152 4 Inactive Ethanol (20) Rotaru 2014b

Co-cultures with other Geobacter G. hydrogenophilus Ms. barkeri 800 Yes/No 7.5 ± 0.8 135 3 Exponential Ethanol (20) Rotaru 2015 G. hydrogenophilus Mtx. harundinacea No/No 2.3 ± 0.4 117 2 Incomplete Ethanol (20) This study G. hydrogenophilus Msp. hungateii Yes/Yes 0.3 ± 0.1 117 3 Inactive Ethanol (20) This study G. bemidjiensis Ms. barkeri 800 Yes/No 0.3 ± 0.0 135 3 Inactive Ethanol (20) Rotaru 2015 G. chapellei Ms. barkeri 800 Yes/No 0.1 ± 0.0 112 3 Inactive Ethanol (20) Rotaru 2015 G. uraniireducens Ms. barkeri 800 Yes/No 0.1 ± 0.0 112 3 Inactive Ethanol (20) Rotaru 2015 G. bremensis Ms. barkeri 800 Yes/No -0.1 ± 0.1 119 3 Inactive Ethanol (20) Rotaru 2015 G. humireducens Ms. barkeri 800 Yes/No 0.0 ± 0.0 119 3 Inactive Ethanol (20) Rotaru 2015 Co-cultures with Rhodoferrax R. ferrireducens Mtx. harundinacea No/No 6.8 ± 0.5 270 3 Stationary Glucose (5) This study R. ferrireducens Ms. barkeri 800 Yes/No 1.8 ± 0.4 270 3 Stationary Glucose (5) This study R. ferrireducens Msp. hungateii Yes/Yes 0.7 ± 0.1 270 3 Inactive Glucose (5) This study R. ferrireducens Mbt. formicicum Yes/Yes 0.7 ± 0.0 270 3 Inactive Glucose (5) This study

Co-cultures with Pelobacter (H2-producing control) P. carbinolicus Ms. barkeri 800 Yes/No 7.5 ± 1.4 16 4 Exponential Ethanol (20) Rotaru 2014b P. carbinolicus Msp. hungateii Yes/Yes 8.4 ± 0.2 5 3 Stationary Ethanol (20) This study P. carbinolicus Mbt. formicicum Yes/Yes 7.8 ± 1.5 5 3 Stationary Ethanol (20) This study

353

354 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

355

356 Table 2. c-type cytochromes in methanogens tested for DIET. a) c-type cytochromes in 13 species of

357 methanogens as predicted by CxxCH motif and/or annotated, this includes multiheme cytochromes. b)

358 Predicted localization of the multiheme c-type cytochromes according to various bioinformatics tools.

DIET methanogens

h m

a .

M M Annotated cytochrome c 0 1 1 0 0 0 0 0 1 2 1 0 0 Predicted c-cytochromes (single CxxCH motifs) 16 10 17 23 16 32 20 21 21 30 28 19 23 Predicted mutliheme (>2 CxxCH motifs) 0 0 0 1 0 1 0 0 0 3 1 0 0

DIET methanogens

c s

b s

M M

Ms. horonobensis M

T 5

Locus tag new/old (Protein) H

)

(

)

(

)

(

M p Predicted Hemes (#CxxCH) 2 2 5 2 5 5 localization TMHMM Outside Outside 2 TMH 1 TMH 1 TMH Outside PSORTB Cytoplasmic Cytoplasmic Unknown Unknown Cytoplasmic Cytoplasmic SOSUI Soluble Soluble Membrane Soluble Membrane Membrane Pred-signal Periplasmic Periplasmic Signal peptide Signal peptide Signal peptide Periplasmic TatP No No Yes No No No SignalP No No Yes Yes Yes No SecretomeP No No Yes Yes Yes Yes 359

360 bioRxiv preprint doi: https://doi.org/10.1101/747485; this version posted August 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

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