bioRxiv preprint doi: https://doi.org/10.1101/556704; this version posted December 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 Opportunistic interactions on Fe0 between methanogens and acetogens 2 from a climate lake 3 Paola Andrea Palacios 1 and Amelia-Elena Rotaru1* 4 1Nordcee, Department of Biology, University of Southern Denmark, Odense, Denmark 5 * Correspondence: 6 Amelia-Elena Rotaru 7 [email protected] 8 Keywords: microbial influenced corrosion, acetogens, methanogens, interspecies interactions, 9 iron corrosion, Clostridium, Methanosarcinales, Methanothermobacter. 10 Abstract 11 Microbial-induced corrosion has been extensively studied in pure cultures. However, Fe0 corrosion 12 by complex environmental communities, and especially the interplay between microbial 13 physiological groups, is still poorly understood. In this study, we combined experimental physiology 14 and metagenomics to explore Fe0-dependent microbial interactions between physiological groups 15 enriched from anoxic climate lake sediments. Then, we investigated how each physiological group 16 interacts with Fe0. We offer evidence for a new interspecies interaction during Fe0 corrosion. We 17 showed that acetogens enhanced methanogenesis but were negatively impacted by methanogens 18 (opportunistic microbial interaction). Methanogens were positively impacted by acetogens. In the 19 metagenome of the corrosive community, the acetogens were mostly represented by Clostridium and 20 Eubacterium, the methanogens by 21 Methanosarcinales, Methanothermobacter and Methanobrevibacter. Within the corrosive 22 community, acetogens and methanogens produced acetate and methane concurrently, however at 0 23 rates that cannot be explained by abiotic H2-buildup at the Fe surface. Thus, microbial-induced 24 corrosion might have occurred via a direct or enzymatically mediated electron uptake from Fe0. The 25 shotgun metagenome of Clostridium within the corrosive community contained several H2-releasing 0 26 enzymes including [FeFe]-hydrogenases, which could boost Fe -dependent H2-formation as 27 previously shown for pure culture acetogens. Outside the cell, acetogenic hydrogenases could 28 become a common good for any H2/CO2-consuming member in the microbial community including 29 methanogens that rely on Fe0 as a sole energy source. However, the exact electron uptake mechanism 30 from Fe0 remains to be unraveled. 31 1 Introduction 32 Steel infrastructure extends for billions of kilometers below ground enabling transport and storage of 33 clean water, chemicals, fuels, sewage, but also protection for telecommunication and electricity 34 cables. Climate change has led to extreme weather conditions like severe storms and rainfall. Urban 35 storm and rainfall management in many countries especially northers countries like Denmark 36 involves so called climate lakes (also known as storm water ponds or retention ponds) harvesting 37 rainfall at large scale thus alleviating stormwater runoff in the cities (Mishra et al., 2020). If 38 stormwater runoff is improperly detained, undergrown steel infrastructure could suffer tremendous 39 damage. Damages induced by microbial induced corrosion (MIC) in the underground are often 40 discovered too late, leading to environmental and economic devastation. Thus, it is important to be 41 able to predict the lifespan of the material if exposed to microbial communities native to the site 42 where steel structures are located. This would lead to effective replacement strategies and bioRxiv preprint doi: https://doi.org/10.1101/556704; this version posted December 6, 2020. 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 available0 under aCC-BY-NC-ND 4.0 InternationalOpportunistic license. microbial interactions on Fe 43 recuperation of the metal prior to accidental spills that may be detrimental to the surrounding 44 environment (Arriba-Rodriguez et al., 2018; Skovhus et al., 2017; Usher et al., 2014a). 45 Corrosion occurring in climate lakes is poorly understood. In this study, we investigate corrosion by 46 microorganisms from the anoxic sediments of a danish climate lake. In such anoxic environments 47 where non-sulfidic conditions prevail, steel was expected to persist unharmed for centuries (Arriba- 48 Rodriguez et al., 2018; Skovhus et al., 2017; Usher et al., 2014a). And yet, researchers showed that 49 certain groups of anaerobes (methanogens and acetogens) strip electrons off the Fe0 surface leading 50 to MIC (Kato et al., 2015; Mand et al., 2014, 2016; Mori et al., 2010; Zhang et al., 2003). Previous 51 studies showed that MIC in non-sulfidic environments is often linked to the presence of acetogens 52 like Clostridium and methanogens like Methanosarcinales on the surface of the corroded steel 53 structure (Kato et al., 2015; Mand et al., 2014, 2016; Mori et al., 2010; Zhang et al., 2003; Zhu et al., 54 2003). It was therefore suggested that Methanosarcinales were indirectly involved in corrosion, 55 growing in a mutualistic relationship with the acetogens, and allegedly both groups were gaining 56 from the interaction (Mand et al., 2016; Zhang et al., 2003). This assumption was based on acetogens 57 producing acetate, which would be then consumed by acetotrophic Methanosarcinales methanogens, 58 acetogens were expected to be favored by the removal of their metabolic product - acetate. Such a 59 mutualistic association on Fe0 between acetogenic Clostridium and methanogenic Methanosarcinales 60 has not been yet demonstrated. In contrast, we recently showed that instead of acting cooperatively 61 on Fe0, acetogens and methanogens competed for Fe0-electrons (Palacios et al., 2019a). Since, 62 interspecies interactions on Fe0 are scantily examined, here we investigated the interplay between 63 climate lake acetogens (reaction 1) and methanogens (reaction 2) when provided solely with Fe0 as 64 their electron donor. 0 - + 65 4Fe + 2CO2 + 4HCO3 + 4H → 4FeCO3 + CH3COOH + 2H2O (∆G0’ = -388 kJ/mol; Reaction 1) 0 - + 66 4Fe + CO2 + 4HCO3 + 4H → 4FeCO3 + CH4 + 2H2O (∆G0’ = -446 kJ/mol; Reaction 2) 67 Theoretically, under standard thermodynamic conditions, methanogens should have an advantage 68 over acetogens when provided with Fe0 as sole electron donor (Reactions 1 & 2). Especially, since 0 69 methanogens, unlike acetogens, are more effective at retrieving abiotic H2 (formed on Fe ) due to 70 their low H2-uptake thresholds (Drake et al., 2002; Kotsyurbenko et al., 2001). Several groups of 71 methanogens could corrode Fe0 independent of acetogenic bacteria, including species of 72 Methanosarcina (Belay and Daniels, 1990; Boopathy and Daniels, 1991; Daniels et al., 1987), 73 Methanobacterium (Belay and Daniels, 1990; Dinh et al., 2004; Lorowitz et al., 1992) and 74 Methanococcus (Lienemann et al., 2018c; Mori et al., 2010; Tsurumaru et al., 2018; Uchiyama et al., 75 2010). The mechanism by which methanogens corrode Fe0, has been debated and includes reports 0 76 which suggest they retrieve abiotic-H2 off the Fe surface (Daniels et al., 1987), retrieve electrons 77 directly using an unknown electron-uptake mechanism (Beese-Vasbender et al., 2015; Dinh et al., 78 2004) or use extracellular enzymes, which stimulate enzymatic H2-evolution or formate generation 79 on the Fe0-surface (Deutzmann et al., 2015a; Lienemann et al., 2018c; Tsurumaru et al., 2018). The 80 later mechanisms were especially relevant for Methanococcus species which harbored an unstable 81 genomic island encoding [NiFe]-hydrogenases along with the heterodisulfide reductase super 82 complex involved in formate generation (Lienemann et al., 2018c; Tsurumaru et al., 2018). 83 And yet, oftentimes acetogens dominate corrosive communities, outcompeting methanogens when 84 concentrations of H2 are high and temperatures are low, presumably due to the higher kinetics (Vmax) 85 of their hydrogenases (Kotsyurbenko et al., 2001). Moreover, unlike methanogens, acetogens 86 contain [FeFe]-hydrogenases (Peters et al., 2015), which could retrieve electrons directly from Fe0 87 for proton reduction to H2 (Mehanna et al., 2008, 2016; Rouvre and Basseguy, 2016). 88 In this study, we were interested to understand the interspecies dynamics on Fe0 of acetogens and 89 methanogens from a climate lake. We used a combination of physiological experiments, inhibition 90 strategies and whole metagenomic analyses to study the interactions of acetogens and methanogens 91 during Fe0 corrosion. In contrast to our previous report on a corrosive coastal community (Palacios et 2 bioRxiv preprint doi: https://doi.org/10.1101/556704; this version posted December 6, 2020. 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 available0 under aCC-BY-NC-ND 4.0 InternationalOpportunistic license. microbial interactions on Fe 92 al., 2019a), in this climate lake we observed a different type of microbial interaction, where 93 acetogens (Clostridium) were negatively impacted by the presence of methanogens whereas 94 methanogens were benefiting from their presence in a parasitic (-/+) type of interaction while 95 inducing Fe0 corrosion. 96 2