An iron corrosion-assisted H2-supplying system: a culture method for methanogens and acetogens under low H2 pressures

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the “iron corrosion-assisted H2-supplying (iCH) system” by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifically enriched in the iCH cultures. We also observed selective enrichment of acetogen species by the iCH system (Acetobacterium spp. and Sporomusa spp.), whereas Clostridium spp. predominated in the high H2 cultures. These results demonstrate that the iCH system facilitates culture of anaerobic microorganisms under low H2 pressures, which will enable the selective culture of microorganisms adapting to low H2 environments.

Molecular hydrogen (H 2 ) is an important intermediary metabolite and an energy carrier in anaerobic environments [1][2][3] . Because H 2 is rapidly turned over in natural anaerobic environments, it occurs at very low partial pressures of only a few to several tens of pascals (Pa) 4 . In conventional studies, however, culture and isolation of H 2 -utilizing microorganisms have commonly been performed under high H 2 partial pressures (100 kPa or more). Under such laboratory conditions, it is difficult to draw conclusions about the ecophysiology of H 2 -utilizing microorganisms in their natural environment, nor can microorganisms that have adapted to conditions with low H 2 be isolated. In fact, the presence of uncultured H 2 -utilizing methanogens and acetogens in anaerobic environments where H 2 concentrations are estimated to be quite low (i.e., environments with low available organic matter, including subsurface environments, peat soils, and deep-sea sediments) has been inferred by molecular environmental analyses such as metagenomics [5][6][7] .
Because H 2 supplied at low partial pressure is rapidly consumed, sufficient microbial growth cannot be obtained in conventional batch culture systems. To date, several research groups have developed methods that can continuously supply H 2 at low partial pressure to elucidate ecophysiology of hydrogenotrophic methanogens in low H 2 environments. Morgan et al. 8 reported a low-H 2 culture of a hydrogenotrophic methanogen by using a continuous culture system with a continuous influx of a mixed gas containing H 2 . By using this system, the authors found that the expression of some metabolic enzymes in the methanogenic pathway is regulated by  11 developed the "coculture method", in which methanogens are cocultured with heterotrophic H 2 -producing bacteria to achieve a continuous supply of H 2 at low concentration. The coculture method enabled selective enrichment of uncultured hydrogenotrophic methanogens that were expected to adapt to low H 2 pressures, which finally resulted in the isolation of phylogenetically novel methanogens such as Methanocella spp. and Methanolinea spp. 12,13 . The coculture method has also been employed to analyze physiological responses of methanogens to low H 2 pressures 14,15 . Although these methods yielded laboratory cultures under low H 2 pressures, several issues still need to be addressed. The continuous gas influx process cannot be carried out in parallel with a large number of cultures because it requires relatively complex systems, including large-or small-scale reactors and gas supply devices. Although the coculture method only requires simple systems, which makes it suitable for enrichment cultures, it cannot be directly utilized for isolation of H 2 -utilizing microorganisms because it relies on coexistence with fermentative bacteria. Furthermore, it cannot be excluded that metabolites other than H 2 (e.g., organic acids such as acetate) affect the growth of H 2 -utilizing microorganisms.
In this study, we aimed to develop a simple method capable of selective culture of microorganisms under low H 2 pressures. The reaction on which we focused was the corrosion of metallic iron in anoxic solution: Fe 0 + 2H + ↔ Fe 2+ + H 2 . The concept of culturing microorganisms using H 2 derived from iron corrosion has already been reported 16,17 . The authors demonstrated that hydrogenotrophic methanogens can be cultured using H 2 derived from metallic iron as sole energy source. However, this method has not been applied to culture microorganisms adapted to low H 2 environments. Considering that iron corrosion proceeds very slowly in anoxic and circumneutral solution because of the small difference in the standard redox potentials of Fe 0 oxidation (E 0 ʹ ≈ − 0.47 V) and the reduction of protons to generate H 2 (E 0 ʹ ≈ − 0.41 V), we can expect that H 2 supply via iron corrosion is suitable to culture hydrogenotrophic microorganisms under low H 2 pressures. Here we report that a culture system based on iron corrosion reactions has been successfully used for the selective enrichment of hydrogenotrophic methanogens and acetogens, which have the potential to adapt to environments with very low H 2 content.

Results and discussion
Validation of continuous H 2 supply by iron corrosion. Because the growth of microorganisms is considered to be very slow under low H 2 pressures, the culture system requires a continuous supply of H 2 over a long period of time. Hence, we first determined whether H 2 can be continuously supplied for a long time by the iron corrosion reaction. Furthermore, to develop a culture system capable of regulating the H 2 supply rate, we added various amounts of Fe 0 with different particle sizes (Fe 0 granules with a diameter of 1-2 mm or Fe 0 powder with a diameter < 45 µm) to the anoxic buffer solution and determined the H 2 production rates (Fig. 1).
We observed continuous H 2 production for more than 6 months in the vials supplemented with Fe 0 granules (Fig. 1A). The H 2 production rates were almost proportional to the amount of Fe 0 granules added (0.03 to 3 g vial −1 ) and were in the range of 0.12 to 4.4 μmol vial −1 day −1 (Fig. 1C). With Fe 0 powder, H 2 production ceased after approximately 2 weeks, 1 month, and 3 months in the vials supplemented with Fe 0 at 3, 1, and 0.3 g vial −1 , respectively (Fig. 1B). The highest H 2 accumulation was approximately 1000 μmol vial −1 , corresponding to approximately 50 kPa. We assume that H 2 production ceased for thermodynamic reasons (increase in H 2 partial pressure and decrease in proton concentration).The H 2 production rates were also proportional to the amount of Fe 0 powder added, with a range of 2.0 to 93 μmol vial −1 day −1 (Fig. 1C). Since the specific surface area of the spherical material is inversely proportional to the diameter, the Fe 0 powder (< 45 µm) has a surface 20 times larger than the Fe 0 granule (1-2 mm), if used particles are assumed to be spherical. The H 2 production from Fe 0 powder was 16-22 times greater than from Fe 0 granule (Fig. 1C), suggesting that surface area is the major determinant of H 2 production rate. These results demonstrate that it is possible to supply H 2 over a long period of time by utilizing the iron corrosion reaction, and also that it is possible to regulate the rate of H 2 supply by altering the size and amount (i.e., total surface area) of Fe 0 particles.   [16][17][18][19][20] . If microorganisms are cultured in the coexistence with Fe 0 , however, some undesirable phenomena can occur. For example, the iron corrosion reaction produces a high concentration of ferrous iron and induces an increase in pH due to the consumption of protons. Furthermore, it can promote growth of anaerobic microorganisms that use Fe 0 itself as the energy source 18,21,22 . These phenomena likely hamper the culture of target microorganisms. We therefore constructed a culture system in which the gas phases in two vials (one for the culture of microorganisms and the other for the iron corrosion reaction) are connected by a stainless-steel tube (Fig. 2), in reference to the system developed by Daniels et al. 16 . This system, hereafter referred to as the "iron corrosion-assisted H 2 -supplying (iCH) system", was expected to allow the culture of microorganisms under low H 2 pressures while avoiding the unfavorable effects of the iron corrosion reaction.
Pure culture of a hydrogenotrophic methanogen in the iCH system. We evaluated the capability of the iCH system to culture microorganisms under low H 2 pressures using a hydrogenotrophic methanogen Methanobacterium formicicum, which is known to be able to grow under both high and low H 2 partial pressures 23 , as a model strain. We inoculated the iCH system with M. formicicum using Fe 0 powder at 1 g vial -1 as the H 2 source, and monitored the amounts of CH 4 and H 2 in the gas phase (Fig. 3). We observed continuous CH 4 production by M. formicicum in the iCH system for almost 1 month. Although there was accumulation of H 2 at the beginning of the culture (around 190 Pa at day 3, corresponding to 7.4 µmol vial -1 ), the partial pressure of H 2 subsequently decreased and remained extremely low (30 to 50 Pa after day 14). The observed H 2 partial pressures are comparable to those observed in natural anaerobic environments and laboratory cocultures of methanogens with fermentative bacteria 4,24,25 . These results demonstrated that the iCH system is capable of long-term culture of H 2 -utilizing microorganisms under low H 2 pressures.
Enrichment cultures of hydrogenotrophic methanogens using the iCH system. We performed enrichment cultures of methanogens to demonstrate the capability of the iCH system to selectively culture  www.nature.com/scientificreports/ microorganisms adapted to low H 2 pressures. We used rice paddy field soil as the microbial source, because it is known as a low H 2 environment 4 and was the isolation source of Methanocella spp. that is known to adapt to low H 2 environments 12,26,27 . In addition to the iCH system (with Fe 0 powder at 1 g vial -1 as the H 2 source), we set up conventional cultures under high H 2 pressures (160 kPa of H 2 in the gas phase, hereafter referred to as "high H 2 enrichments") and cultures under iron corrosion conditions (microorganisms cultured in the same vial with 1 g vial -1 of Fe 0 powder, hereafter referred to as "Fe 0 enrichments") as control experiments. Hydrogenotrophic methanogens were enriched in inorganic medium supplemented with rifampicin to suppress growth of bacteria. After three successive subcultures, the metabolic products (CH 4 and H 2 ) were analyzed during incubation (Fig. 4A-C). In the high H 2 enrichments, we observed CH 4 production almost equal to the theoretical value calculated from the consumption of H 2 (Fig. 4A). In the Fe 0 enrichments, we observed CH 4 production comparable to the theoretical value (broken line in Fig. 4B) calculated from the H 2 production via iron corrosion in the early phase of the incubation (day 0-14). However, CH 4 production levelled off in the later phase (after day 14). The pH of the culture solution of Fe 0 enrichments increased from 7.0 to around 8.1 during the incubation, whereas the pH of the culture solution remained around 7.0 in the other enrichments. The increase in pH, and possibly the increase in concentration of ferrous iron, might have inhibited the growth of methanogens in the Fe 0 enrichments. In the iCH enrichments, CH 4 was produced almost proportional to the theoretical value (broken line in Fig. 4C) without levelling off. We observed accumulation of H 2 at the beginning of culture (around day 10), which then decreased below the detection limit (< 10 Pa). These results indicate that enrichment cultures of methanogens under low H 2 pressures were achieved in the iCH system.

Microbial community analysis of the methanogenic enrichment cultures.
To confirm the capability of the iCH system to specifically enrich microorganisms adapting to low H 2 pressures, we assessed the microbial community structures of the enrichment cultures and the inoculum soil by high throughput sequencing analysis of 16S rRNA gene amplicons. A total of 56,388 16S rRNA gene reads (3744-5531 reads per sample) were retrieved and classified into 2949 operational taxonomic units (OTUs) using a 97% sequence identity cut- www.nature.com/scientificreports/ off. The microbial composition is displayed in Fig. S1A. All OTUs dominant in the enrichment cultures (relative abundance > 3% in at least one enrichment culture) had low abundance in the inoculum soil (~ 1.2%), suggesting that H 2 -utilizing microorganisms were sufficiently enriched. Principal component analysis was performed to quantitatively evaluate the similarity of microbial community structures of each sample (Fig. S1B). The results showed that the microbial community patterns of the duplicate enrichment cultures were very similar, and that the microbial community composition differs between the different experimental setups. Therefore, for further analysis we used the average values of community analysis data of the duplicate cultures. We plotted the relative abundances of the dominant archaea (> 3% under at least one set of culture conditions) in the methanogenic enrichments (Fig. 5A). Different types of methanogens were enriched depending on the culture conditions. In the high H 2 enrichments, two OTUs closely related to Methanobacterium spp. (OTU183, with 99% identity to Methanobacterium oryzae and OTU192, with 100% identity to Methanobacterium lacus) predominated. By contrast, OTU173 and OTU1307 (100% identity to Methanocella arvoryzae and Methanoculleus chikugoensis, respectively) were specifically enriched in the iCH cultures. The genera Methanocella and Methanoculleus have been frequently detected as dominant hydrogenotrophic methanogens in various anaerobic environments with low H 2 concentrations, including rice paddy fields, peat bogs, marine and freshwater sediments, and subsurface environments [28][29][30][31][32][33] . Furthermore, methanogens closely related to these genera have been selectively enriched from rice paddy field soils and marine/freshwater sediments under the low H 2 pressures achieved by the coculture method 11 . These findings suggest that the iCH system can selectively culture hydrogenotrophic methanogens adapting to low H 2 pressures. By contrast, a different type of methanogen (OTU153, with 100% identity to Methanobacterium flexile) was enriched in the Fe 0 enrichment cultures. This suggests that factors other than H 2 concentration (e.g., increase in pH and/or high concentration of ferrous iron) were the main selective pressures in the Fe 0 enrichments. Acetogen enrichment culture and its microbial community analysis. In addition to methanogenic archaea, acetogenic bacteria are also one of the important H 2 -utilizing microorganisms in anaerobic environments 34,35 . Hence, we set up enrichment cultures of H 2 -utilizing acetogens with inorganic medium supplemented with 2-bromoethanesulphonate (BES) to inhibit methanogens. We followed the transitions of www.nature.com/scientificreports/ metabolites during the incubations of enrichment cultures of acetogens in the high H 2 , Fe 0 , and iCH cultures (Fig. 4D-F). As described below, the trends were similar to those observed with the enrichment of methanogens.
In the high H 2 enrichments, we observed acetate production with concomitant consumption of H 2 , and the H 2 consumption rate was much higher than in enrichment cultures of methanogens (Fig. 4D). In the Fe 0 enrichments, we observed acetate production comparable to the theoretical value from day 0 to day 14, after which acetate production ceased (Fig. 4E), suggesting that the Fe 0 cultures have inhibitory effects on acetogens as observed in the methanogenic enrichments. In the iCH enrichments, acetate was produced at a rate comparable with theoretical values (Fig. 4F). Although H 2 initially accumulated in the iCH enrichments, it was below the detection limit (< 10 Pa) after day 14. These results indicate that acetogens adapting to low H 2 pressures could be enriched in the iCH system. We plotted the relative abundances of the dominant bacteria (> 3% under at least one set of conditions) in the enrichments for acetogens (Fig. 5B). As with the methanogen enrichments, the acetogen community structures were completely different for each culture condition. Only one phylotype (OTU898) closely related to Clostridium magnum, which is well known as an acetogen 33 , was enriched in the high H 2 enrichments. In contrast, two phylotypes closely related to other acetogen species (OTU2305 and OTU2514, with 100% identities to Acetobacterium carbinolicum and Sporomusa sphaeroides, respectively) were selectively enriched in the iCH cultures. The phylotypes closely related to Clostridium glycolicum and Romboutsia lituseburensis (OTU316 and OTU1895, respectively) predominated in the Fe 0 enrichments, in addition to OTU898 (C. magnum) and OTU2514 (S. sphaeroides), which were also detected in other enrichments. Clostridium glycolicum is a well-known acetogen species 34 . Romboutsia spp. have often been detected in enrichment cultures of H 2 -utilizing acetogens 36 , although there is no report of their acetogenic metabolism.
It has been reported that affinities for H 2 and kinetics of H 2 consumption differ depending on the species of acetogens, mainly due to the differences in their energy acquisition efficiencies 37 . In contrast to methanogens, however, there are only few studies of the response of acetogens to low H 2 pressures. Generally, acetogens have lower affinity for H 2 than methanogens for thermodynamic reasons 38 , which may be one of the reasons that the ecophysiology of acetogens under low H 2 pressures has not attracted much attention. Our result shows that different types of acetogens can be selectively enriched under conditions with different H 2 availability. This suggests that environmental H 2 concentration provides an ecological niche not only for methanogens but also for acetogens.

Conclusion
We constructed a simple system for culturing anaerobic microorganisms under low H 2 pressures by using the iron corrosion reaction as the source of H 2 . The system, which we call the iCH system, can continuously supply H 2 for several months, and it is possible to control the H 2 supply rate by changing the amount and size of Fe 0 particles. We demonstrated that the iCH system can selectively enrich anaerobic microorganisms adapting to low H 2 pressures. Although this study focused only on methanogens and acetogens, the iCH system is applicable to cultures of other H 2 -utilizing microorganisms such as nitrate, iron, and sulfate reducers. The iCH system is also applicable to colony isolation using agar-solidified media (e.g., a roll-tube method), which is an on-going study in our research group. This culture method should enable selective enrichment and isolation of unidentified microorganisms adapting to or even specialized for low H 2 pressures from anaerobic environments with low H 2 availability, such as subsurface environments, peat soils, and deep-sea sediments, which would shed light on the novel ecophysiology of hydrogenotrophic microorganisms in anaerobic environments.

Materials and methods
Bacterial strains and culture conditions. To culture microorganisms we used a freshwater basal medium containing (per liter) 0.3 g KH 2 PO 4 , 1 g NH 4 Cl, 0.1 g MgCl 2 ·6H 2 O, 0.08 g CaCl 2 ·2H 2 O, 0.6 g NaCl, 2 g KHCO 3 , 0.02 g MgSO 4 ·7H 2 O, 9.52 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES), 0.1 g yeast extract, and 10 ml each of trace metal and vitamin solutions 39 . The pH of the medium was adjusted to 7.0 by adding 6 N KOH solution. Methanobacterium formicicum (DSM1535 T ) was cultured in the freshwater basal medium supplemented with 0.1 g l -1 of sodium acetate and reducing agents (0.3 g l -1 each of cysteine·HCl·H 2 O and Na 2 S·9H 2 O) at 37 °C without shaking under an atmosphere of 200 kPa of H 2 :CO 2 (80:20). CH 4 and H 2 in the gas phases were measured using a gas chromatograph (GC-2014; Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector (for quantification of H 2 ) and a flame ionization detector (for quantification of CH 4 ) as described previously 40 . The concentration of acetate was determined using high-performance liquid chromatography (D-2000 LaChrom Elite HPLC system; Hitachi, Tokyo, Japan) equipped with an ion exclusion column (Aminex HPX-87H; Bio-Rad Laboratories, Hercules, CA, USA) and UV detector (L2400; Hitachi). The culture experiments were conducted in triplicate.
The iron corrosion-assisted H 2 -supplying (iCH) system. The iCH system consists of two vials (68 ml in capacity). One vial ("corrosion vial" in Fig. 2) was filled with 20 ml of the freshwater basal medium and supplemented with Fe 0 granules (1-2 mm, 99.98% purity; Alfa Aesar, Ward Hill, MA, USA) or Fe 0 powder (< 45 µm, 99.9% purity; Wako Pure Chemical, Osaka, Japan). The second vial ("culture vial" in Fig. 2) was also filled with 20 ml of the freshwater basal medium. After removing the air from the medium by bubbling with N 2 :CO 2 (80:20) gas for 5 min, the vials were sealed with butyl rubber stoppers and aluminum seals, and sterilized by autoclaving. After the cultivation vials were supplemented with reducing agents, inhibitor chemicals, and/or microorganisms, the gas phases of the two vials were connected by sterile, stainless-steel tube with an inner diameter of 1.8 mm (Swagelok, Solon, OH, USA), which was separately sterilized by autoclaving, through a guiding hole made by a gauge 18 syringe needle. Before incubation, the vials were again purged with N 2 :CO 2 (80:20) gas for www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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