1. Introduction
In the European Union, around 70% of primary energy is generated by the combustion of fossil fuels, contributing about 78% (3367 Tg-CO
2 equ.) of the total emitted greenhouse gases [
1,
2]. A transition to a carbon dioxide (CO
2)-neutral and sustainable energy production system is urgently needed. One of the possibilities is to utilize the power-to-gas process [
3,
4,
5]. Within this process, biomethanation of CO
2 to methane (CH
4) offers a sustainable opportunity to enable the transition from fossil fuels, as it is an autobiocatalytic process. Therefore, it is envisioned that biomethanation will become an essential part of future energy production systems, as CH
4 could be produced at a stable pace and stored in vast amounts in the natural gas grid network [
6]. Pure cultures of methanogenic archaea (methanogens) [
7,
8,
9,
10,
11,
12] and enrichment cultures containing methanogens [
13,
14,
15,
16,
17] can be utilized for in situ or ex situ biomethanation [
18,
19,
20].
Carbon monoxide (CO)-containing rich waste gases are a by-product of industrial processes such as steelmaking [
21]. CO-containing gases can also be obtained by gasification of carbon-rich materials, such as domestic organic waste or lignocellulose conversion to syngas [
22]. The fact that biofuel production directly from lignocellulose is still costly and biotechnologically challenging makes microbial gas to gas conversion from CO a promising alternative bioprocess [
7,
23]. Developments in the transformation of gaseous waste products to energetically valuable compounds emphasize the potential for syngas as a substrate [
24,
25,
26,
27]. CO has a high potential for donating electrons, making it a favourable substrate for lithotrophic microorganisms [
28,
29]. Besides CO, waste and syngas mainly consist of molecular hydrogen (H
2), CO
2, and CH
4. As early as 1990, it was demonstrated that methanogens can metabolize some components from syngas [
30].
The direct conversion of pure CO to CH
4 was performed by hydrogenotrophic methanogens and subsequently analysed [
31,
32,
33,
34]. Furthermore, growth adaption to CO did not change the CH
4 production rates significantly in the case of
Methanothermobacter marburgensis, and the specific growth rate (µ) of
Methanothermobacter thermautotrophicus on pure CO was only a hundredth of the growth rate achieved using H
2:CO
2 as a substrate [
31]. This led to the assumption that an artificial co-culture, where CO is converted to CH
4 in a successive bioprocess, would lead to higher efficiency, as microorganisms specifically adapted to the task of converting CO and producing CH
4 can be selected. Studies showed that the independent performance of the water gas shift reaction (WGSR) and biomethanation by two different organisms in the same vessel resulted in a more than 20-fold faster conversion than direct conversion by a single organism [
34]. Therefore, we hypothesized that an artificial co-culture of a carboxydotrophic, hydrogenogenic microbe with a hydrogenotrophic, autotrophic organism would drive favourable thermodynamic conditions for the WGSR. These conditions are created by direct removal of the gaseous metabolic end products of the WGSR, that is H
2:CO
2, by the methanogen. The conversions that are successively performed by the two organisms can be summarized with the following equation:
Overall, studies using a co-culture approach showed promising results for biomethane production rates [
34,
35]. Based on a previous study where a bacterial/archaeal co-culture was utilized [
36], we wanted to investigate the potential of artificial archaeal co-cultures consisting of a carboxydotrophic, hydrogenogenic archaeon, and different hydrogenotrophic, autotrophic, and methanogenic archaea for biomethanation. We selected
Thermococcus onnurineus for performing the catalysis of the WGSR, as it was shown that µ and H
2 productivity on CO was substantially higher compared to other carboxydotrophic and hydrogenogenic microbes [
37,
38,
39,
40]. To catalyse the second part of the reaction,
Methanocaldococcus jannaschii, Methanocaldococcus vulcanius, and
Methanocaldococcus villosus were selected, because of their similar cultivation requirements to
T. onnurineus with respect to temperature, salt concentration, and pH optimum. All four organisms were isolated from deep-sea hydrothermal vents and belong to the Euryarchaeota. They are able to grow in a temperature range of 63 to 86 °C, a salt concentration of 1 to 5%, and a pH of 5.5 to 7.0.
T. onnurineus is a heterotroph, while the methanogens are chemolithoautotrophs [
41,
42,
43,
44]. Moreover, hyperthermophilic organisms are more advantageous over mesophiles, since at higher temperatures, a faster conversion of CO by the carboxydotrophic microorganism occurs, and a three times faster removal of H
2 is obtained by the methanogen [
34,
45]. Therefore, the properties of a hyperthermophilic environment positively affects growth and conversion rates and is, thus, advantageous over mesophilic conditions. Here, we analysed whether
T. onnurineus together with one of the three methanogens can be grown as a powerful artificial archaeal co-culture to efficiently generate biological CH
4 from synthetic waste gases.
2. Materials and Methods
2.1. Chemicals
CO (99.999 Vol.-%), H2:CO (60 Vol.-% in CO), H2:CO2 (80 Vol.-% in CO2), and an artificial CO-containing syngas (CO2 16.7 Vol.-%, H2 Vol.-% 16.8%, CH4 Vol.-%14.7, N2 Vol.-% 14.5%, and CO 37.3 Vol.-%) were used for closed batch experiments. For gas chromatography (GC), H2 (99.999 Vol.-%), CO2 (99.999 Vol.-%), CO (99.999 Vol.-%), H2/CO2 (80 Vol.-% in CO2), H2/N2 (4.5 Vol.-% H2 in N2), CH4 (99.995 Vol.-%), and the standard test gas (Messer GmbH, Wien, Austria) (containing 0.01 Vol.-% CH4, 0.08 Vol.-% CO2 in N2) were used in addition to the gases mentioned above. All gases, except the standard test gas, were purchased from Air Liquide (Air Liquide GmbH, Schwechat, Austria). All other chemicals were of the highest grade available.
2.2. Media
Medium A is a modified version of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) medium 282. The exact composition of medium A and medium B can be found in
Supplementary Materials, Tables S1 and S2. Balch’s vitamin solution was used [
46]. For
M. marburgensis and
M. thermautotrophicus, a phosphate-buffered medium was used [
47].
2.3. Strains and Cultivation Conditions
The strains M. jannaschii JAL-1, M. villosus KIN24-T80, M. vulcanius M7, M. marburgensis DSM 2133 (Marburg), and M. thermautotrophicus DSM 1053 (delta H) were purchased from the DSMZ. T. onnurineus NA1 was provided by Prof. Dr. Sung Gyun Kang (Korea Institute of Ocean Science and Technology (KIOST), Ansan, Korea).
Every cultivation was performed in closed batch mode [
48]. Experiments were conducted in 120 mL serum bottles (Ochs Glasgeraetebau, Langerwehe, Germany), sealed with a 20 mm butyl rubber stopper and an aluminium crimp cap (Chemglass Life Science LLC, Vineland, NJ, USA). Serum bottles were filled with the corresponding medium and sealed. Anaerobic conditions were created by evacuating and re-pressurizing with H
2:CO
2 (4:1) to 0.5 barg five times. The bottles were autoclaved and stored at 4 °C until further use. Unless otherwise stated, the medium was augmented with the following sterile filtered stock solutions before inoculation: NaHCO
3, L-Cysteine-HCL, and Balch’s Vitamin solution [
46] (see
Supplementary Materials, Tables S1 and S2). Afterwards, the bottles were flushed with H
2:CO
2 (4:1) to 0.5 barg, before the addition of autoclaved Na
2S·9H
2O.
The inoculum, 1 mL (2% v/v) of an actively grown culture, was added anaerobically. The final liquid phase in each serum bottle added up to 50 mL. Depending on the experiment, the headspace gas phase was exchanged with the corresponding gas. Bottles were incubated at 80 °C in either a double-layer shaking incubator at 100 rpm (LABWIT ZWYR-2102C, Labwit Scientific Pty Ltd., Burwood East, Australia) or in a water bath at ~100 lateral shakes per minute (GFL 1083, LAUDA-GFL, Burgwedel, Germany).
2.4. Pure-Culture Closed Batch Experiments
The methanogens M. villosus, M. vulcanius, and M. jannaschii were grown under 2 barg H2:CO2 in either medium A or B. A reduced version of them without vitamins, yeast extract, trace elements, or a combination of the three was also tested. The incubation rhythm consisted of 13 and 7 h incubation periods, with 2 h of sampling in between every period. The same rhythm was applied for cultivation of T. onnurineus. It was grown in the same medium, but under 1 barg CO. These incubation periods did not apply for M. marburgensis and M. thermautotrophicus, because of their slow growth. Furthermore, they were grown in a phosphate-buffered M. marburgensis medium with either 1.7 barg CO, 1.7 barg H2:CO (4:1), or 1.7 H2:CO2 (4:1).
2.5. Co-Culture Closed Batch Experiments
The co-cultures consisted of T. onnurineus and either M. villosus, M. vulcanius, or M. jannaschii. The experiments were conducted according to two general schemes. In the first setup, the experiment started by growing the respective Methanocaldococcus strain under 2 barg H2:CO2 first. After 13 h in the incubator, T. onnurineus was added. The bottles were then pressurized with CO, H2:CO, or artificial syngas to 1 barg. In the second setup, T. onnurineus grew 13 h under pure CO, H2:CO, or artificial syngas before one of the Methanocaldococcus strains was added. From this point onward, both schemes followed the same incubation rhythm, as described in the pure-culture experiments, and ended after a maximum of 80 h of cumulative incubation time. Every experiment was performed in hexuplicates (n = 6) on media A and B, as well as on reduced versions of them.
2.6. Sampling
The routinely performed sampling consisted of removing the cultures from the incubator and measuring the pressure as soon as the bottles cooled down to room temperature (~60 min). To analyse growth, 0.7 mL of the cultures were withdrawn for optical density (OD) measurements (λ = 578 nm). Lastly, the bottles were flushed and re-pressurized with the corresponding gas.
2.7. Analytical Procedures
OD of the cultures was measured via a spectrophotometer at λ = 578 nm (Specord 200 Plus, Analytik Jena, Jena, Germany). The headspace gas composition before and after inoculation in pure cultures was determined via the gas headspace pressure difference [
18,
47,
49]. Gas evolution and uptake rates were calculated according to methods described in refs. [
47,
50,
51]. Samples that were withdrawn for gas chromatography (GC) analysis did not undergo the sampling procedure. The headspace gas composition in co-culture experiments was analysed via GC, and the evolution and uptake rates were calculated [
52]. Some of the negative controls revealed “air contamination” and were removed from the calculations of the results. To maintain the correct atmosphere, OD was measured after completion of the GC run and as such is only an estimate of the true OD.
4. Discussion
This study is a new brick in the emerging research field of Archaea Biotechnology [
54] and artificial microbial ecosystem design and engineering [
55]. We investigated the conversion of one-carbon substrates (CO, H
2:CO, and CO-rich waste gases) by the artificially designed co-cultures of either
M. villosus,
M. jannaschii, or
M. vulcanius together with
T. onnurineus. Our results showed fast and reliable gas conversion, with a reduction in pure CO to about 50 mol% and simultaneous production of ~10 mol% CH
4 within 7 h. The most efficient conversion of the artificial syngas was performed by the co-culture
M. villosus with
T. onnurineus, inoculated in this order. This culture showed a CO reduction of 7%, starting from 37.3 mol% and an increase in CH
4 by ~10 mol% within 7 h. This proved the ability of the co-cultures to convert a variety of different CO-containing gas compositions with a lower proportion of CO.
A study with a similar experimental setup reported an ~6% CH
4 increase after 22 h [
35]. We obtained 10% increase in 7 h. This suggests that the established co-culture (
T. onnurineus/
M. villosus) is of higher catalytic power. Nonetheless, most up-to-date published co-cultures were tested in different setups than the one reported in this study, making a direct comparison of evolution and uptake rates rather difficult [
26,
30,
34]. Establishing different co-cultures in a bioreactor setup will, thus, be of great importance to fully understand their growth and production kinetics.
Pure-culture closed batch experiments of the methanogens in a defined medium on H
2:CO
2 showed a higher MER (4.2 ± 0.1 mmol L
−1 h
−1) than the co-cultures (2.0 ± 0.3 mmol L
−1 h
−1) (
Figure 1,
Table 3). Therefore, the potential for achieving a higher MER in co-cultures is possible if the necessary gas supply can be performed, and the inhibitory concentration of CO would not be surpassed [
56]. Although the main limitation of the co-culture grown on pure CO was the availability of H
2 for methanogenesis and, hence, the conversions of CO to H
2 and CO
2 by
T. onnurineus (
Figure 3 and
Supplementary Materials, Table S7). The addition of H
2 to the gas phase did not provide an increase in the MER. Rather, it led to a limitation of CO
2 availability (
Supplementary Materials, Table S7), most likely due to the reduced performance of
T. onnurineus under the lower CO partial pressure. Consequently, application of the artificial syngas led to an increase in MER, as biomethanation seems to be neither limited by CO
2 nor H
2 availability. Nonetheless, it did not reach the same values as in pure CO (
Table 3). This can be explained by the concentration reduction in CO, CO
2, and H
2 in syngas by the other initially present gases, reducing substrate availability.
A change in the cultivation method to a bioreactor setup with higher pressure, agitation, and a constant gassing, resulting in having a higher gas solubility and higher gas transfer rate to the liquid phase, might increase the conversion of CO by
T. onnurineus and the potential of the co-culture [
37,
39,
48]. However, the ratio of the gas in the liquid phase has to be considered carefully, as high agitation might also lead to an excessive CO availability for the methanogens [
34]. As recently more and more genetic tools for archaea become available, an overexpression of the carbon monoxide dehydrogenase could also be a solution to debottleneck CO conversion by
T. onnurineus [
39,
57].
Unfortunately,
T. onnurineus is still dependent on a complex medium containing yeast extract, which limits the potential industrial applicability. However, co-cultivation could be used to gain advantages or improve growth of both microorganisms through improving their syntrophic relationships [
34]. Finding a suitable defined medium for
T. onnurineus or replacing it by a different organism that can catalyse the WGSR from CO in minimal conditions is one suggested avenue of research.
Carboxydocella thermautotrophica or
Carboxydocella sporoproducens would fall into the same pH and temperature conditions as the herein employed methanogens [
35].
A direct conversion of CO to CH
4 by a single organism such as
M. marburgensis or
M. thermautotrophicus is likely not the most suitable biotechnological approach, as the inhibitory concentration of CO for these methanogens is very low [
31,
34]. The artificially created archaeal co-cultures consisting of one of the hyperthermophilic methanogenic archaea
M. villosus,
M. jannaschii, and
M. vulcanius together with
T. onnurineus, as performed in this study, are highly efficient and reliable for biomethanation. By adaptation to a minimal medium and by performing targeted bioprocess development, artificial archaeal co-cultures could be of environmental, economic, and industrial value in renewable energy production and storage.