Efficient whole cell biocatalyst for formate-based hydrogen production
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Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute significantly to the future energy mix. However, biological H2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H2 production, as it can be produced itself from various sources including electrochemical reduction of CO2 or from synthesis gas. Many microbes that can produce H2 from formate have been isolated; however, in most cases H2 production rates cannot compete with other H2 production methods.
We established a formate-based H2 production method utilizing the acetogenic bacterium Acetobacterium woodii. This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO2 reductase that catalyzes oxidation of formate to H2 and CO2. Cell suspensions reached specific formate-dependent H2 production rates of 71 mmol g protein −1 h−1 (30.5 mmol g CDW −1 h−1) and maximum volumetric H2 evolution rates of 79 mmol L−1 h−1. Using growing cells in a two-step closed batch fermentation, specific H2 production rates reached 66 mmol g CDW −1 h−1 with a volumetric H2 evolution rate of 7.9 mmol L−1 h−1. Acetate was the major side product that decreased the H2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H2 production phase.
Acetobacterium woodii reached one of the highest formate-dependent specific H2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H2. This makes this organism a very promising candidate for sustainable H2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H2 storage.
KeywordsHydrogen production Biohydrogen Acetobacterium woodii Formate dehydrogenase Hydrogenase
Fossil fuel limitation and increasing atmospheric CO2 concentrations necessitate alternative energy carriers. Molecular hydrogen (H2) is an attractive carbon-free alternative that can be converted to energy without CO2 emission. It can be used as energy carrier for mobile applications (i.e., fuel cell powered vehicles) or as an intermediate energy storage system to store excess electrical energy that is produced in peak times from renewable sources . Currently, H2 is produced mainly from fossil fuels by steam reforming and thus unsustainable and environmentally harmful . Hence, new H2 production methods are required.
Microbial formate oxidation is catalyzed by multiple enzyme systems. Organisms such as some enterobacteria use a membrane-bound formate-hydrogen lyase system composed of membrane-associated hydrogenase and formate dehydrogenase subunits [8, 9]. Clostridiaceae or archaea such as Methanococcus can produce H2 from formate by the action of separate cytoplasmic formate dehydrogenases and hydrogenases . The observed HERs for these organisms are typically very low and do not reach the levels for H2 production from other feedstocks . One exception is the recently characterized organism Thermococcus onnurineus. This organism requires 80 °C for growth and formate-dependent H2 formation reached HERs that outcompete other dark fermentations for the first time [11, 12]. H2 production in this organism depends on a membrane-bound enzyme complex of formate dehydrogenase, hydrogenase, and Na+/H+ antiporter subunits that couples H2 formation to formate oxidation as well as energy conservation [13, 14].
A new enzyme of the bacterial formate metabolism has been discovered recently in the strictly anaerobic bacterium Acetobacterium woodii . The enzyme named hydrogen-dependent CO2 reductase (HDCR) was the first described soluble enzyme complex that reversibly catalyzes the reduction of CO2 to formate with H2 as electron donor. CO2 reduction is catalyzed at ambient conditions with rates far superior to chemical catalysis [15, 16, 17]. Therefore, it could not only be used for H2 production but, depending on the application, for H2 storage as well. In the form of formate, the explosive gas could be stored and handled much easier and with an increased volumetric energy density . H2-dependent CO2 reduction to formate by the HDCR has also been shown to be very efficient in whole cell catalysis with A. woodii . However, the reverse reaction has not been addressed in detail so far.
In the present report, we describe the first characterization of formate-based H2 production with an organism harboring an HDCR complex. The results show that A. woodii has H2 production rates from formate of 66 mmol H2 g CDW −1 h−1 at ambient temperatures that are among the highest reported so far for an organism without genetic modification. Therefore, A. woodii is an efficient catalyst for H2 production and, considering the reversibility of the whole cell system, a potent catalyst for reversible H2 storage. In addition, A. woodii can grow with formate as sole carbon and energy source making it possible to produce cell mass and H2 with the same substrate.
H2 production with resting cells
H2 production in batch fermentation
In summary, A. woodii and the corresponding enzyme HDCR turned out to be a very promising catalyst for formate-based H2 production and storage, as it operates at ambient temperatures with very similar reaction rates in the forward and reverse reaction. The specific H2 productivity (qH2) from formate observed with whole cells of A. woodii (66 mmol g CDW −1 h−1) is among the highest reported at ambient temperatures for an organism without genetic modification, highlighting the H2 production potential of this organism [4, 5]. Much higher qH2 are reported at 80 °C utilizing the thermophile T. onnurineus . This organism uses a different enzyme system for formate-based H2 production, namely a membrane-bound enzyme complex consisting of a hydrogenase, formate dehydrogenase, and Na+/H+ antiporter subunits . If T. onnurineus can also catalyze, the reverse reaction has not been shown so far. At ambient temperatures, the best results have been achieved using E. coli or other Enterobacteria such as Citrobacter in non-growing conditions . Without genetic modification, E. coli has typically a low formate-dependent H2 productivity. However, by metabolic engineering including overexpression of the formate-hydrogen lyase enzyme, deletion of inhibitory pathways such as uptake hydrogenases and process optimization, the H2 productivity could be increased dramatically (144.2 mmol g−1 h−1 when products was removed continuously from the medium) [27, 28]. On the other hand, E. coli is inhibited by low concentrations of approximately 50 mM formate. This was addressed by using agar-embedded immobilized cells that were able to tolerate higher concentrations .
This study demonstrated that A. woodii is an efficient H2 producer from the very flexible and inexpensive substrate formate. Together with our recent study on the reverse reaction, the results show that A. woodii can also be used as whole cell biocatalyst for the reversible storage of H2, by binding it to CO2 to produce formate and vice versa. Future studies need to address the process in a larger scale and in a continuous fermentation to analyze the stability and investigate alternatives to the expensive inhibitor ETH2120. Since any inhibition of the metabolism that does not affect the HDCR should be sufficient, other inhibitors or a genetic modification of the organism should be easy to find to improve the cost of the process.
Growth of A. woodii
Acetobacterium woodii (DSM 1030) was cultivated at 30 °C under anaerobic conditions. The defined carbonate buffered medium was prepared as described . For closed batch fermentation, defined phosphate buffered medium was used and prepared as described . Fructose (20 mM), formate (100 mM), or H2 + CO2 (80:20 [v/v]) was used as substrates. Growth was followed by measuring the optical density at 600 nm (OD600).
Preparation of cell suspensions
The medium and all buffers were prepared using the anaerobic techniques described [32, 33]. All preparation steps were performed under strictly anaerobic conditions at room temperature in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) filled with 95–98% N2 and 2–5% H2 as described . A. woodii (DSM 1030) was grown in carbonate buffered medium till late exponential phase, harvested by centrifugation, and washed two times with imidazole buffer (50 mM imidazole–HCl, 20 mM MgSO4, 20 mM KCl, 4 mM DTE, 1 mg L−1 resazurin, pH 7.0). Cells were resuspended in imidazole buffer and transferred to Hungate tubes. The protein concentration of the cell suspension was determined as described previously . To remove remaining H2 from the Hungate tube, the gas phase of the cell suspension was changed to N2 and the cells were stored on ice until use. For the experiments, the cells were suspended in the same buffer to a concentration of 1 mg mL−1 in 115-mL glass bottles. The bottles contained a final volume of 10 mL buffer under an N2 atmosphere and were incubated at 30 °C in a shaking water bath. Samples for substrate/product determination were taken with a syringe, cells were removed by centrifugation (15,000g, 2 min), and the supernatant was stored at − 20 °C until further analysis. For determination of H2, gas samples were taken with a gas tight syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) and analyzed by gas chromatography.
Closed batch fermentations
Acetobacterium woodii (DSM 1030) was grown at 30 °C in 50 mL phosphate buffered medium in 115-mL glass bottles containing an initial gas phase of 100% N2. Samples for substrate/product determination were taken with a syringe and handled as described for the cell suspension experiments.
Determination of hydrogen, formate, and acetate
For determination of H2, the gas samples were analyzed by gas chromatography on a Clarus 580 GC (Perkin Elmer, Waltham, USA) with a ShinCarbon ST 80/100 column (2 m × 0.53 mm, PerkinElmer, Waltham, MA, USA). The samples were injected at 100 °C with nitrogen as carrier gas with a head pressure of 400 kPa and a split flow of 30 mL min−1. The oven was kept at 40 °C and H2 was determined with a thermal conductivity detector at 100 °C. The peak areas were proportional to the concentration of H2 and calibrated with standard curves.
The concentration of formate was determined with an enzymatic assay using the formate dehydrogenase from Candida boidinii (Sigma-Aldrich, Munich, Germany). The assay contained in addition to the sample 1 U of enzyme in 50 mM potassium phosphate buffer (pH 7.5) and 2 mM NAD+. Formation of NADH was measured photometrically at 340 nm. Sodium formate was used for preparation of standard curves.
Acetate was measured using a commercially available enzymatic assay kit from R-Biopharm (Darmstadt, Germany).
All chemicals were supplied by Sigma-Aldrich Chemie GmbH (Munich, Germany) and Carl Roth GmbH & Co KG (Karlsruhe, Germany). All gases were supplied by Praxair (Düsseldorf, Germany).
VM and KS designed and supervised the research, analyzed the data, and wrote the manuscript. PK performed the experiments and analyzed the data. All authors read and approved the final manuscript.
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No 741791).
The authors declare that they have no competing interests.
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