Efficient Whole Cell Biocatalyst for Formate-Based Hydrogen Production
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Kottenhahn et al. Biotechnol Biofuels (2018) 11:93 https://doi.org/10.1186/s13068-018-1082-3 Biotechnology for Biofuels RESEARCH Open Access Efcient whole cell biocatalyst for formate‑based hydrogen production Patrick Kottenhahn, Kai Schuchmann and Volker Müller* Abstract Background: Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute signifcantly 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 com- pound 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. Results: 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 spe- 1 1 1 1 cifc formate-dependent H2 production rates of 71 mmol gprotein− h− (30.5 mmol gCDW− h− ) and maximum volumet- 1 1 ric H2 evolution rates of 79 mmol L− h− . Using growing cells in a two-step closed batch fermentation, specifc H 2 1 1 1 1 production rates reached 66 mmol gCDW− h− with a volumetric H2 evolution rate of 7.9 mmol L− h− . 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 specifc switch from a growing phase to a H2 production phase. Conclusions: Acetobacterium woodii reached one of the highest formate-dependent specifc H2 productivity rates at ambient temperatures reported so far for an organism without genetic modifcation 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. Keywords: Hydrogen production, Biohydrogen, Acetobacterium woodii, Formate dehydrogenase, Hydrogenase Background renewable sources [1]. Currently, H2 is produced mainly Fossil fuel limitation and increasing atmospheric CO 2 from fossil fuels by steam reforming and thus unsustain- concentrations necessitate alternative energy carriers. able and environmentally harmful [2]. Hence, new H2 Molecular hydrogen (H2) is an attractive carbon-free production methods are required. alternative that can be converted to energy without CO 2 Biologically produced H2 provides a promising alter- emission. It can be used as energy carrier for mobile native for a sustainable H2-based energy economy. H2 applications (i.e., fuel cell powered vehicles) or as an production by biological systems can generally be classi- intermediate energy storage system to store excess fed into four diferent mechanisms: direct and indirect electrical energy that is produced in peak times from biophotolysis, photofermentation, and dark fermentation [3]. From these processes, the latter mechanism has so far the highest H evolution rates (HER). However, the *Correspondence: [email protected]‑frankfurt.de 2 Molecular Microbiology & Bioenergetics, Institute of Molecular major drawback of dark fermentations, e.g., from glucose, Biosciences, Johann Wolfgang Goethe University, Max‑von‑Laue‑Str. 9, is the low H2 yield per substrate consumed and the limi- 60439 Frankfurt am Main, Germany tations of agricultural production of the substrate [4]. A © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kottenhahn et al. Biotechnol Biofuels (2018) 11:93 Page 2 of 9 recently considered alternative substrate is formic acid/ storage. In addition, A. woodii can grow with formate as formate that could be produced from electrochemical sole carbon and energy source making it possible to pro- reduction of CO 2 or from synthesis gas, a very fexible duce cell mass and H2 with the same substrate. substrate that can derive as by-product from steel mills or from waste gasifcation [5–7]. Conversion of formate Results H production with resting cells to H2 proceeds according to the reaction: 2 − ⇋ − G0′ −1 Te acetogenic bacterium A. woodii can utilize, among HCOO + H2O HCO3 + H2 =+1.3 kJ mol . others, H2 + CO2, formate, or monosaccharides such Microbial formate oxidation is catalyzed by multiple as fructose as substrates for growth. In all three cases, enzyme systems. Organisms such as some enterobacte- acetate (or acetate + CO2 in the case of formate) is the ria use a membrane-bound formate-hydrogen lyase sys- major end product [19, 20]. Recently, we could show tem composed of membrane-associated hydrogenase and that the addition of the sodium ionophore ETH2120 formate dehydrogenase subunits [8, 9]. Clostridiaceae or (sodium ionophore III) led to a complete inhibi- archaea such as Methanococcus can produce H 2 from for- tion of acetate formation from H2 + CO2 and the two mate by the action of separate cytoplasmic formate dehy- gases were completely converted to formate [15]. Tis drogenases and hydrogenases [10]. Te observed HERs opened the possibility to utilize A. woodii as catalyst for these organisms are typically very low and do not for H2 storage. Te hydrogen-dependent CO 2 reduction reach the levels for H 2 production from other feedstocks activity could be addressed to a novel enzyme complex [4]. One exception is the recently characterized organ- of a formate dehydrogenase and hydrogenase, named ism Termococcus onnurineus. Tis organism requires HDCR. Experiments with the purifed enzyme showed 80 °C for growth and formate-dependent H2 formation that the catalyzed reaction proceeds with almost the reached HERs that outcompete other dark fermentations same rate in the reverse reaction as well, making A. for the frst time [11, 12]. H2 production in this organ- woodii a potential candidate for formate-based H 2 ism depends on a membrane-bound enzyme complex of production [15]. In this study, we analyzed this poten- formate dehydrogenase, hydrogenase, and Na +/H+ anti- tial using whole cells of A. woodii. First, we grew the porter subunits that couples H2 formation to formate oxi- organism with fructose, a substrate to reach high cell dation as well as energy conservation [13, 14]. densities relatively quickly (doubling time tD = 4.7 h A new enzyme of the bacterial formate metabolism has compared to 11 h with formate as substrate), harvested been discovered recently in the strictly anaerobic bac- the cells, and incubated them in reaction bufer at a terium Acetobacterium woodii [15]. Te enzyme named protein concentration of 1 mg mL−1 (corresponding −1 hydrogen-dependent CO2 reductase (HDCR) was the to 2.3 mgCDW mL ). After addition of sodium formate frst described soluble enzyme complex that reversibly to a fnal concentration of 300 mM, the cells produced catalyzes the reduction of CO2 to formate with H2 as H2 with an initial specifc H2 productivity (qH2) of −1 −1 −1 −1 electron donor. CO 2 reduction is catalyzed at ambient 52.2 ± 3 mmol gprotein h (22.5 mmol gCDW h ) (Fig. 1). conditions with rates far superior to chemical cataly- 0.6 mmol H2 was produced from 2.14 mmol formate sis [15–17]. Terefore, it could not only be used for H 2 consumed leading to a yield of H 2 consumed per sub- Y −1 production but, depending on the application, for H2 strate consumed (H2/formate) of 0.28 mol mol . It was storage as well. In the form of formate, the explosive gas surprising to observe these high H2 production rates could be stored and handled much easier and with an since H is typically no major product from cells grow- 2 Y increased volumetric energy density [18]. H2-dependent ing on formate; however, (H2/ formate) was signifcantly CO2 reduction to formate by the HDCR has also been decreased by the high amount of 0.45 mmol acetate shown to be very efcient in whole cell catalysis with A. produced alongside H2. Te produced acetate results woodii [15]. However, the reverse reaction has not been from the assimilation of CO 2 or formate via the Wood– addressed in detail so far. Ljungdahl pathway for autotrophic CO2 fxation of A. In the present report, we describe the frst characteri- woodii [20, 21]. As shown recently for the reverse reac- zation of formate-based H2 production with an organism tion of formate formation from H2 + CO2, we tried to harboring an HDCR complex. Te results show that A. decrease acetate formation by addition of the sodium woodii has H2 production rates from formate of 66 mmol ionophore ETH2120. Acetate formation in A. woodii −1 −1 H2 gCDW h at ambient temperatures that are among the is coupled to a sodium ion gradient for energy conser- highest reported so far for an organism without genetic vation across the cytoplasmic membrane that can be modifcation. Terefore, A. woodii is an efcient catalyst specifcally diminished by the sodium ionophore.