Hydrogen Production by the Hyperthermophilic Bacterium
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Boileau et al. Biotechnol Biofuels (2016) 9:269 DOI 10.1186/s13068-016-0678-8 Biotechnology for Biofuels RESEARCH Open Access Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth Céline Boileau, Richard Auria, Sylvain Davidson, Laurence Casalot, Pierre Christen, Pierre‑Pol Liebgott and Yannick Combet‑Blanc* Abstract Background: Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H2/glucose yields. However, to develop economically sustainable bio-processes, the culture medium for‑ mulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen. Results: Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glu‑ 1 1 cose 60 mmol L− and yeast extract 1 g L− ), the cellular growth was limited by thiosulfate concentrations lower 1 than 0.06 mmol L− . Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 1 3617 mg mmol− . In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysac‑ charides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium signifi‑ cantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production. Conclusions: The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present 1 at low concentrations (about 0.3 mmol g− of cells), and (2) as both sulfur source and detoxifying agent for H2 when thiosulfate is present at higher concentrations and, when, simultaneously, the pH2 is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosul‑ fate in the culture medium. Keywords: Thermotoga maritima, Hydrogen, Thiosulfate, Productivity, Growth, Yields, Sulfured nutriments, Glucose, Metabolism *Correspondence: yannick.combet‑[email protected] Aix Marseille Université, CNRS, Université de Toulon, IRD, MIO UM 110, 13288 Marseille, France © The Author(s) 2016. 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. Boileau et al. Biotechnol Biofuels (2016) 9:269 Page 2 of 17 Background be preserved. The use of marine microorganisms, which Today, the amount of energy derived from fossils fuels produce hydrogen in seawater from a wide variety of sug- (petroleum, natural gas, and coal) represents about ars, appears, in this case, to be a promising approach. 80% of the world energy consumption. It is hence- Thermotoga spp. are hyperthermophile or thermophile forth recognized that their use has induced very seri- fermentative anaerobic bacteria belonging to a deep- ous environmental pollutions. The accumulation of the branching lineage within the domain Bacteria [31–34]. greenhouse-gas carbon dioxide in the atmosphere and They inhabit various hot ecosystems, including hot the depletion of fossil fuels, altogether with the high springs, hydrothermal vents, and oil reservoirs [35–37]. prices and the ever-increasing demand have forced most Among Thermotoga species, T. maritima, originally iso- of the countries to start looking for cleaner and renew- lated from a geothermal-heated marine sediment at able energy sources. Therefore, major research efforts Vulcano, Italy [32], has received considerable interest as focusing on solar and wind energy, geothermal resources, potential hydrogen producer [38]. Indeed, T. maritima and energy derived from biomass were undertaken to is able to produce hydrogen with high productivity and develop new technologies suitable for industrial use. yield [29, 39] from a wide variety of sugars, ranging from In this context, although dihydrogen (H2) is not a pri- hexose and pentose monomers to starch and xylan poly- mary energy source, it is currently seen as a very prom- mers [40]. In addition, compared to the other hydrogen- ising carbonless “energy carrier” which may be used to producing microorganisms, Thermotogales, including store energy and provide an efficient alternative to fossil T. maritima, exhibit the highest H2 yields, close to the fuels. Up to now, most of the H2 is currently industri- Thauer limit (4 mol H2 per mol glucose). For instance, a ally produced by steam reforming of natural gas or by yield of 4 mol of H2 per mol of glucose has been reported alternative processes based on electrolysis or thermoly- by Schöder et al. [39] when T. maritima growth was sis of water [1]. However, in the last decade, biological limited by glucose, the energy source, and under very processes employing bacteria for H2 production have low hydrogen partial pressure (down to 1.3% as hydro- received a significant and increasing attention [2–11]. gen partial pressure). Actually, T. maritima harvests Indeed, like with other biofuels such as ethanol, butanol, energy by glycolysis via the Embden–Meyerhof pathway fatty acids, and methane, bio-hydrogen can be produced (EMP) as the main route and via the Entner–Doudor- by processes using living organisms such as green algae off (ED) pathway to a lesser extent (about 85 and 15% of (photolysis of water), phototrophic and anaerobic micro- consumed glucose, respectively) [39, 41]. The ultimate organisms (photofermentation of organic acids), and pyruvate-reduction steps resulted in acetate, H2 and CO2 anaerobic fermentative microorganisms (dark fermen- (1:2:1 as molar proportions, respectively) as major end- tation of organic substrates) [2, 7, 8, 11–19]. Among products of glucose fermentation, and in lactate, alanine, these, due to the large spectrum of catabolic activities and extracellular polysaccharides (EPS) as minor end- of H2-producing microorganisms, dark fermentation is products. The production of these fermentation prod- considered as one of the most promising route. The fer- ucts was shown to depend on culture conditions (culture mentation processes of these microorganisms poten- medium, nutritional or oxidative stress, operating condi- tially allow producing hydrogen from renewable energy tions such as pH, pH2, stirring, Eh,…) [35, 39, 42–46]. In sources derived from biomass or various carbohydrate- agreement with this classical fermentation model, the H2 rich waste streams [2, 20–26]. yield is optimal (4 H2:2 CO2:2 acetate as molar propor- Depending on the microorganism species used, dark tions per mol of glucose or other hexose) only when all fermentations can be performed at either moderate or the glucose is converted to acetate because the lactate, elevated temperature. In the former case, hydrogen pro- alanine, and EPS productions are bypassing pathways ductivities (QH2/time) were generally higher whereas impairing the H2 production. Furthermore, it has been higher yields (YH2/substrate) were reached in the latter reported in the literature that Thermotogales reduced [4]. Moreover, the advantages of high versus low tem- sulfur-containing compounds such as elemental sulfur, peratures for the bio-hydrogen production include bet- polysulfide, and thiosulfate to hydrogen sulfide [47–49]. ter pathogen destruction, reduced risks of methanogen When T. maritima and T. neapolitana, two very closely or acetogen growth (hydrogen consuming), and less related species [50], were cultivated in the presence of sensitivity of hyperthermophiles to the H2 partial pres- elemental sulfur, and with glucose as energy source, final sure [27–30]. However, in order to develop economically cell yields were significantly enhanced but their growth sustainable hydrogen-producing bio-processes, both pro- rate remained unaffected [39, 51, 52]. In addition, it has ductivities and yields should be significantly increased. been shown that, in the presence of sulfur, glucose was Another economic aspect for these bio-processes is the not more efficiently used. Indeed fermentation–carbon– high fresh water requirement, a resource which needs to product pattern remained similar (e.g., acetate and CO2 Boileau et al. Biotechnol Biofuels (2016) 9:269 Page 3 of 17 as major fermentation