Biohydrogen production from barley straw hydrolysate through sequential dark and photofermentation

Highlights

• Photobiological H₂ production on barley straw dark fermenter effluent was realized.

• High efficiency biohydrogen production is possible on the lignocellulosic feedstock.

• Fe and Mo addition improves photofermentative hydrogen production by Rhodobacter capsulatus.

• Omitting yeast extract from dark fermentation does not affect photofermentation.

• The highest H₂ production of 0.58 mmol/(L_c·h) was achieved with R. capsulatus YO3.

Abstract

Biohydrogen production by sequential operation of dark and photo-fermentation processes is a promising method to produce hydrogen from renewable resources, in a sustainable way. In this study, barley straw hydrolysate (BSH) dark fermenter effluent (DFE) was used as the biomass feedstock for biohydrogen production through photofermentation. Two different dark fermentation effluents were obtained by performing fermentation with or without addition of yeast extract (YE), using hyperthermophilic dark fermentative bacteria, Caldicellusiruptor saccharolyticus. Photofermentative hydrogen production on BSH DFEs was carried out using purple non-sulfur (PNS) bacterial strains Rhodobacter capsulatus DSM1710 and Rhodobacter capsulatus YO3 (an uptake hydrogenase deleted MT1131 strain). The effects of YE addition during dark fermentation and Fe/Mo supplementation to DFEs on subsequent photofermentation were investigated. Presence of YE in DFEs did not influence the photofermentative hydrogen production. Fe/Mo supplementation of the effluents improved the H₂ production on both DFEs, regardless of the strain used. The highest productivity of 0.58 mmol/(L_c·h) was achieved by R. capsulatus YO3 on Fe/Mo supplemented BSH DFE that had no YE. Biohydrogen production on BSH DFE was achieved at a high rate and yield, showing that it can be used as a feedstock for photofermentative hydrogen production.

Introduction

Increasing demand in energy and rapid exhaustion of fossil fuel reserves highly accelerated the research in renewable energy sources. Provided that it is produced in a renewable way, hydrogen is a good candidate as a clean energy carrier of the future, since it has a high energy density per weight and produces only water upon combustion.

Renewable hydrogen production can be realized biologically from biomass feedstock using fermentative microorganisms. Dark and photo-fermentative bacteria are utilized for biohydrogen production from various biomass resources including agro-industrial wastes. Dark fermentative bacteria can produce hydrogen at a high rate from various biomass resources through anaerobic degradation of carbohydrates, and liberates organic acids as end products. The hydrogen generation is mainly attributed to hydrogenase enzyme, which reversibly catalyzes reduction of protons. However, due to incomplete degradation of carbohydrates to CO₂ and H₂O, the yield of dark fermentative hydrogen production process is low; from a six-carbon sugar only one third of theoretical hydrogen yield can be achieved. Photofermentative PNS bacteria produce hydrogen under anaerobic, nitrogen-limited conditions using light as energy source, and organic acids as electron donors. Hydrogen production is catalyzed by nitrogenase enzyme, which is active anaerobically under nitrogen limitation. In the absence of N₂, the enzyme catalyzes the H₂ production by reduction of protons, at the expense of 4 ATP (Eq. (1)).

$$2{\text{H}}^{+}+2{\text{e}}^{-}+4\text{ATP}\to {\text{H}}_2+4\text{ADP}+{\text{P}}_i$$

Purple non-sulfur bacteria (PNS) bacteria also exhibit a membrane-bound uptake hydrogenase, which reversibly catalyzes the conversion of H₂ into protons and electrons. For this reason, uptake hydrogenase deleted (hup-) strains of PNS bacteria were developed for various hydrogen producing strains (Zhu et al., 2006; Kars et al., 2008; Öztürk et al., 2006). Although the rate of photofermentative hydrogen production is slow compared to dark fermentation, photofermentation offers high hydrogen yields on various organic acids including acetate (Asada et al., 2008; Özgür et al., 2010a), lactate, malate (Barbosa et al., 2001), butyrate (Su et al., 2009), propionate, formate and mixtures of them (Shi and Yu, 2006; Uyar et al., 2009a).

Coupled operation of dark and photo-fermentation has long been recognized as a promising route to increase the overall yield of biohydrogen production from biomass. Dark fermentation effluents (DFEs), which contain high concentrations of organic acids such as acetate, lactate and propionate, can be readily used as substrates for photofermentative bacteria. An overall yield of 9 mol H₂/mol hexose can be predicted based on 75% substrate conversion efficiency. Using this strategy, biohydrogen productions from glucose (Nath et al., 2005; Redwood and Macaskie, 2006), sucrose (Tao et al., 2007) and from various agricultural wastes, including sugar beet molasses (Özgür et al., 2010b), potato steam peels hydrolysate (Afsar et al., 2011), cheese whey (Azbar et al., 2009), ground wheat starch (Ozmihci and Kargi, 2010), cassava and food waste (Zong et al., 2009), miscanthus hydrolysate (Uyar et al., 2009b) have been documented in batch cultures. Continuous operations of photofermentative hydrogen production on dark fermentation effluents of molasses (Avcioglu et al., 2011) and thick juice (Boran et al., 2012) have also been reported.

Barley straw is a lignocellulosic agricultural residue of barley production. It has a dry matter content of 91.1%: 38.9% being glucan, 23.7% xylan, 3.5% other hemicelluloses and 22.8% lignin (Foglia et al., 2011). The annual barley straw production was estimated to be 114 million tons in Europe, which corresponds to 511 TWh, assuming a lower heating value of 16.3 MJ/kg dry matter (Ljunggren et al., 2011). It has been considered as a feedstock for biofuel production through biochemical processes in several studies (Qureshi et al., 2010; Sigurbjornsdottir and Orlygsson, 2012). A techno-economical analysis on the integrated biohydrogen production on barley straw hydrolysate (BSH) in comparison with bioethanol production has been carried out (Ljunggren et al., 2011). The authors reported that, with current technologies, biohydrogen production is not a competitive process to bioethanol production. However, biohydrogen production is not a mature process and open to major improvements in terms of bacterial strain and bioreactor developments, which will lead to decreased cost. Given the superior properties of biohydrogen in terms of its environmental benefits and suitability for fuel cell applications, research efforts on biohydrogen production should continue.

Within the context of FP6 HYVOLUTION (Non-Thermal Production of Pure Hydrogen from Biomass) project (Claassen et al., 2010), we have investigated the photofermentative hydrogen production by PNS bacterial strain Rhodobacter capsulatus DSM1710 and Rhodobacter capsulatus YO3 (a hup-mutant of MT1131) on BSH DFE. Dark fermentation was carried out on alkaline-pretreated BSH using hyperthermophilic dark fermentative bacteria, Caldicellulosiruptor saccharolyticus, which is known for its very high hydrogen yield (de Vrije et al., 2007). One of the problems in integrating dark and photofermentation is the optimization of operational conditions of photofermentative bacteria on DFEs. The addition of NH₄Cl during dark fermentation represents one of the obstacles for the integration of two processes, since ${\text{NH}}_4^{+}$ is the feedback inhibitor of the nitrogenase enzyme, which catalyzes the hydrogen production in PNS bacteria. Nutrient requirement, need for dilution or centrifugation, and adjustment of ammonia concentration have to be considered for optimal efficiency. Dark fermentation broths usually contain yeast extract (YE) to stimulate the bacterial growth on defined media. However, real feedstocks like molasses, thick juice and barley straw are by themselves rich in nutrients. Hence, YE can be omitted from dark fermentation to reduce the cost. However, presence of YE in DFEs may have stimulatory effect on subsequent photofermentation (Oh et al., 2004; Chen et al., 2010). For this reason, DFEs with or without YE supplementation were prepared, and experiments were carried out using both effluents.