Sequential dark and photo-fermentative hydrogen gas production from agar embedded molasses

Highlights

• Molasses and dark fermentation effluent were solidified using agar for H₂ production.

• Slow-release of sugar in DF enabled efficient H₂ production.

• TVFA release in photo-fermentation from the solid matrix was not convenient.

• Embedding of molasses enabled to start DF with 36.2 g total sugar/L.

• Yields of DF and PF were 226.24 mL H₂/g TS and 870.26 mL H₂/g TVFA, respectively.

Abstract

In this study, molasses and dark fermentation effluent were solidified using agar and used for H₂ production by dark and photo-fermentation. During dark fermentation, the solid jelly form of molasses enabled a slow release of the substrate to the liquid broth hindering fast pH decreases. The initial total sugar concentration, H₂ yield, H₂ rate and lag phase in dark fermentation were 36.2 g/L, 226.24 mL H₂/g TS, 29.85 mL H₂/h and 4.37 h, respectively. Photo-fermentation of 5.77 g TVFA/L embedded dark fermentation effluent did not lead to efficient H₂ production. The best performance in photo-fermentation was obtained with 1.55 g TVFA/L containing diluted dark fermentation effluent. The H₂ yield, H₂ rate and lag phase in photo-fermentation were 870.26 mL H₂/g TVFA, 0.913 mL H₂/h and 54.07 h, respectively. Embedding concentrated substrate using agar can enhance H₂ production performance but only if the release of the substrate does not exceed inhibitory levels and if the rate of diffusion is tolerable for microbial activity.

Introduction

Global warming due to greenhouse gas emissions (GHGEs) is one of the most important problems that our planet is facing today [1]. And fossil fuel combustion is addressed to be one of the main contributors to the release of GHGEs [[2], [3], [4], [5]]. Therefore there is an essential need for finding renewable energy resources that could provide sustainable and clean energy [6]. Among many options, Hydrogen gas (H₂) is foreseen to have a wide use as a fuel in the future due to its promising futures such as high energy content per mass, effective use in fuel cells, no GHGEs and vast availability [7]. Even, hydrogen is the amplest element [7] its presence in the form of H₂ is not more than 1% in the atmosphere [8]. Therefore, H₂ is produced in the industry by chemical processes [9]. Most of the H₂ in the market is produced from natural gas by steam reforming which is followed by gasification and water electrolysis [10,11]. On the other hand, H₂ production from wastes by microbial routes is gaining attention during the last decades [[12], [13], [14], [15], [16], [17]]. However, the microbial route is not able to compete with conventional technologies because of microbial constraints [18,19]. The known microbial H₂ production methods are dark fermentation of carbohydrates, photo-fermentation of volatile fatty acids and water splitting by algae [[20], [21], [22]]. The last is the most promising because of the immense availability of water and the formation of only oxygen and hydrogen [23]. However, the enzymatic activity of algal water splitting is inhibited by the oxygen that is formed [24,25]. Dark fermentation is the fastest among the others but the hydrogen embedded in carbohydrates can not completely be transformed into H₂ instead some of it is stored in volatile fatty acids in the broth medium [26]. On the other hand, photo-heterotrophic bacteria have the ability to convert volatile fatty acids into H₂ and CO₂ under light illumination [[26], [27], [28], [29]]. Therefore, the integration of dark and photo-fermentation in a combined or sequential mode of operation promises a whole conversion of carbohydrates into H₂ and CO₂ [21,30]. However, in practice, this conversion occurs with lower yields than expected [31]. Theoretically, it is possible to get 4 and 2 mol of H₂ and acetic acid, respectively per mole glucose in dark fermentation [7]. And a further step in photo-fermentation of 2 mol acetic acid allows getting 8 mol of H₂. So, the overall process leads to 12 mol of H₂ per mole glucose [32].

Dark fermentation can be accomplished using pure or mixed cultures [33]. The most known microbial consortia are spore-forming Clostridia species having the ability to dissipate excess protons from the cell by hydrogenase enzymes [34]. The process requires strictly anaerobic conditions [35] and a controlled pH around 5.5–6.5 [[36], [37], [38]]. Different types of carbohydrates can be used as substrate and the fermentation efficiency depends on the availability of readily decomposable sugar monomers [[39], [40], [41], [42]]. Most of the H₂ producers prefer glucose as a carbon source. However, glucose is not always present for microbial utilization and needs to be made available by a proper pre-treatment of poly-saccharides [43]. Substrate pre-treatment can be accomplished enzymatically or chemically [21]. The enzymatic route does not produce toxic by-products but it is slow and expensive. The latter is faster and cheaper but suffers mostly from toxic by-product formation [44,45] and requirements of excessive energy, neutralization and special reactors difficult to be operated at an industrial scale [46]. Other factors affecting the efficient conversion of the substrate into H₂ and CO₂ in dark fermentation are substrate and product inhibitions [47,48]. The initial sugar concentration is usually kept less than 20 g glucose/L due to the fast formation of VFA and sharp pH decreases shifting the metabolism towards solvent production [34,47,49]. Moreover, more water is needed to dilute wastewater streams to ensure low substrate concentration.

Anoxygenic Rhodobacter species are the most known photo-fermentation bacteria [50]. The media needs to be illuminated at 400–950 nm due to positive Gibbs free energy value of VFA to H₂ and CO₂ conversion reaction [51]. Neutral pH ensures more efficient H₂ production in photo-fermentation [52]. The responsible enzyme for H₂ production is nitrogenase which requires an ammonia deficient media [50]. If the ammonia concentration is more than 47 mg/L then the bacteria shift their metabolism for growth instead of H₂ production [53]. VFA concentration between 1.5 and 2.3 g VFA/L is reported to be suitable [53]. Therefore ammonia and VFA concentrations need to be adjusted accordingly for efficient H₂ formation. Moreover, the broth is essential to be supplemented with proper micronutrients [54]. Turbidity due to microbial growth creates a shading effect that is to be considered to prevent light deficiency [50,55].

If H₂ is intended to be produced from waste streams then all the above-mentioned factors along with waste disposal criteria need to be considered, accordingly. The integration of photo-fermentation to dark fermentation offers to evaluate the VFA containing effluent for H₂ production [56] and thus completing the task for water purification. Otherwise, VFA containing dark fermentation effluent is still wastewater that needs purification.

In this study, it was aimed to investigate the possibility to start dark and photo-fermentation at higher initial substrate concentrations than the state of the art values by embedding the substrate into a solid matrix using agar. This approach enabled the bacteria to use the substrate slowly as needed instead of direct contact with high substrate concentration in the liquid limiting microbial activity and allowing sharp pH decreases.