Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

Hydrogen productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate

Hydrogen productivities of different photosynthetic bacteria have been searched on real thermophilic dark fermentation effluents (DFE). The results obtained with potato steam peels hydrolysate (PSP) DFE were compared to glucose DFE. Photobiological hydrogen production has been carried out in indoor, batch photobioreactors using several strains of purple non-sulfur (PNS) bacteria such as Rhodobacter capsulatus (DSM1710), Rhodobacter capsulatus hup- (YO3), Rhodobacter sphaeroides O.U.001 (DSM5864), Rb. sphaeroides O.U.001 hup- and Rhodopseudomonas palustris.The efficiency of photofermentation depends highly on the composition of the effluent and the PNS bacterial strain used. Rb. sphaeroides produced the highest amount of hydrogen on glucose DFE. Rb. capsulatus gave better results on PSP DFE. This study demonstrates that photobiological hydrogen production with high efficiency and productivity is possible on thermophilic dark fermentation effluents. Consequently, a sequential operation of dark fermentation and photofermentation is a promising route to produce hydrogen, and it provides a higher hydrogen yield compared to single step processes.     

Replacing incandescent lamps with an LED panel for hydrogen production by photofermentation: Visible and NIR wavelength requirements

Hydrogen production by Rhodobacter capsulatus is a photobiological anaerobic process requiring light as energy source. In this study, the influence of visible and near-infrared (NIR) parts of light spectra from incandescent lamp and LED panels on hydrogen production was investigated. The results showed that the lack of the visible part of the incandescent lamp light spectrum (17% of the lamp light intensity) reduced hydrogen production by 50%. NIR wavelength only partially sustained photofermentation due to light limitation reached at low bacterial concentration. Hydrogen production with NIR light source was only 58% of hydrogen obtained with an incandescent lamp used at the same irradiance. To maximize hydrogen production and flow rate, visible and NIR wavelength should be used concomitantly as light source. Using an energy-efficient LED panel with light spectrum designed to promote photofermentation, hydrogen production and flow rate were equivalent to the ones reached with incandescent lamp as light source.     

Hydrogen production by co-cultures of Clostridium butyricum and Rhodospeudomonas palustris: Optimization of yield using response surface methodology

Both dark and photo-fermentation can be used for biological hydrogen production; either performed separately, in two-stage systems, or in co-culture. A single stage process is less laborious and costly; however, the two types of microorganisms have different nutritional requirements requiring optimization of culture conditions. Here a response surface methodology (RSM) with a Box-Behnken design was used to optimize microorganism ratio and substrate and buffer concentrations, and to evaluate their interactive effects for maximization of hydrogen yield. Clostridium butyricum and Rhodopseudomonas palustris were grown on a potato starch/glucose base medium at 30 °C under continuous illumination (40 W m^?2 light intensity). The highest hydrogen yield, 6.4 ± 1.3 mol H₂/mol glucose, was obtained with a substrate concentration of 15 g/L, buffer concentration of 50 mM, and microorganism ratio of 3. The observed strong interaction between buffer and substrate concentration is most likely due to the need to optimize the pH for co-cultures.     

Non-thermal production of pure hydrogen from biomass: HYVOLUTION

HYVOLUTION is the acronym of an Integrated Project “Non-thermal production of pure hydrogen from biomass” which has been granted in the Sixth EU Framework Programme on Research, Technological Development and Demonstration, Priority 6.1.ii, Sustainable Energy Systems. The aim of HYVOLUTION: “Development of a blue-print for an industrial bioprocess for decentral hydrogen production from locally produced biomass” adds to the number and diversity of _H2${\mathrm{H}}_2$ production routes giving greater security of supply at the local and regional level. Moreover, this IP contributes a complementary strategy to fulfil the increased demand for renewable hydrogen expected in the transition to the Hydrogen Economy. The novel approach in HYVOLUTION is based on a combined bioprocess employing thermophilic and phototrophic bacteria, to provide the highest hydrogen production efficiency in small-scale, cost-effective industries. In HYVOLUTION, 11 EU countries, Turkey and Russia are represented to assemble the critical mass needed to make a breakthrough in cost-effectiveness.     

Effect of arabinose concentration on dark fermentation hydrogen production using different mixed cultures

Dark fermentation hydrogen production from arabinose at concentrations ranging between 0 and 100 g/L was examined in batch assays for three different mixed anaerobic cultures, two suspended sludges (S1, S2) obtained from two different sludge digesters and one granular sludge (G) obtained from a brewery wastewater treatment plant. After elimination of the methanogenic activity by heat treatment, all mixed cultures produced hydrogen, and optimal hydrogen rates and yields were generally observed for concentrations between 10 and 40 g/L of substrate. Higher concentrations of arabinose up to 100 g/L inhibited hydrogen production, although the effect was different from inoculum to inoculum. It was evident that the granular biomass was less affected by increased initial arabinose concentrations when calculating the rate of decrease in hydrogen yields versus arabinose concentrations, compared against the two suspended sludges.     

Biohydrogen production from biomass and industrial wastes by dark fermentation

Hydrogen is a clean energy carrier which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. This article summarizes the dark fermentative biohydrogen production from biomass. Types of potential biomass that could be the source for biohydrogen generation such as food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol are discussed in this article. Moreover, the microorganisms, factors affecting biohydrogen production such as undissociated acid, hydrogen partial pressure and metal ions are also discussed.     

Hydrogen production from glucose by a mutant strain of Rhodovulum sulfidophilum P5 in single-stage photofermentation

Single-stage hydrogen production from glucose was investigated using the marine photosynthetic bacterium Rhodovulum sulfidophilum TH-79, a Tn7 transposon mutant of strain P5. The mutation in strain TH-79 did not affect its cell growth in glucose medium compared with the parent strain. TH-79 displayed improved photoheterotrophic hydrogen production performance when the medium contained glucose or galactose as the sole carbon source. The mutant produced about 7.07 mol H₂/mol glucose, which is similar to the yields of more complicated integration systems. A one-stage photofermentation system using a seawater culture medium appears to be a promising alternative to the integration of dark- and photofermentation systems.     

Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H₂ yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H₂ generation potential (171.3 NL H₂/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.     

Hydrogen production from agricultural waste by dark fermentation: A review

The degradation of the natural environment and the energy crisis are two vital issues for sustainable development worldwide. Hydrogen is considered as one of the most promising candidates as a substitute for fossil fuels. In this context, biological processes are considered as the most environmentally friendly alternatives for satisfying future hydrogen demands. In particular, biohydrogen production from agricultural waste is very advantageous since agri-wastes are abundant, cheap, renewable and highly biodegradable. Considering that such wastes are complex substrates and can be degraded biologically by complex microbial ecosystems, the present paper focuses on dark fermentation as a key technology for producing hydrogen from crop residues, livestock waste and food waste. In this review, recent findings on biohydrogen production from agricultural wastes by dark fermentation are reported. Key operational parameters such as pH, partial pressure, temperature and microbial actors are discussed to facilitate further research in this domain.     

Photofermentative hydrogen production using dark fermentation effluent of sugar beet thick juice in outdoor conditions

In the present study, photofermentative hydrogen production on thermophilic dark fermentation effluent (DFE) of sugar beet thick juice was investigated in a solar fed-batch panel photobioreactor (PBR) using Rhodobacter capsulatus YO3 (hup⁻) during summer 2009 in Ankara, Turkey. The DFE was obtained by continuous dark fermentation of sugar beet thick juice by extreme thermophile Caldicellulosiruptor saccharolyticus and it contains acetate (125 mM) and NH₄⁺ (7.7 mM) as the main carbon and nitrogen sources, respectively. The photofermentation process was done in a 4 L plexiglas panel PBR which was daily fed at a rate of 10% of the PBR volume. The DFE was diluted 3 times to adjust the acetate concentration to approximately 40 mM and supplemented with potassium phosphate buffer, Fe and Mo. In order to control the temperature, cooling was provided by recirculating chilled water through a tubing inside the reactor. Hydrogen productivity of 1.12 mmol/L_c/h and molar yield of 77% of theoretical maximum over consumed substrate were attained over 15 days of operation. The results indicated that Rb. capsulatus YO3 could effectively utilize the DFE of sugar beet thick juice for growth and hydrogen production, therefore facilitating the integration of the dark and photo-fermentation processes for sustainable biohydrogen production.     

Dark and photofermentation H2 production from hydrolyzed biomass of the potent extracellular polysaccharides producing cyanobacterium Nostoc commune and intracellular polysaccharide (glycogen) enriched Anabaena variabilis NIES-2095

The biological H₂ production industry would be independent from other industries if it has its own supply of organic materials especially in non-agricultural countries. In this study, acid hydrolyzed biomass of the potent extracellular polysaccharides (EPSs) producing cyanobacterium Nostoc commune and glycogen (as intracellular polysaccharide) enriched Anabaena variabilis NIES-2095 were used as a cheap organic carbon feedstock for biological H₂ production by two stages dark fermentation by Escherichia coli strain MWW and Clostridium acetobutylicum DSM-792 or Clostridium beijerinckii DSM-1820 and photofermentation by Rhodobacter capsulatus JCM-21090 under anaerobic conditions. Acid hydrolysis of air dried cyanobacterial biomass was conducted at optimum conditions of 4 M HCl at 120 °C in an autoclave for 30 min and subsequently neutralized and used as an organic carbon source for first stage dark fermentation followed by a second stage photofermentation. The facultative anaerobe Escherichia coli strain MWW was used for maintaining anaerobiosis. Escherichia coli strain MWW was isolated and identified by morphological and biochemical characterizations as well as molecular biological phylogenetic analysis of its 16S rDNA sequence. Nostoc commune was identified by morphological and microscopic characterizations and by 16S rDNA sequence phylogenetic analysis. The two stages dark fermentation by Escherichia coli and Clostridium acetobutylicum or Clostridium beijerinckii and photofermentation by Rhodobacter capsulatus produced in total 5.9 and 5.6 mol H₂/mole reducing sugars of acid hydrolyzed Nostoc commune EPSs/biomass, respectively and 5.43 and 5 mol H₂/mole reducing sugars of acid hydrolyzed biomass of glycogen enriched Anabaena variabilis, respectively. These results indicate a high potency of using cyanobacterial polysaccharides/biomass (extracellular polysaccharides and intracellular glycogen) as an organic carbon source for H₂ production which would be of importance for non-agricultural countries.     

Hydrogen rich fuel gas production by gasification of wet biomass using a CO2 sorbent

Hydrogen rich fuel gas production by gasification of wet biomass accompanied by CO₂ absorption is proposed. The paper addressed this topic, and experiments were conducted to investigate the effects of the moisture content (M), the molar ratio of Ca(OH)₂ to carbon in the biomass ([Ca]/[C]) and the reactor temperature (T) on hydrogen production and CO₂ absorption by CaO. Measurement of the calcium compounds in solid residues was carried out with XRD and SEM. The results show that directly gasifying of wet biomass not only favors hydrogen production but also promotes CO₂ absorption by CaO. For the experiment with wet biomass (M = 0.90), the H₂ yield is increased by 51.5% while the CO₂ content is decreased by 28.4% than that for experiments with dry biomass (M = 0.09). CaO plays the dual role of catalyst and sorbent. It is noteworthy that CaO reveals a stronger effect on the water gas shift reaction than on the steam reforming of methane. The increase of the reactor temperature contributes to produce more H₂, but goes against CO₂ absorption by CaO. XRD spectrum and SEM image of the solid residues further confirmed that high temperature is unfavorable to CO₂ absorption by CaO. For the new method, the optimal operating temperature is in the 923–973 K range.     

Optimization of culture conditions for biohydrogen production from sago wastewater by Enterobacter aerogenes using Response Surface Methodology

Sago wastewater (SWW) causes pollution to the environment due to its high organic content. Annually, about 2.5 million tons of SWW is produced in Malaysia. In this study, the potential of SWW as a substrate for biohydrogen production by Enterobacter aerogenes (E. aerogenes) was evaluated. Response Surface Methodology (RSM) was employed to find the optimum conditions. From preliminary optimization, it was found that the most significant factors were yeast extract, temperature, and inoculum size. According to Face Centered Central Composite Design (FCCCD), the maximum hydrogen concentration and yield were 630.67 μmol/L and 7.42 mmol H₂/mol glucose, respectively, which is obtained from the sample supplemented with 4.8 g/L yeast extract concentration, 5% inoculum, and incubated at the temperature of 31 °C. Cumulative hydrogen production curve fitted by the modified Gompertz equation suggested that H_max, R_max, and λ from this study were 15.10 mL, 2.18 mL/h, and 9.84 h, respectively.     

Modeling of biohydrogen production using generalized multi-scale kinetic model: Impacts of fermentation conditions

This paper presents a new multi-scale kinetic model built upon the multi-stage growth Hypothesis for predicting biohydrogen production. The proposed model represents the significant factors affecting biohydrogen production using a sum of first-order kinetic terms with varying dynamics from slow to fast one. The current work investigates 52 case studies of biohydrogen production that show the double first-order kinetic model provides the best modeling fitness (R² > 0.99). This result suggests two prevalent pathways or microbial groups with distinct dynamics (i.e., fast and slow modes) in biohydrogen production. An increase in temperature (30 °C–43 °C) or substrate concentration (10 g/L to 40 g/L) and the use of simple substrates or mixed cultures can increase the fast-mode dominance up to 100% contribution. Model analysis suggests that the fast mode corresponds to the butyrate production pathway, the growth-associated hydrogen-producing activity, the easily-biodegradable substrates, or the quick hydrogen-producing groups.     

Hydrogen production by Chlamydomonas reinhardtii under light-driven and sulfur-deprived conditions: Using biomass grown in outdoor photobioreactors at the Yucatan Peninsula

Photosynthesis is the ultimate natural process that supports all the biofuels generation. Photosyntetic production of hydrogen by microalgae is very attractive from the renewability point of view. Moreover, it faces several challenges: since the process itself has a low yield, a large number of considerations should be studied to optimize the hydrogen production at the lowest cost. In this work, wild-type Chlamydomonas reinhartii was grown outdoors in the Yucatán peninsula. Three different diameters of tubular photobioreactors (PBRs), two autotrophic culture media, as well as two seasons of the year were analyzed. From these variables, it was determined that the best biomass yield was during the winter season and with the Sueoka culture medium. Statistical significance differences were not found for the diameters of the PBRs. During growth, the biomass was exposed to natural light–dark cycles and at the end of the exponential phase of growth it was harvested with superabsorbent polymers. This biomass was able to produce hydrogen under anaerobic conditions in Tris-Acetate-Phosphate culture medium in indoor PBRs exposed to continuous artificial illumination. Experiments with different initial biomass concentrations in the anaerobic PBRs showed direct relationship with the hydrogen production profile.     

Continuous dark fermentative hydrogen production by mesophilic microflora: Principles and progress

Continuous, dark fermentative hydrogen production technology using mixed microflora at mesophilic temperatures may be suitable for commercial development. Clostridial-based cultures from natural sources have been widely used, but more information on the need for heat treatment of inocula and conditions leading to germination and sporulation are required. The amount of nutrients given in the literature vary widely. Hydrogen production is reported to proceed without methane production in the reactor in the pH range 4.5–6.7, with hydraulic retention times optimally between a few hours and 3 days depending on substrate. Higher substrate concentrations should be more energy-efficient but there are product inhibition limitations, for example from unionised butyric acid. Inhibition by H₂ can be reduced by stirring, sparging or extraction through membranes. Of the reactor types investigated, while granules have the best performance with soluble substrate, for particulate feedstock biofilm reactors or continuous stirred tank reactors may be most successful. A second stage is required to utilise the fermentation end products which, when cost-effective reactors are developed, may be photofermentation or microbial fuel cell technologies. Anaerobic digestion is a currently-available technology and the two-stage process is reported to give greater conversion efficiency than anaerobic digestion alone.     

Kinetics of cotton cellulose hydrolysis using concentrated acid and fermentative hydrogen production from hydrolysate

The kinetics of cotton cellulose hydrolysis using concentrated sulfuric acid and the performance of fermentative hydrogen production from the hydrolysate in the batch system was carried out in this study. Effects of sulfuric acid concentrations, cotton cellulose concentrations and operating temperatures on the cotton cellulose hydrolysis were investigated. It was found that cotton cellulose can dissolve completely in sulfuric acid concentration above 55% (by volume) at room temperature. The reduced sugar yields were varied from 64.3 to 73.9% (g R-sugar/g cotton cellulose) with the initial cotton cellulose concentrations of 30-70 g/L at a temperature of 40 °C.The reduced sugar concentrations and the initial pH of biohydrogen production were investigated at 37 °C. It was found that the optimal values of the hydrogen yield and substrate utilization were 0.95 mol H₂/mol R-sugar and 98% with an initial pH of 8.2, when substrate concentration was fixed at 20 g R-sugar/L. The maximum hydrogen yield was 0.99 mol H₂/mol R-sugar at a substrate concentration of 15 g R-sugar/L. Using the Gompertz Equation Model simulation, the maximum hydrogen production rate was 253 mL H₂/h/L at a substrate of 30 g/L and initial pH of 8.4.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.     

Hydrogen production characteristics from dark fermentation of maltose by an isolated strain F.P 01

A hydrogen producing strain F.P 01 was newly isolated from cow dung sludge in an anaerobic bioreactor. The strain F.P 01 was a mesophilic and facultative anaerobic bacterium, which exhibited gram-negative staining in both the exponential and stationary growth phases, and a regular long rod-shaped bacteria with the size of 0.6–0.9 μm × 1.2–2.5 μm, and also could biodegrade a variety of carbohydrates such as glucose, xylose, maltose, etc. The effects of important process parameters on hydrogen producing of F.P 01 were further investigated from hydrogen fermentation of maltose by strain F.P 01, including substrate concentration, medium pH, etc. And the results showed that hydrogen production potential and hydrogen production rate from maltose of this strain F.P 01 was180 mLH₂/g-maltose and 4.0 mLH₂/h, respectively. The corresponding hydrogen concentration of 58–73% was also be observed. Both butyric acid and acetic acid as main by-product was left in the reactor.