Effects of sludge pre-treatment method on bio-hydrogen production by dark fermentation of waste ground wheat

Three different pre-treatment methods were applied on two different anaerobic sludge cultures and their mixtures in order to investigate the effects of pre-treatment methods on bio-hydrogen production from dark fermentation of waste ground wheat solution. Repeated heat, chloroform and combinations of heat and chloroform pre-treatment methods were applied to anaerobic sludges from different sources. Repeated heat treatment (2 × 5 h) was found to be more effective in selecting hydrogen producing bacteria compared to the other treatment methods tested on the basis of cumulative hydrogen production. The highest hydrogen formation (652 ml) and specific hydrogen production rate (SHPR = 25.7 ml H₂ g⁻¹ cells h⁻¹) were obtained with the anaerobic sludge pre-treated by repeated boiling. Both the type of anaerobic sludge and the pre-treatment method had considerable effects on bio-hydrogen production from wheat powder solution (WPS) by dark fermentation.     

Effects of the substrate and cell concentration on bio-hydrogen production from ground wheat by combined dark and photo-fermentation

Effects of the substrate and cell concentration on bio-hydrogen production from ground wheat solution were investigated in combined dark-light fermentations. The ratio of the dark to light bacteria concentration (D/L) was kept constant at 1/10 while the wheat powder (WP) concentration was changed between 2.5 and 20 g L⁻¹ with a total cell concentration of 0.41 g L⁻¹ in the first set of experiments. Cell concentration was changed between 0.5 and 5 g L⁻¹ in the second set of experiments while the wheat powder concentration was constant at 5 g L⁻¹ with a D/L ratio of 1/7. The highest cumulative hydrogen (135 ml) and formation rate (3.44 ml H₂ h⁻¹) were obtained with the 20 g L⁻¹ wheat powder concentration. However, the highest yield (63.9 ml g⁻¹ starch) was obtained with the 2.5 g L⁻¹ wheat powder. In variable cell concentration experiments, the highest cumulative hydrogen (118 ml) and yield (156.8 ml H₂ g⁻¹ starch) were obtained with 1.1 g L⁻¹ cell concentration yielding an optimal biomass/substrate ratio of 0.22 g cells/g WP.     

Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation

Hydrogen formation performances of different anaerobic bacteria were investigated in batch dark fermentation of waste wheat powder solution (WPS). Serum bottles containing wheat powder were inoculated with pure cultures of Clostridium acetobutylicum (CAB), Clostridium butyricum (CB), Enterobacter aerogenes (EA), heat-treated anaerobic sludge (ANS) and a mixture of those cultures (MIX). Cumulative hydrogen formation (CHF), hydrogen yield (HY) and specific hydrogen production rate (SHPR) were determined for every culture. The heat-treated anaerobic sludge was found to be the most effective culture with a cumulative hydrogen formation of 560 ml, hydrogen yield of 223 ml H₂ g⁻¹ starch and a specific hydrogen production rate of 32.1 ml H₂ g⁻¹ h⁻¹.     

Bio-hydrogen production from acid hydrolyzed waste ground wheat by dark fermentation

Waste ground wheat was subjected to acid hydrolysis (pH = 3.0) at 90 °C for 15 min using an autoclave. The sugar solution obtained from acid hydrolysis was subjected to dark fermentation for hydrogen gas production after neutralization. In the first set of experiments, initial total sugar concentration was varied between 3.9 and 27.5 g L⁻¹ at constant biomass (cell) concentration of 1.3 g L⁻¹. Biomass concentration was varied between 0.28 g L⁻¹ and 1.38 g L⁻¹ at initial total sugar concentration of 7.2 ± 0.2 g L⁻¹ in the second set of experiments. The highest hydrogen yield (1.46 mol H₂ mol⁻¹ glucose) and the specific formation rate (83.6 ml H₂ g⁻¹ cell h⁻¹) were obtained with 10 g L⁻¹ initial total sugar concentration. Biomass (cell) concentration affected the specific hydrogen production rate yielding the highest rate (1221 ml H₂ g⁻¹ cell h⁻¹) and the yield at the lowest (0.28 g L⁻¹) initial biomass concentration. The most suitable X_o/S_o ratio, maximizing the yield and specific rate of hydrogen gas formation was X_o/S_o = 0.037. Dark fermentation of acid hydrolyzed ground wheat was found to be more beneficial as compared to simultaneous bacterial hydrolysis and fermentation.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of ground wheat solution: Effects of light source and light intensity

Dark fermentation effluent of wheat powder solution was subjected to light fermentation for bio-hydrogen production using different light sources and intensities. Tungsten, fluorescent, infrared (IR), halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Pure culture of Rhodobacter sphaeroides-RV was used in batch light fermentation experiments. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (CHF, 252 ml) and yield (781 ml H₂ g⁻¹ TVFA). In the second set of experiments, light fermentations were performed at different light intensities (1–10 klux) using halogen lamp. The optimum light intensity was found to be 5 klux (approx. 176 Wm⁻²) resulting in the highest CHF (88 ml) and hydrogen yield (1037 ml H₂ g⁻¹TVFA). Hydrogen formation was limited by the availability of light at low light intensities below 5 klux and was inhibited by the excess light above 5 klux.     

Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation

Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were compared in batch dark fermentations under mesophilic (37 °C) and thermophilic (55 °C) conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 °C and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H₂ h⁻¹) was obtained with the acid-hydrolyzed and the lowest (1.12 ml H₂ h⁻¹) with the boiled wheat at 55 °C. The highest hydrogen gas yield (333 ml H₂ g⁻¹ total sugar or 2.40 mol H₂ mol⁻¹ glucose) and final total volatile fatty acid (TVFA) concentration (10.08 g L⁻¹) were also obtained with the acid-hydrolyzed wheat under thermophilic conditions (55 °C). Dark fermentation of acid-hydrolyzed ground wheat under thermophilic conditions (55 °C) was proven to be more beneficial as compared to mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.     

Hydrogen production from melon and watermelon mixture by dark fermentation

Hydrogen gas production from melon and watermelon mixture by dark fermentation was studied with and without inoculum addition. In this context, hydrogen production performance of natural and external inoculation was compared in batch experiments by varying fruit mixture concentration between 0.74 and 37 g TS/L. Hydrogen production increased by increasing the substrate concentration due to higher initial total sugar content at elevated TS (total solids) concentrations. Hydrogen productivity at 37 g TS/L for natural microflora was 80.62 mLH₂/L_reactor.h. However, this value significantly increased to 351.12 mLH₂/L_reactor.h at same solid concentration when the fruit mixture was externally inoculated with heat treated anaerobic sludge. Most favorable nutrient and inoculum composition for hydrogen gas production were at 37 g TS/L. Moreover, the presence of the natural microflora in the fruit mixture led to less inoculum requirement and contribution for hydrogen formation.     

Bio-hydrogen production from ground wheat starch by continuous combined fermentation using annular-hybrid bioreactor

Continuous combined fermentation of ground wheat starch was realized in an annular-hybrid bioreactor (AHB) for hydrogen gas production. A mixture of pure cultures of Clostridium beijerinkii (DSMZ-791) and Rhodobacter sphaeroides-RV were used as seed cultures in combined fermentation. The feed contained 5 g L⁻¹ ground wheat with some nutrient supplementation. Effects of hydraulic residence time (HRT) on the rate and yield of hydrogen gas formation were investigated. Steady-state daily hydrogen production decreased but, hydrogen yield increased with increasing HRT. The highest hydrogen yield was 90 ml g⁻¹ starch at HRT of 6 days. Hydrolysis of starch and fermentation of glucose to volatile fatty acids (VFA) were readily realized at all HRTs. However, slow conversion of VFAs to H₂ and CO₂ by photo-fermentation caused accumulation of VFAs in the medium. Specific and volumetric rates of hydrogen formation also decreased with increasing HRT. High hydrogen yields obtained at high HRTs are due to partial fermentation of VFAs by Rhodobacter sp. The system should be operated at HRTs longer than 5 days for effective hydrogen gas formation by the dark and photo-fermentation bacteria.     

Thermophilic dark fermentation of acid hydrolyzed waste ground wheat for hydrogen gas production

Batch dark fermentation experiments were performed to investigate the effects of biomass and substrate concentration on bio-hydrogen production from acid hydrolyzed ground wheat at 55 °C. In the first set of experiments, the substrate concentration was constant at 20 g total sugar L⁻¹ and biomass concentration was varied between 0.52 and 2.58 g L⁻¹. Total sugar concentration was varied between 4.2 and 23.7 g L⁻¹ in the second set of experiments with a 1.5 g L⁻¹ constant biomass concentration. The highest cumulative hydrogen formation (582 mL, 30 °C, 1 atm), formation rate (5.43 mL h⁻¹) and final total volatile fatty acid (TVFA) concentration (6.54 g L⁻¹) were obtained with 1.32 g L⁻¹ biomass concentration. In variable substrate concentration experiments, the highest cumulative hydrogen (365 mL) and TVFA concentration (4.8 g L⁻¹) were obtained with 19.25 g L⁻¹ initial total sugar concentration while hydrogen gas formation rate (12.95 mL h⁻¹) and the yield (200 mL H₂ g⁻¹ total sugar) were the highest with 4.2 g L⁻¹ total sugar concentration.     

Effects of light source,intensity and lighting regime on bio-hydrogen production from ground wheat starch by combined dark and photo-fermentations

Combined dark and photo-fermentation of ground wheat starch was carried out by using different light sources, intensities and lighting regime. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides-RV with a certain light/dark bacteria ratio was used in batch experiments. Tungsten, fluorescent, infrared (IR), tungsten + infrared, halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (178 ml) and yield (218 ml g⁻¹ starch). Combined fermentations were performed at different light intensities (1–10 klux) using the halogen lamp in the second set of experiments. The optimum light intensity was found to be 10 klux (approx. 352 Wm⁻²) resulting in the highest cumulative hydrogen (111 ml) and hydrogen yield (139 ml H₂ g⁻¹ starch). Hydrogen formation was limited by the availability of light at low light intensities below 10 klux. Durations of dark/light cycles were changed to determine the most suitable lighting regime. Hydrogen gas formation increased with increasing cycle time and continuous lighting resulted in the highest cumulative hydrogen formation and hydrogen yield.     

Dark fermentation of ground wheat starch for bio-hydrogen production by fed-batch operation

Ground wheat solution was used for bio-hydrogen production by dark fermentation using heat-treated anaerobic sludge in a completely mixed fermenter operating in fed-batch mode. The feed wheat powder (WP) solution was fed to the anaerobic fermenter with a constant flow rate of 8.33 mL h⁻¹ (200 mL d⁻¹). Cumulative hydrogen production, starch utilization and hydrogen yields were determined at three different WP loading rates corresponding to the feed WP concentrations of 10, 20 and 30 g L⁻¹. The residual starch (substrate) concentration in the fermenter decreased with operation time while starch consumption was increasing. The highest cumulative hydrogen production (3600 mL), hydrogen yield (465 mL H₂ g⁻¹ starch or 3.1 mol H₂ mol⁻¹ glucose) and hydrogen production rate (864 mL H₂ d⁻¹) were obtained after 4 days of fed-batch operation with the 20 g L⁻¹ feed WP concentration corresponding to a WP loading rate of 4 g WP d⁻¹. Low feed WP concentrations (10 g L⁻¹) resulted in low hydrogen yields and rates due to substrate limitations. High feed WP concentrations (30 g L⁻¹) resulted in the formation of volatile fatty acids (VFAs) in high concentrations causing inhibition on the rate and yield of hydrogen production.     

Hydrogen gas production from cheese whey powder (CWP) solution by thermophilic dark fermentation

Hydrogen gas production from cheese whey powder (CWP) solution by thermophilic dark fermentation was investigated at 55 °C. Experiments were performed at different initial total sugar concentrations varying between 5.2 and 28.5 g L⁻¹ with a constant initial bacteria concentration of 1 g L⁻¹. The highest cumulative hydrogen evolution (257 mL) was obtained with 20 g L⁻¹ total sugar (substrate) concentration within 360 h while the highest H₂ formation rate (2.55 mL h⁻¹) and yield (1.03 mol H₂ mol⁻¹ glucose) were obtained at 5.2 and 9.5 g L⁻¹ substrate concentrations, respectively. The specific H₂ production rate (SHPR = 4.5 mL h⁻¹ g⁻¹cells) reached the highest level at 20 g L⁻¹ total sugar concentration. Total volatile fatty acid (TVFA) concentration increased with increasing initial total sugar content and reached the highest level (14.15 g L⁻¹) at 28.5 g L⁻¹ initial substrate concentration. The experimental data was correlated with the Gompertz equation and the constants were determined. The optimum initial total sugar concentration was 20 g L⁻¹ above which substrate and product (VFA) inhibitions were observed.     

A sequential process of anaerobic solid-state fermentation followed by dark fermentation for bio-hydrogen production from Chlorella sp.

Microalgal biomass has recently been one of the most widely studied feedstocks for bio-hydrogen production, owing to its richness in fermentable components, e.g. polysaccharides and proteins, and high biomass productivity. In this study, biomass of microalga Chlorella sp. TISTR 8411 was converted to hydrogen through a sequential process consisting of an anaerobic solid-state fermentation (ASSF) followed by a dark fermentation. The microalga was grown photoautothrophically in 80-L rectangular glass tanks and then scaled-up to a 240-L open pond for the production of biomass. The highest biomass concentration attained was 4.45 g L⁻¹. The biomass was harvested with over 90% flocculation efficiency at pH 11.5 and a biomass concentration of 2.6 g/L. The sequential process gave a total hydrogen yield (HY) of 16.2 mL/g-volatile-solid (VS), of which 11.6 mL/g-VS was from ASSF. The high HY obtained from the ASSF indicated that it was effective and could be integrated with a conventional hydrogen production process to improve energy recovery from biomass.     

Bio-hydrogen production by different operational modes of dark and photo-fermentation: An overview

This article overviews reported studies on bio-hydrogen production from different raw materials by dark and photo-fermentations operated with different modes. Sequential and combined dark and photo-fermentations operated in batch, continuous and fed-batch modes were compared. Operating conditions and modes resulting in the highest hydrogen yield and formation rate were revealed. Relative advantages of sequential and combined dark and photo-fermentations were discussed. Sequential fermentation was found to be preferable due to high H₂ yields and productivities. High cell density fed-batch culture with controlled feeding and simultaneous product removal was concluded to be the most suitable operation mode at the optimum environmental conditions.     

Hydrogen gas production from distillery wastewater by dark fermentation

The influence of concentration of distillery wastewaters, concentration of inoculum and pH value on hydrogen generation in batch dark fermentation process was studied. Anaerobic digested sludge from municipal purification unit was applied as the source of bacteria mixture. The best specific yield was obtained in system containing 10% v/v of inoculum and 20% v/v of the waste (S₀/X₀ = 2.8), whereas the maximum amount of hydrogen and the highest rate of reaction was achieved in system containing 25% v/v inoculum and 40% v/v of waste (S₀/X₀ = 2.2). The content of generated hydrogen in biogas was always higher than 62%. Maximum amount of generated hydrogen was 1 l H₂/l medium and the rate was 0.12 l/l/h. Liquid metabolites of hydrogen generation process were mainly acetic and butyric acids. Ethanol and propionic acid were in traces. The ratio of HBu/HAc in medium influenced the yield of generated hydrogen.     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal was investigated at different feeding intervals. Ground wheat was acid hydrolyzed at pH = 3 and T = 121 °C for 30 min using an autoclave. The resulting sugar solution was subjected to dark fermentation with periodic feeding and effluent removal. The feed solution contained 9 ± 0.5 g L⁻¹ total sugar supplemented with some nutrients. Depending on the feeding intervals hydraulic residence time (HRT) was varied between 6 and 60 h. Steady-state daily hydrogen production increased with decreasing HRT. The highest daily hydrogen production (305 ml d⁻¹) and volumetric hydrogen production rate (1220 ml H₂ L⁻¹ d⁻¹) were obtained at HRT of 6 h. Hydrogen yield (130 ml H₂ g⁻¹ total sugar) reached the highest level at HRT = 24 h. Effluent total sugar concentration decreased, biomass concentration and yield increased with increasing HRT indicating more effective sugar fermentation at high HRTs. Dark fermentation end product profile shifted from acetic to butyric acid with increasing HRT. High acetic/butyric acid ratio obtained at low HRTs resulted in high hydrogen yields.     

Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: An unexpected deviation from the dark fermentation model

The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of organic substrates. The process is referred to as dark fermentation and is typically complemented by production of acetic acid. Here we show that synthesis of products derived by reductive metabolism of pyruvate, mainly lactic acid, occurs to the detriment of acetic acid fermentation when the cultures of the thermophilic bacterium are flushed by saturating level of CO₂. Sodium bicarbonate in a very narrow range of concentrations (∼14 mM) also causes the same metabolic shift. The capnophilic (CO₂-requiring) re-orientation of the fermentative process toward lactic acid does not affect hydrogen productivity thus challenging the currently accepted dark fermentation model that predicts reduction of this gas when glucose is converted into organic products different from acetate.     

Hydrogen production via dark fermentation by bacteria colonies on porous PDMS-scaffolds

The development of biofuels and the question of finding renewable energy sources are important issues nowadays, due to the increasing shortage of other supplies. Hydrogen has gained very much attention as biofuel, as it is highly energetic and a clean energy source. A very interesting method to produce hydrogen is dark fermentation. It generates a clean energy from organic wastes with low value and at low energy requirements. The production of hydrogen and bio-hydrogen from waste and wastewaters can have a positive environmental impact in terms of creation of highly effective energy fuel and reduction of waste. Due to their nutrients, organic waste and wastewaters are suitable substrates to obtain bio-hydrogen. In this paper we investigate the behaviour and the stability of porous scaffolds containing iron oxide particles in a dark fermentation environment and explore the possibility of hosting mixed cultures of clostridia on them, aiming to an increase in hydrogen production. We address the effect of embedding hematite particles (in different concentrations) in the scaffolds, to see whether there is an increase in bio-hydrogen-production. This latter can be enhanced, if particles of various metal oxides are present, as they can increase bacterial growth and encourage the bioactivity of species that produce hydrogen. The scaffolds analysed consist of polydimethylsiloxane (PDMS) containing Fe₂O₃ particles and were produced via the sugar template method. X-ray diffraction patterns, SEM images as well as dark fermentation tests in batch procedure are presented and discussed. Bacteria colonies could be detected after long treatment in municipal wastewater and production of biohydrogen was ascertained for all samples investigated.     

Inhibition of hydrogen-yielding dark fermentation by ascomycetous yeasts

Hydrogen-yielding fermentation conducted in bioreactors is an alternative method of hydrogen production. However, unfavourable processes can seriously inhibit bio-hydrogen generation during the acidogenic step of anaerobic digestion. Here, ascomycetous yeasts were identified as a major factor inhibiting the production of bio-hydrogen by fermentation. Changes in the performance of hydrogen-producing bioreactors including metabolic shift, quantitative changes in the fermentation products, decreased pH, instability of the microbial community and consequently a dramatic drop in bio-hydrogen yield were observed following yeast infection. Ascomycetous yeasts from the genera Candida, Kazachstania and Geotrichum were isolated from hydrogen-producing bioreactors. Yeast metabolites secreted into the growth medium showed antibacterial activity. Our studies indicate that yeast infection of hydrogen-producing microbial communities is one of the serious obstacles to use dark fermentation as an alternative method of bio-hydrogen production. It also explains why studies on hydrogen fermentation are still limited to the laboratory or pilot-scale systems.