Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

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.     

Hydrogen gas production from electrohydrolysis of industrial wastewater organics by using photovoltaic cells (PVC)

Electrical current generated by a photovoltaic cell (PVC) was supplied to wastewater in a mechanically mixed and sealed reactor using stainless steel electrodes. Hydrogen gas was generated by reaction of protons released from decomposition of organic compounds and electrons provided by electrical current. Gas phase was composed of 75–99% H₂ gas.     

Photofermentative hydrogen production from volatile fatty acids present in dark fermentation effluents

In the present study, the growth and hydrogen production of Rhodobacter sphaeroides O.U. 001, was investigated in media containing five different volatile fatty acids (VFA) individually (malate, acetate, propionate, butyrate and lactate) and in media containing mixtures of these acids that reflect the composition of dark fermentation effluents. The highest hydrogen production rate was obtained in malate (24 ml_hydrogen/l_reactor h) and the highest biomass concentration was obtained in acetate containing media (1.65 g/l). The substrate conversion efficiencies for different volatile fatty acids were found to vary between 14 and 50%. The malate and butyrate consumption rates were first order with consumption rate constants of 0.026 h⁻¹ and 0.015 h⁻¹, respectively. In the case of substrate mixtures, it was observed that the bacteria consumed acetate first, followed by propionate and then butyrate. It was also found that the consumption rate of the main substrate significantly increased when the minor substrates were depleted.     

Hydrogen gas production from olive mill wastewater by electrohydrolysis with simultaneous COD removal

Diluted olive mill wastewater (OMW) was subjected to direct current (DC) voltages (0.5-4.0 V) for hydrogen gas production with simultaneous chemical oxygen demand (COD) removal by electrohydrolysis. The highest cumulative hydrogen production (3020 ml) and hydrogen yield (2500 ml H₂ g⁻¹ COD) were obtained with 3 V DC voltage while the highest current intensity (80 mA), percent hydrogen (95%) in the gas phase, hydrogen gas formation rate (614 ml d⁻¹), percent COD removal (44%) and energy conversion efficiency (95%) were realized with 2 V. Hydrogen gas production by electrolysis of water was negligible for all voltages. COD removal from OMW with no DC voltage application was usually lower than that obtained with DC power application. Hydrogen gas production by electrohydrolysis of OMW was proven to be a fast and effective method with simultaneous COD removal.     

Enhanced hydrogen and volatile fatty acid production from sweet sorghum stalks by two-steps dark fermentation with dilute acid treatment in between

This study investigated the potential of hydrogen and volatile fatty acid coproduction from two steps dark fermentation with dilute acid treatments of the residual slurry after 1st step fermentation. Sweet sorghum stalks (SS) was used as substrate along with Clostridium thermosaccharolyticum as production microbe. Residual lignocelluloses after 1st step fermentation were treated for 1 h by sulfuric acid concentration of 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5% (w/v) with different reaction temperature of 120, 90 and 60 °C were studied. The optimum severity conditions for the highest yield of products found from the treatment acid concentration of 1.5% (w/v) at 120 °C for 10 g/L of substrate concentration. Experimental data showed that two-step fermentation increased 76% hydrogen, 84% acetic acid and 113% of butyric acid production from single step. Maximum yields of hydrogen, acetic acid and butyric acid were 5.77 mmol/g-substrate, 2.17 g/L and 2.07 g/L respectively. This two-step fermentation for hydrogen and VFA production using the whole slurry would be a promising approach to SS biorefinery.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

Hydrogen gas production from food wastes by electrohydrolysis using a statical design approach

Hydrogen gas production was investigated by electrohydrolysis of food waste due to its high organic content. Different voltages generated by DC power supply were applied to food waste in order to produce hydrogen gas. Effects of the DC voltage, reaction time and initial solid content on cumulative hydrogen gas production, hydrogen gas content in the gas phase and total organic carbon (TOC) removal were investigated by using a Box-Behnken statistical experiment design approach. The most suitable voltage/reaction time/solid content values resulting in the highest hydrogen gas content (99%), the highest cumulative hydrogen gas formation (7000 mL) and total organic carbon removal (33%) were determined as 5 V/75 h/20%. The results indicated that food wastes constitute a good source for H₂ gas production by electrohydrolysis. Hydrogen gas produced by electrohydrolysis of food waste can be directly used in fuel cells due to its high putrity.     

Hydrogen gas production from vinegar fermentation wastewater by electro-hydrolysis: Effects of initial COD content

Vinegar fermentation wastewater with different initial COD contents (9.66–48.6 g L⁻¹) were used for hydrogen gas production with simultaneous COD removal by electro-hydrolysis. The applied DC voltage was constant at 4 V. The highest cumulative hydrogen production (3197 ml), hydrogen yield (2766 ml H₂ g⁻¹ COD), hydrogen formation rate (799 ml d⁻¹), and percent hydrogen (99.5%) in the gas phase were obtained with the highest initial COD of 48.6 g COD L⁻¹. The highest energy efficiency (48%) was obtained with the lowest COD content of 9.66 g L⁻¹. Hydrogen gas production by water electrolysis was less than 250 ml and wastewater control resulted in less than 25 ml H₂ in 96 h. The highest (12%) percent COD removal was obtained with the lowest COD content. Hydrogen gas was produced by reaction of (H⁺) ions present in raw WW ( pH = 3.0) and protons released from acetic acid with electrons provided by electrical current. Electro-hydrolysis of vinegar wastewater was proven to be an effective method of H₂ gas production with some COD removal.     

Hydrogen gas production from waste anaerobic sludge by electrohydrolysis: Effects of applied DC voltage

Waste anaerobic sludge was subjected to different DC voltages (0.5-5 V) for hydrogen gas production by using aluminum electrodes and a DC power supply. Effects of applied DC voltage on the rate and extent of hydrogen gas production were investigated. The highest cumulative hydrogen production (2775 ml), daily hydrogen gas formation (686.7 ml d⁻¹), hydrogen yield (96 ml H₂ g⁻¹ COD) and percent hydrogen (94.3%) in the gas phase were obtained with 2 V DC voltage. Energy conversion efficiency (H₂ energy/electrical energy) also reached the highest level (74%) with 2 V DC voltage application. Control experiments with no voltage application to the sludge yielded almost the same level of COD removal, but no hydrogen gas production. Voltage application to water resulted in much lower hydrogen gas production as compared to sludge indicating negligible electrolysis of water. The results indicated that the sludge was naturally decomposed by the active cells removing COD and releasing hydrogen ions to the medium which reacted with the electrons provided by DC current to produce hydrogen gas. Hydrogen gas production from electrohydrolysis of waste sludge was found to be a fast and effective method with high energy efficiency.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

Improved hydrogen gas production in electrohydrolysis of vinegar fermentation wastewater by scrap aluminum and salt addition

Scrap aluminum particles and salt (NaCl) were added to the vinegar fermentation wastewater to improve hydrogen gas formation by electrohydrolysis of the wastewater organics. The applied DC voltage and initial COD of the wastewater were constant at 4 V and 33.16 g L⁻¹, respectively. The highest cumulative hydrogen gas formation (2877 mL) was obtained with scrap Al (1 g L⁻¹) and NaCl (1 g L⁻¹) additions within 72 h as compared to 1925 mL H₂ gas formation from raw wastewater. Hydrogen gas formation from Al and NaCl added water was 302 ml as compared to 260 ml from raw water. The highest H₂ gas formation rate (952 mL d⁻¹), the yield (1660 mL H₂ g⁻¹ COD) and the highest current intensity (163 mA) were also obtained with combined effects of scrap Al and NaCl additions. Almost pure hydrogen gas (99%) was produced using the raw wastewater. Initial conductivity of the raw wastewater increased from 1.80 mS cm⁻¹ to 5.01 mS cm⁻¹ with the addition of scrap Al and salt for which the final conductivities were 4.0 mS cm⁻¹ and 6.91 mS cm⁻¹, respectively. The highest energy conversion efficiency was obtained with only scrap Al addition (37.8%) as compared to 30.5% efficiency obtained with Al and salt additions. Additions of NaCl and scrap Al particles was found to be very beneficial for H₂ gas formation by electrohydrolysis of vinegar fermentation wastewater.     

Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

Comparison of different electrodes in hydrogen gas production from electrohydrolysis of wastewater organics using photovoltaic cells (PVC)

Electrical power generated by a photovoltaic cell (PVC) was supplied to diluted industrial wastewater in a mechanically mixed and sealed stainless-steel reactor for hydrogen gas production. Three different electrodes, graphite, stainless steel and aluminum rods were used for comparison. Protons released from decomposition of organic compounds and electrons provided by the DC current reacted to form hydrogen gas. The highest cumulative hydrogen gas formation (CHF) was obtained with the aluminum electrode (120 L in 8 days) and the lowest was with the graphite electrode (4 L). Hydrogen gas production from wastewater was 2.4 times higher than that produced from water when aluminum electrodes were used. TOC content of wastewater was reduced from 2400 to 1700 mg L⁻¹ with nearly 29% TOC removal within 6 days. CHF from wastewater was 76 L within 18 days with the stainless-steel electrodes while CHF from water was only 9.5 L. Fermentative hydrogen gas production from wastewater was negligible in the absence PVC. Energy conversion efficiency for hydrogen gas production (hydrogen energy/electric energy) was found to be 74% with the aluminum electrodes.     

Hydrogen production by combined dark and light fermentation of ground wheat solution

Ground waste wheat was subjected to combined dark and light batch fermentation for hydrogen production. The dark to light biomass ratio (D/L) was changed between 1/2 and 1/10 in order to determine the optimum D/L ratio yielding the highest hydrogen formation rate and the yield. Hydrogen production by only dark and light fermentation bacteria was also realized along with the combined fermentations. The highest cumulative hydrogen formation (CHF = 76 ml), hydrogen yield (176 ml H₂ g⁻¹ starch) and formation rate (12.2 ml H₂ g⁻¹ biomass h⁻¹) were obtained with the D/L ratio of 1/7 while the lowest CHF was obtained with the D/L ratio of 1/2. Dark–light combined fermentation with D/L ratio of 1/7 was faster as compared to the dark and light fermentations alone yielding high hydrogen productivity and reduced fermentation time. Dark and light fermentations alone also yielded considerable cumulative hydrogen, but slower than the combined fermentation.     

Maximizing performance of microbial electrolysis cell fed with dark fermentation effluent from water hyacinth

The performance of microbial electrolysis cell (MEC) fed with dark fermentation effluent (DEF) from water hyacinth (WH) was enhanced in this study. First, the single effects of the auxiliary processes, including centrifugation, dilution, buffering, and external power input, were investigated. Then, the interaction of these processes was further evaluated using response surface methodology (RSM) and a combination of artificial neural network (ANN) and particle swarm optimization (PSO). Statistical analysis results revealed that ANN-PSO outperformed RSM in predictability. Consequently, the ANN-PSO approach determined that a 2.2-fold dilution of centrifuged-DFE (～1.64 g of soluble metabolite products per L), buffer concentration of 75 mM, and an applied voltage of 0.7 V were the optimal conditions for simultaneously maximizing H₂ production yield and energy efficiency of DFE@WH-fed MEC. Under co-optimized conditions, H₂ yield (560.8 ± 10.8 mL/g-VS) and electrical energy recovery (162.2 ± 4.7%) significantly improved compared to unoptimized conditions.     

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.     

Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production

The production of biohydrogen through anaerobic fermentation has received increasingly attention and has great potential as an alternative process for clean fuel production in the future. The monitoring of the stages of anaerobic fermentation provides relevant information about the bioprocess. The objective of this study is to propose a novel methodology for simultaneous analysis of sucrose, glucose, fructose and volatile fatty acids (VFAs), such as, acetic, propionic, isobutyric and butyric during anaerobic fermentation by using high-performance liquid chromatography (HPLC). The following chromatographic conditions were optimized: column Aminex HPX-87H, mobile phase consisting of H₂SO₄ 0.005 mol/L, flow rate of 1.0 mL/min and temperature of 55 °C. Sucrose, glucose and fructose were analyzed by refractive index detector (RI) while acetic, propionic, isobutyric and butyric acids were analyzed by ultraviolet (UV) detection at 210 nm. Some analytical parameters of validation, such as, linearity, selectivity, repeatability, intermediate precision, limit of detection and quantification, accuracy and robustness were evaluated. The proposed methodology was successfully applied in the determination of substrates and metabolites during different stages of biohydrogen production.     

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.     

Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge

This paper is a comprehensive review of H₂ consumption during anaerobic mixed culture H₂ dark fermentation with a focus on homoacetogenesis. Homoacetogenesis consumed from 11% to 43% of the H₂ yield in single and repeated batch fermentations, respectively. However, its quantification and extent during continuous fermentation are still not well understood. Models incorporating thermodynamic and kinetic controls are required to provide insight into the dynamic of homoacetogenesis during H₂ dark fermentation. Currently, no adequate method exists to eliminate homoacetogenesis because it does not depend on the culture's source, pre-treatment, substrate, type of reactor, or operation conditions. Controlling CO₂ concentrations during dark fermentation needs further investigation as a potential strategy towards controlling homoacetogenesis. Incorporating radioactive labeling technique in H₂ fermentation research could provide information on simultaneous production and consumption of H₂ during dark fermentation. Genetic studies investigating blocking H₂ consuming pathways and enhancing H₂ evolving hydrogenases are suggested towards controlling homoacetogenesis during dark fermentation.