Effects of starch loading rate on performance of combined fed-batch fermentation of ground wheat for bio-hydrogen production

Ground wheat powder solution (10 g L⁻¹) was subjected to combined dark and light fermentations for bio-hydrogen production by fed-batch operation. A mixture of heat treated anaerobic sludge (AN) and Rhodobacter sphaeroides-NRRL (RS-NRRL) were used as the mixed culture of dark and light fermentation bacteria with an initial dark/light biomass ratio of 1/2. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. The highest cumulative hydrogen formation (CHF = 3460 ml), hydrogen yield (201 ml H₂ g⁻¹ starch) and formation rate (18.1 ml h⁻¹) were obtained with a starch loading rate of 80.4 mg S h⁻¹. Complete starch hydrolysis and glucose fermentation were achieved within 96 h of fed-batch operation producing volatile fatty acids (VFA) and H₂. Fermentation of VFAs by photo-fermentation for bio-hydrogen production was most effective at starch loading rate of 80.4 mg S h⁻¹. Hydrogen formation by combined fermentation took place by a fast dark fermentation followed by a rather slow light fermentation after a lag period.     

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 acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

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.     

Potential of bio-hydrogen production from dark fermentation of crop residues: A review

This study provided an estimate of the potential of bio-hydrogen production from dark fermentation of crop residues on a worldwide scale. The different crop residues reviewed included sugarcane tops, leaves and bagasse, corn straw, corn cob and corn stover, wheat straw, rice straw and husk, soybean straw, oil palm trunk and empty fruit bunch, sugar beet pulp, cassava residue, barley straw and sweet sorghum bagasse. Among these crop residues, wheat and rice straws are produced in the highest amount although sugarcane dominates crop production on a worldwide scale. Based on the bio-hydrogen yields reported in literature, estimated worldwide bio-hydrogen potential is highest for untreated rice straw at 58,002 Mm³/year followed by untreated wheat straw at 34,680 Mm³/year. This corresponds to a bio-energy potential of 623 PJ/year and 373 PJ/year for raw rice straw and wheat straw respectively while pre-treatment of the crop residues significantly increases the bio-hydrogen and bio-energy potential. While dark fermentation of crop residues offers a huge bio-energy potential, the process suffers from several constraints that hinder its implementation. As such, coupling of the dark fermentation process with the anaerobic digestion process as a two-stage process seems the most economically viable option for large-scale implementation.     

Aerobic and anaerobic sequential culture fermentation (AASF) to produce bio-hydrogen from steam-exploded cornstalk

A novel aerobic and anaerobic sequential culture fermentation (AASF) method was designed to improve the conversion efficiency of steam-exploded cornstalk during bio-hydrogen production. The enzyme activities of cellulase and β-glucosidase produced by Trichoderma viride ACCC 30169 were 76.79 FPU g⁻¹ dry weight and 45.23 IU g⁻¹ dry weight after 6-days steam-exploded cornstalk fermentation, respectively. The aerobic fermentation residue was used as the substrate for bio-hydrogen production by Clostridium butyricum AS1.209 anaerobic fermentation. The optimum solid-to-liquid ratio of the anaerobic fermentation substrate was 1:5. The maximum bio-hydrogen yield was attained on the medium with addition of 0.1 g g⁻¹ substrate urea after 2 days of aerobic fermentation. Compared with simultaneous saccharification and fermentation (SSF), AASF for bio-hydrogen production could shorten the fermentation period by at least 66% and the hydrogen yield reached 83% of the total volume after 24 h of anaerobic fermentation. AASF from steam-exploded cornstalk was an effective way for bio-hydrogen production without additional commercial cellulase.     

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.     

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.     

Influence of seed sludge and pretreatment method on hydrogen production in packed-bed anaerobic reactors

Several studies have reported on the effects of inoculums source and pretreatment on biological hydrogen production. However, there have been few studies on continuous reactors. This paper investigated the influence of different seed sludge sources and pretreatment methods on biohydrogen production in up-flow anaerobic fixed-bed reactors fed with sucrose. The following inoculum sources were included in the study: (1) anaerobic sludge from an up-flow anaerobic sludge blanket (UASB) reactor used to treat poultry slaughterhouse wastewater (Sl), (2) anaerobic sludge from a UASB reactor used to treat swine wastewater (Sw) and (3) autofermentation (A). Heat (He) and acid (Ac) shock were used to increase hydrogen production and suppress hydrogen consumption. The average hydrogen yields (HY) in the experiment were 2.1 (A), 2.0 (SlHe), 2.0 (SlAc), 1.0 (Sl), 1.0 (SwAc), 0.7 (SwHe) and 0.7 (Sw) mol H₂ mol⁻¹ sucrose. Although heat shock produced the maximum HY value (SlHe), acid pretreatment (SlAc) resulted in more stable hydrogen production with the largest average value, which could be an advantage of using pH shock. The autofermentation process presented HY values similar to those produced with SlAc and SlHe, making it a suitable seed sludge for biohydrogen production because pretreatment was not required.     

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.     

Hydrogen gas production by electrohydrolysis of volatile fatty acid (VFA) containing dark fermentation effluent

Dark fermentation effluents of wheat powder (WP) solution containing different concentrations of volatile fatty acids (VFAs) were subjected to low voltage (1–3 V) DC current to produce hydrogen gas. Graphite and copper electrodes were tested and the copper electrode was found to be more effective due to higher electrical conductivity. The effects of solution pH (2–7), applied voltage (1–3 V) and the total VFA (TVFA) concentration (1–5 g L⁻¹) on hydrogen gas production were investigated. Hydrogen production increased with decreasing pH and became maximum at pH = 2. Increases in applied voltage and the TVFA concentration also increased the cumulative hydrogen formation. The most suitable conditions for the highest cumulative hydrogen production was pH = 2, with 3 V applied voltage and 5 g TVFA L⁻¹. Up to 110 ml hydrogen gas was obtained with 5 g L⁻¹ TVFA at pH = 5.8 and 2 V applied voltage within 37.5 h. The highest energy efficiency (56%) was obtained with the 2 V applied voltage and 10.85 g L⁻¹ TVFA. Hydrogen production by electrolysis of water in control experiments was negligible for pH > 4. Hydrogen production by electrohydrolysis of VFA containing anaerobic treatment effluents was found to be an effective method with high energy efficiency.     

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.     

Evaluation and simultaneous optimization of bio-hydrogen production using 3 factorial design and the desirability function

Various mixtures incorporating a simulated organic fraction of municipal solid wastes and blood from a poultry slaughterhouse were used as substrate in a dark fermentation process for the production of hydrogen. The individual and interactive effects of hydraulic retention time (HRT), solid content in the feed (%TS) and proportion of residues (%Blood) on bio-hydrogen production were studied in this work. A central composite design and response surface methodology were employed to determine the optimum conditions for the hydrogen production process. Experimental results were approximated to a second-order model with the principal effects of the three factors considered being statistically significant (P < 0.05). The production of hydrogen obtained from the experimental point at conditions close to best operability was 0.97 L Lr⁻¹ day⁻¹. Moreover, a desirability function was employed in order to optimize the process when a second, methanogenic, phase is coupled with it. In this last case, the optimum conditions lead to a reduction in the production of hydrogen when the optimization process involves the maximization of intermediary products.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

A comparison of treatment techniques to enhance fermentative hydrogen production from piggery anaerobic digested residues

To accelerate the start-up process and enhance the efficiency of a hydrogen production system, piggery anaerobic digested residues (PADRs) were subjected to several different treatment methods to enrich the hydrogen-producing bacteria. Eight treatment methods were performed on the PADRs, including acid, alkali, heat, drying, ultrasound, aeration, sodium 2-bromoethanesulfonate (BES), and chloroform. The best method was found to be drying at 60 °C for 48 h, which maximised the total biogas production and the hydrogen fraction without causing any methane production. The volatile fatty acids (VFAs) found after the drying treatment were acetate and butyrate, which together accounted for 91.9% of all VFAs, indicating that butyric acid fermentation was established. Due to the drying treatment, the metabolites produced from the biodegradable DOM were utilised more rapidly, more completely, and with the least amount of hard-degradation organic matter content obtained, according to EEM fluorescence spectra. This drying treatment offers a promising method to¹ improve bio-hydrogen production.     

Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge

An anaerobic fermentation process to produce hydrogen from cornstalk wastes was systematically investigated in this work. Batch experiments numbered series I, II and III were designed to investigate the effects of acid pretreatment, enzymatic hydrolysis (enzymatic temperature, enzymatic time and enzymatic pH) on hydrogen production by using the natural sludge as inoculant. A maximum cumulative H₂ yield of 126.22 ml g⁻¹-CS (Cornstalk, or 146.94 ml g⁻¹-TS, Total Solid) and an average H₂ production rate of 9.58 ml g⁻¹-CS h⁻¹ were obtained from fermentation cornstalk with a concentration of 20 g/L and an initial pH of 7.0 at 36 °C through an optimal pretreatment process. The optimal process was that the substrate was soaked with an HCl concentration of 0.6 wt% at 90 °C for 2 h, and subsequently enzymatic hydrolysis for 72 h at 50 °C and pH 4.8 before fermentation. The biogas consisted of only H₂ and CO₂. In addition, the fermentation system was the typical ethanol-type fermentation according to ethanol and acetate as the main liquid by-products.     

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.     

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH₄⁺, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H₂ photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L⁻¹), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H₂ production. The influence of various fermentation end products at different concentrations (up to 30 g L⁻¹) on dark, H₂ production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L⁻¹) resulted in 5.7 L H₂ L⁻¹ of culture (1.6 mol H₂ per mole of hexose) with the co-production mainly of butyrate and acetate.     

Potential improvement to a citric wastewater treatment plant using bio-hydrogen and a hybrid energy system

Treatment of highly concentrated organic wastewater is characterized as cost-consuming. The conventional technology uses the anaerobic-anoxic-oxic process (A²/O), which does not produce hydrogen. There is potential for energy saving using hydrogen utilization associated with wastewater treatment because hydrogen can be produced from organic wastewater using anaerobic fermentation. A 50 m³ pilot bio-reactor for hydrogen production was constructed in Shandong Province, China in 2006 but to date the hydrogen produced has not been utilized. In this work, a technical-economic model based on hydrogen utilization is presented and analyzed to estimate the potential improvement to a citric wastewater plant. The model assesses the size, capital cost, annual cost, system efficiency and electricity cost under different configurations. In a stand-alone situation, the power production from hydrogen is not sufficient for the required load, thus a photovoltaic array (PV) is employed as the power supply. The simulated results show that the combination of solar and bio-hydrogen has a much higher cost compared with the A²/O process. When the grid is connected, the system cost achieved is 0.238 US$ t⁻¹ wastewater, which is lower than 0.257 US$ t⁻¹ by the A²/O process. The results reveal that a simulated improvement by using bio-hydrogen and a FC system is effective and feasible for the citric wastewater plant, even when compared to the current cost of the A²/O process. In addition, lead acid and vanadium flow batteries were compared for energy storage service. The results show that a vanadium battery has lower cost and higher efficiency due to its long lifespan and energy efficiency. Additionally, the cost distribution of components shows that the PV dominates the cost in the stand-alone situation, while the bio-reactor is the main cost component in the parallel grid.     

Optimization of media composition for hydrogen gas production from hydrolyzed wheat starch by dark fermentation

Effects of N/C, P/C and Fe(II)/C ratios in fermentation medium on biohydrogen production by dark fermentation of acid-hydrolyzed wheat starch was investigated. The powdered wheat was autoclaved at pH = 3 and 90 °C for 15 min and the resulting sugar solution was fermented after external addition of N, P and Fe(II) to overcome nutrient limitations. Box–Wilson statistical experiment design was used by considering the N/C (0–0.05, w w⁻¹), P/C (0–0.02) and Fe(II)/C (0–0.03) ratios as the independent variables while the hydrogen yield and specific hydrogen production rate (SHPR) were the objective functions to be optimized. A quadratic response function was used to correlate the response functions with the independent variables. Low levels of the variables (N/C < 0.02, P/C < 0.01, Fe(II)/C < 0.01) resulted in low hydrogen yield and SHPR due to nutrient limitations and high levels of nutrients caused inhibitions. The optimum conditions yielding the maximum hydrogen yield (Y = 2.84 mol H₂ mol⁻¹ glucose) were N/C = 0.02, P/C = 0.008 and Fe(II)/C = 0.015. The maximum SHPR (96 mL H₂ g⁻¹ biomass h⁻¹) was obtained at N/C = 0.025, P/C = 0.008 and Fe(II)/C = 0.015 (w w⁻¹).