Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor

The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of downcomer and riser (A_d/A_r) in range of 0.4–3.2 were considered. Maximum biomass concentration of 1.63 g L⁻¹ in 9 d under light intensity of 120 μE m⁻² s⁻¹ was observed at A_d/A_r of 1.6. The mixing time of the reactors was inversely proportional to A_d/A_r. Maximal H₂ production was found to be 1600 mL L⁻¹ upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L⁻¹), autoclave (600 mL L⁻¹) and HCl treatment (1230 mL L⁻¹). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L⁻¹ as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H₂ production.     

Hydrogen-producing purple non-sulfur bacteria isolated from the trophic lake Averno (Naples,Italy)

Seventeen purple non-sulfur bacterial strains, isolated from the trophic lake Averno, Naples, Italy, were phylogenetically classified and their H₂-producing performances were tested utilizing various synthetic substrates and the fermentation broth derived from the spontaneous fermentation of vegetable residues. All the strains showed the capability to produce hydrogen on at least one of the four carbon substrates tested (malic, lactic, acetic and succinic acid). On lactate, Rhodopseudomonas palustris strain AV33 showed the best maximum production rate (50.7 ± 2.6 mL (H₂) L⁻¹ h⁻¹), with a mean rate, calculated on the whole period of production, of 17.9 mL ± 0.7 (H₂) L⁻¹ h⁻¹. In the presence of acetate, AV33 produced only few mL of H₂, but intracellularly accumulated poly-β-hydroxybutyrate up to a concentration of 21.4 ± 3.4% (w/w) of cell dry weight. Rp. palustris AV33 also produced H₂ on the fermentation broth supplemented with Fe, with a maximum production rate of 16.4 ± 2.3 mL (H₂) L⁻¹ h⁻¹ and a conversion yield of 44.2%.     

Hydrogen production from acid hydrolyzed molasses by the hydrogen overproducing Escherichia coli strain HD701 and subsequent use of the waste bacterial biomass for biosorption of Cd(II) and Zn(II)

This study was devoted to investigate production of hydrogen gas from acid hydrolyzed molasses by Escherichia coli HD701 and to explore the possible use of the waste bacterial biomass in biosorption technology. In variable substrate concentration experiments (1, 2.5, 5, 10 and 15 g L⁻¹), the highest cumulative hydrogen gas (570 ml H₂ L⁻¹) and formation rate (19 ml H₂ h⁻¹ L⁻¹) were obtained from 10 g L⁻¹ reducing sugars. However, the highest yield (132 ml H₂ g⁻¹ reducing sugars) was obtained at a moderate hydrogen formation rate (11 ml H₂ h⁻¹ L⁻¹) from 2.5 g L⁻¹ reducing sugars. Subsequent to H₂ production, the waste E. coli biomass was collected and its biosorption efficiency for Cd²⁺ and Zn²⁺ was investigated. The biosorption kinetics of both heavy metals fitted well with the pseudo second-order kinetic model. Based on the Langmuir biosorption isotherm, the maximum biosorption capacities (q_max) of E. coli waste biomass for Cd²⁺ and Zn²⁺ were 162.1 and 137.9 (mg/g), respectively. These q_max values are higher than those of many other previously studied biosorbents and were around three times more than that of aerobically grown E. coli. The FTIR spectra showed an appearance of strong peaks for the amine groups and an increase in the intensity of many other functional groups in the waste biomass of E. coli after hydrogen production in comparison to that of aerobically grown E. coli which explain the higher biosorption capacity for Cd²⁺ or Zn²⁺ by the waste biomass of E. coli after hydrogen production. These results indicate that E. coli waste biomass after hydrogen production can be efficiently used in biosorption technology. Interlinking such biotechnologies is potentially possible in future applications to reduce the cost of the biosorption technology and duplicate the benefits of biological H₂ production technology.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

Continuous thermophilic hydrogen production and microbial community analysis from anaerobic digestion of diluted sugar cane stillage

The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 °C using a mixture of sugar cane stillage and glucose at approximately 5000–5300 mg COD L⁻¹. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H₂ yields of 5.73 mmol g COD_added⁻¹ were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m⁻³ d⁻¹). The maximum volumetric H₂ production of 0.78 L H₂ h⁻¹ L⁻¹ was achieved using stillage as carbon source. In all operational phases, the H₂ average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacterium sp. and Clostridium sp. were identified.     

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.     

Optimization of thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH₂) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH₂ production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L⁻¹ bromoethanesulphonic acid, 24 h) on bioH₂ production from starch powder (1% w/v) showed highest yield (323.4 mL g⁻¹ starch) in heat-treatment and peak production rate (144.5 mL L⁻¹ h⁻¹) in acid-treatment. Acetate (1.07 g L⁻¹) and butyrate (1.21 g L⁻¹) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH₂ production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H₂ yielding conditions. Peak H₂ yield and production rate were 412.6 mL g⁻¹ starch and 78.6 mL L⁻¹ h⁻¹, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH₂ yield of 126.5 mL g⁻¹ COD and 456 mL g⁻¹ starch. The net energy was calculated to be +2.97 kJ g⁻¹ COD and +0.57 kJ g⁻¹ COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 10¹³ kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH₂ production with low energy input.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Hydrogen production using Rhodobacter sphaeroides (O.U. 001) in a flat panel rocking photobioreactor

Photofermentative hydrogen production is challenged by the photobioreactor design that can overcome poor light penetration, agitation and temperature control. Flat panel reactors have been reported to have several advantages over other reactors. But they are limited to a suitable type of agitation system when using it for hydrogen production. The aim of the present study is to develop and improve a flat panel reactor that can overcome the problem of agitation with a rocking motion. Studies with Rhodobacter sphaeroides O.U. 001 resulted in a cumulative hydrogen production of 492 ± 10 mL with maximum production rate of 11 mL L⁻¹ h⁻¹, substrate (malic acid) conversion efficiency of 44.4% and light conversion efficiency of 3.31%. The mixing time of the reactor was found to be around 17 s with a power input of 100-275 W/m³. Though the entire reactor was in motion the energy spent for the rocking motion was found to be quite low.     

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.     

Hydrogen production and end-product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or α-cellulose

Hydrogen (H₂) production and end-product synthesis were characterized in a novel, mesophilic, cellulolytic, anaerobic bacterium, Clostridium termitidis strain CT1112, isolated from the gut of the termite, Nasutitermes lujae. Growth curves, pH patterns, protein content, organic acid synthesis, and H₂ production were determined. When grown on 2 g l⁻¹ cellobiose and 2 g l⁻¹ α-cellulose, C. termitidis displayed a cell generation time of 6.5 h and 18.9 h, respectively. The major end-products synthesized on cellobiose included acetate, hydrogen, CO₂, lactate, formate and ethanol, where as on cellulose, the major end-products included hydrogen, acetate, CO₂ and ethanol. The concentrations of acetate were greater than ethanol, formate and lactate on both cellobiose and α-cellulose throughout the entire growth phase. Maximum yields of acetate, ethanol, hydrogen and formate on cellobiose were 5.9, 3.7, 4.6 and 4.2 mmol l⁻¹ culture, respectively, where as on cellulose, the yields were 7.2, 3.1, 7.7 and 2.9 mmol l⁻¹ culture, respectively. Hydrogen and ethanol production rates were slightly higher in C. termitidis cultured on cellobiose when compared to α-cellulose. Although, the generation time on α-cellulose was longer than on cellobiose, H₂ production was favored corresponding to acetate synthesis, thereby restricting the carbon flowing to ethanol. During log phase, H₂, CO₂ and ethanol were produced at specific rates of 4.28, 5.32, and 2.99 mmol h⁻¹ g dry weight⁻¹ of cells on cellobiose and 2.79, 2.59, and 1.1 mmol h⁻¹ g dry weight⁻¹ of cells on α-cellulose, respectively.     

The effects of seed sludge and hydraulic retention time on the production of hydrogen from a cassava processing wastewater and glucose mixture in an anaerobic fluidized bed reactor

The potential for co-fermentation of a cassava processing wastewater and glucose mixture was studied in anaerobic fluidized bed reactors. The effects of different hydraulic retention times (HRTs) (10–2 h) and varying sources of inoculum are reported. The sludge from a UASB reactor that had been used to treat poultry slaughterhouse wastewater (SP) resulted in the highest yields of hydrogen (HY) and ethanol (EtOHY) of 1.0 mmol H₂ g⁻¹ COD (10 h) and 3.0 mmol EtOH g⁻¹ COD (6 h). The sludge from a UASB reactor used for the treatment of swine wastewater (SW) resulted in a maximum HY of 0.65 mmol H₂ g⁻¹ COD (6 h) and EtOHY of 2.1 mmol g⁻¹ COD (10 and 8 h). Methane was produced with a maximum production of 9.68 L CH₄ d⁻¹ L⁻¹. Based on phylogenetic analysis of 16S rRNA, bacteria and methanogenic archaea similar to Lactobacillus and Methanobacterium, respectively, were identified.     

Improvement of hydrogen production by newly isolated Thermoanaerobacterium thermosaccharolyticum IIT BT-ST1

Efficient H₂ producing bacterial strain Thermoanaerobacterium thermosaccharolyticum IIT BT-ST1 was isolated from the anaerobic digester. Taguchi design of experiment was applied to evaluate the influence of the temperature, pH, glucose, FeSO₄ and yeast extract on H₂ production with three levels of orthogonal array in the experimental design. Temperature showed most significant influence on the H₂ production process. Investigation of mutual interaction between the process parameters was studied employing Box–Behnken design. Experimentally optimized process parameters (60 °C, pH 6.5, 20 mM FeSO₄, 4 g L⁻¹ yeast extract and 12 g L⁻¹ glucose) gave the maximum H₂ production of 3930 mL L⁻¹ in 24 h, which have close resemblance with the theoretical values. Continuous H₂ production using packed bed reactor was studied. Maximum H₂ production rate of 1691 mL L⁻¹ h⁻¹ at a dilution rate of 0.6 h⁻¹ was observed which is about 10 times higher than the batch process.     

Hydrogen production from rotten dates by sequential three stages fermentation

This study was devoted for H₂ production from rotten fruits of date palm (Phoenix dactylifera L.) by three fermentation stages. A facultative anaerobe, Escherichia coli EGY was used in first stage to consume O₂ and maintain strict anaerobic conditions for a second stage dark fermentative H₂ production by the strictly anaerobic Clostridium acetobutylicum ATCC 824. Subsequently, a third stage photofermentation using Rhodobacter capsulatus DSM 1710 has been conducted for the H₂ production. The maximum total H₂ yield of the three stages (7.8 mol H₂ mol⁻¹ sucrose) was obtained when 5 g L⁻¹ of sucrose was supplemented to fermentor as rotten date fruits. A maximum estimated cumulative H₂ yield of the three stages (162 LH₂ kg⁻¹ fresh rotten dates) was estimated at the (5 g L⁻¹) sucrose concentration. These results suggest that rotten dates can be efficiently used for commercial H₂ production. The described protocol did not require addition of a reducing agent or flashing with argon which both are expensive.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

Biohydrogen production from ricebran using Clostridium saccharoperbutylacetonicum N1-4

Treated ricebran hydrolysate was fermented anaerobically using Clostridium saccharoperbutylacetonicum N1-4 at an initial pH of 6 ± 0.2 and an operating temperature of 30 °C for production of hydrogen. The effects of different pretreatment methods on the liberation of sugar from 100 g of ricebran per litre of medium (distilled water) were investigated. In addition, the effects of the pretreatment method on ricebran hydrolysates of different initial ricebran concentrations on liberated sugar as well as the effects of the initial inoculum concentration, ricebran (substrate) concentration, and FeSO₄·7H₂O concentration on the yield as well as the productivity of hydrogen were investigated. The combination of enzymatic hydrolysis and a boiling pretreatment method produced the most fermentable sugar, 29.03 ± 0.0 g/L from 100 g of ricebran per litre of medium (distilled water), while the amount of sugar liberated by ricebran hydrolysates of different initial ricebran concentrations upon pretreatment monotonically increased with the initial ricebran concentration. The increment in substrate, inoculum, and FeSO₄·7H₂O concentrations had a significantly positive effect (p < 0.05) on both the yield and productivity of hydrogen. The maximum hydrogen gas yield (Y_P/S) and productivity of 3.37 mol-H₂ per mol-sugar consumed and 7.58 mmol/(L h), respectively, were obtained from ricebran hydrolysate with a 100 g/L ricebran concentration (equivalent to 28.59 ± 1.27 g sugar/L). In other experiments, 0.03 g/L FeSO₄·7H₂O and 1.5 g/L inoculum resulted in the best hydrogen gas yield and productivity from ricebran hydrolysates.     

Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture

Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H₂ from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H₂ yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H₂ mol⁻¹ xylose_consumed and 3.2 ± 0.16 mol-H₂ mol⁻¹ glucose_consumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H₂ yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H₂ mol⁻¹ sucrose_consumed. The effects of stirring speed and different pH levels on growth and H₂ production were studied in the CSABR for highly efficient H₂ production. Growth and H₂ production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H₂ production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H₂ L⁻¹ h⁻¹ at 32, 52, 67, and 86 h, respectively.     

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.