Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.     

Application of desirability function based on neural network for optimizing biohydrogen production process

A fractional factorial design was carried out to investigate the effects of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures in batch tests and then the experimental data of substrate degradation efficiency, hydrogen yield and average hydrogen production rate were described by a neural network, based on which the simultaneous optimization of the three responses was performed by the method of desirability function. The analysis showed that the neural network could successfully describe the effects of temperature, initial pH and glucose concentration on the substrate degradation efficiency, hydrogen yield and average hydrogen production rate of this study. The maximum substrate degradation efficiency of 95.3%, hydrogen yield of 305.3 mL/g glucose and average hydrogen production rate of 23.9 mL/h were all obtained at the optimal temperature of 39.0 °C, initial pH of 7.0 and glucose concentration of 24.6 g/L identified by the method of desirability function based on a neural network. In sum, the method of desirability function based on a neural network was a useful tool to optimize several responses for fermentative hydrogen production processes simultaneously.     

Inhibitory effect of ethanol,acetic acid,propionic acid and butyric acid on fermentative hydrogen production

The inhibitory effect of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate. The experimental results showed that, at 35 °C and initial pH 7.0, during the fermentative hydrogen production, the substrate degradation efficiency, hydrogen production potential, hydrogen yield and hydrogen production rate all trended to decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effect of added ethanol on fermentative hydrogen production was smaller than those of added acetic acid, propionic acid and butyric acid. The modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study successfully. The modified Logistic model could describe the progress of cumulative hydrogen production.     

Combined effects of temperature and pH on biohydrogen production by anaerobic digested sludge

A full factorial design was conducted to investigate the combined effects of temperatures and initial pH on fermentative hydrogen production by mixed cultures in batch tests. The experimental results showed that the modified Logistic model can be used to describe the progress of cumulative hydrogen production in the batch tests of this study. The modified Ratkowsky model can be used to describe the combined effects of the temperatures and initial pH on the substrate degradation efficiency, hydrogen yield and average hydrogen production rate. The temperatures and initial pH had interactive impact on fermentative hydrogen production. The maximum substrate degradation efficiency, the maximum hydrogen yield and the maximum average hydrogen production rate was predicted at the temperature of 37.8 °C and the initial pH of 7.1, 37.4 °C and 6.9, and 38.2 °C and 7.2, respectively. In general, the optimal temperature for the fermentative hydrogen production was around 37.8 °C and the optimal initial pH for the fermentative hydrogen production was around 7.1.     

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 °C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57–70%, 41–55% and 37–67%.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria

Beer lees are the main by-product of the brewing industry. Biohydrogen production from beer lees using anaerobic mixed bacteria was investigated in this study, and the effects of acidic pretreatment, initial pH value and ferrous iron concentration on hydrogen production were studied at 35 °C in batch experiments. The hydrogen yield was significantly enhanced by optimizing environmental factors such as hydrochloric acid (HCl) pretreatment of substrate, initial pH value and ferrous iron concentration. The optimal environmental factors of substrate pretreated with 2% HCl, pH = 7.0 and 113.67 mg/l Fe²⁺ were observed. A maximum cumulative hydrogen yield of 53.03 ml/g-dry beer lees was achieved, which was approximately 17-fold greater than that in raw beer lees. In addition, the degradation efficiency of the total reducing sugar, and the contents of hemicellulose, cellulose, lignin and metabolites are presented, which showed a strong dependence on the environmental factors.     

Feasibility of biohydrogen production at low temperatures in unbuffered reactors

Feasibility of biohydrogen production by dark fermentation at two temperatures (22 °C and 37 °C) in unbuffered batch reactors was evaluated using heat-treated compost as inocula and sucrose as substrate, without any initial pH adjustment or inorganic nutrient supplements. Gas production was quantified by two different pressure release methods – intermittent pressure release (IPR) and continuous pressure release (CPR). Hydrogen production (47.2 mL/g COD/L) and sucrose-to-hydrogen conversion efficiency (53%) were both found to be highest at the lower temperature and IPR conditions. Hydrogen production was higher at the lower temperature irrespective of the pressure release condition. The high yield of 4.3 mol of hydrogen/mole of sucrose obtained in this study under IPR conditions at 22 °C is equivalent to or better than the literature values reported for buffered reactors. Even though literature reports have implied potential inhibition of hydrogen production at high hydrogen partial pressures resulting from IPR conditions, our results did not show any negative effects at hydrogen partial pressures exceeding 5.0 × 10⁴ Pa. While our findings are contrary to literature reports, they make a strong case for cost-effective hydrogen production by dark fermentation.     

High yield simultaneous hydrogen and ethanol production under extreme-thermophilic (70 °C) mixed culture environment

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO₄ (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H₂/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.     

Gamma irradiation as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge

Gamma irradiation was used as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The experimental results demonstrated that 5.0 kGy was optimal dose among the different doses (0.5–10 kGy) applied in this study. The maximum cumulative hydrogen production, hydrogen yield, hydrogen production rate and substrate degradation efficiency of the sludge irradiated at such dose were 529.4 mL, 267.7 mL/g glucose, 37.25 mL/h and 98.9%, respectively when the fermentation conditions were as follows: at 36 °C, initial pH 7.0 and 10 g/L glucose as substrate. In comparison with the conventional pretreatment methods, such as heat-shock, acid, base, aeration and chloroform, gamma irradiation was more powerful pretreatment method for enriching hydrogen-producing bacteria. The effect of Gamma irradiation on the microbial community structure of the pretreated sludge needs further study.     

Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures

Batch tests were conducted to evaluate the enhancement of hydrogen/ethanol (EtOH) productivity using cow dung microflora to ferment α-cellulose and saccharification products (glucose and xylose). Hydrogen/ethanol production was evaluated based on hydrogen/ethanol yields (HY/EY) under 55 °C at various initial pH conditions (5.5–9.0). Our test results indicate that cow dung sludge is a good mixed natural-microflora seed source for producing biohydrogen/ethanol from cellulose and xylose. The heat-pretreatment, commonly used to produce hydrogen more efficiently from hexose, applied to mixed anaerobic cultures did not help cow dung culture convert cellulose and xylose into hydrogen/ethanol. Instead of heat-pretreatment, the mixed culture received enrichments cultivated at 55 °C for 4 days. Positive results were observed: hydrogen/ethanol production from fermenting cellulose and xylose was effectively enhanced at increases of 4.8 (ethanol) to 8 (hydrogen) and 2.4 (ethanol) to 15.6 (hydrogen) folds, respectively. In which, the ethanol concentration produced from xylose reached 4–4.4 g/L, an output comparable to that of using heat-treated sewage sludge and better than that (1.25–3 g/L) using pure cultures. Our test results show that for the enriched cultures the initial cultivation pH can affect hydrogen/ethanol production including HY, EY and liquid fermentation product concentration and distribution. These results were also concurred using a denaturing gradient gel electrophoresis analysis saying that both cultivation pH and substrate can affect the enriched cow dung culture microbial communities. The enriched cow dung culture had an optimal initial cultivation pH range of 7.6–8.0 with peak HY/EY values of 2.8 mmol-H₂/g-cellulose, 5.8 mmol-EtOH/g-cellulose, 0.3 mol-H₂/mol-xylose and 1 mol-EtOH/mol-xylose. However, a pH change of 0.5 units from the optimal values reduced hydrogen/ethanol production efficiency by 20%. Strategies based on the experimental results for optimal hydrogen/ethanol production from cellulose and xylose using cow dung microflora are proposed.     

Optimization of fermentative hydrogen production process using genetic algorithm based on neural network and response surface methodology

A central composite design was carried out to investigate the effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures in batch test. The modeling abilities of the response surface methodology model and neural network model, as well as the optimizing abilities of response surface methodology and the genetic algorithm based on a neural network model were compared. The results showed that the root mean square error and the standard error of prediction for the neural network model were much smaller than those for the response surface methodology model, indicting that the neural network model had a much higher modeling ability than the response surface methodology model. The maximum hydrogen yield of 289.8 mL/g glucose identified by response surface methodology was a little lower than that of 360.5 mL/g glucose identified by the genetic algorithm based on a neural network model, indicating that the genetic algorithm based on a neural network model had a much higher optimizing ability than the response surface methodology. Thus, the genetic algorithm based on a neural network model is a better optimization method than response surface methodology and is recommended to be used during the optimization of fermentative hydrogen production process.     

Optimization and evaluation of fermentative hydrogen production and wastewater treatment processes using data enveloping analysis (DEA) and Taguchi design of experimental (DOE) methodology

The combined process efficiency with respect to fermentative hydrogen (H₂) production and wastewater treatment was evaluated in a series of batch experiments to enumerate the role of selected factors viz., origin of inoculum, pre-treatment, inlet pH and feed composition under anaerobic microenvironment using mixed culture. H₂ production and substrate degradation were found to depend significantly on the selected factors with individual conditions to achieve effective process performance. Significantly diverse operational conditions were observed for both H₂ production and substrate degradation with respect to process efficiency. However, while dealing with H₂ production in association with wastewater treatment, both the parameters are important and balancing the conditions for combined effective performance is critical. Data enveloping analysis (DEA) was applied to evaluate the combined system performance with respect to the two output parameters (H₂ production and substrate degradation) based on relative efficiency. Among various experimental combinations studied, those with untreated anaerobic mixed inoculum under acidophilic conditions (inlet pH 5.5) using simple wastewater as fermentative substrate illustrated combined process efficiency with respect to H₂ production (1.919 m mol H₂/day; 0.52 mol H₂/kg COD_R-day) and substrate degradation (substrate degradation rate, 4.56 kg COD/m³-day). DEA methodology provide the relative efficiency of the system by integrating two output parameters. Further, design of experimental methodology (DOE) by Taguchi approach was applied to enumerate the role of selected factors on the H₂ production and substrate degradation with the final aim of optimizing the process. By adapting the derived optimum conditions, the performance with respect to H₂ production and substrate degradation could be enhanced by three fold.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Effect of temperature on anaerobic fermentative hydrogen gas production from feedlot cattle manure using mixed microflora

Hydrogen production from the anaerobic fermentation of feedlot cattle manure was examined in batch cultures over a temperature range from 36 to 60 °C at a pH of 5.2. The amount of hydrogen produced increased with temperature to a maximum of 65 L H₂ kg TS⁻¹ at 52 °C. At temperatures > 52 °C, acetate was the main volatile fatty acid (VFA) accumulated, while at <52 °C butyrate accumulated the most. Formate was detected in the 56 and 60 °C treatments but was absent in all others. Thermophilic conditions resulted in the highest hydrogen production rates, with maximum hydrogen production occurring 52 °C. Changing incubation temperature by small (4 °C) increments up or down from 52 °C resulted in changes in the metabolic flux (conversion of substrate to VFA and gaseous products) of the anaerobic digestion system. These findings indicate that the hydrogen production potential of anaerobic systems utilizing heat treated cattle manure as inoculum is affected greatly by incubation temperature.     

Fermentative hydrogen production by diverse microflora

In this study, hydrogen production with activated sludge, a diverse bacterial source has been investigated and compared to microflora from anaerobic digester sludge, which is less diverse. Batch experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The hydrogen production yields with activated sludge at 37 °C and 55 °C were 0.56 and 1.32 mol H₂/mol glucose consumed, respectively. While with anaerobically digested sludge hydrogen yield was 2.18 mol H₂/mol glucose consumed at 37 °C and 1.25 mol H₂/mol glucose consumed at 55 °C. The results of repeated batch experiments for 615 h resulted in average yields of 1.21 ± 0.62 and 1.40 ± 0.16 mol H₂/mol glucose consumed for activated sludge and anaerobic sludge, respectively. The hydrogen production with activated sludge was not stable during the repeated batches and the fluctuation in hydrogen production was attributed to formation of lactic acid as the predominant metabolite in some batches. The presence of lactic acid bacteria in microflora was confirmed by PCR-DGGE.     

The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures

The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H₂/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.     

Enhancement of batch biohydrogen production from prehydrolysate of acid treated oil palm empty fruit bunch

Carbohydrates from hydrolyzed biomass has been a potential feedstock for fermentative hydrogen production. In this study, oil palm empty fruit bunch (OPEFB) was treated by sulfuric acid in different concentrations at 120 °C for 15 min in the autoclave. The optimal condition for pretreatment was obtained when OPEFB was hydrolyzing at 6% (w/v) sulfuric acid concentration, which gave the highest total sugar of 26.89 g/L and 78.51% of sugar production yield. However, the best conversion efficiency of OPEFB pretreatment was 39.47 at sulfuric acid concentration of 4%. A series of batch fermentation were performed to determine the effect of pH in fermentation media and the potential of this prehydrolysate was used as a substrate for fermentative hydrogen production under optimum pretreatment conditions. The prehydrolysate of OPEFB was efficiently converted to hydrogen via fermentation by acclimatized mixed consortia. The maximum hydrogen production was 690 mL H₂ L⁻¹ medium, which corresponded to the yield of 1.98 molH₂/mol_xylose achieved at pH 5.5 with initial total sugar concentration of 5 g/L. Therefore, the results implied that OPEFB prehydrolysate is prospective substrate for efficient fermentative hydrogen conducted at low controlled pH. No methane gas was detected throughout the fermentation.