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.     

Effect of temperature on fermentative hydrogen production by mixed cultures

Effect of temperatures ranging from 20 °C to 55 °C on fermentative hydrogen production by mixed cultures was investigated in batch tests. The experimental results showed that, at initial pH 7.0, during the fermentative hydrogen production using glucose as substrate, the substrate degradation efficiency and hydrogen production potential increased with increasing temperatures from 20 °C to 40 °C. The maximal substrate degradation efficiency was 98.1%, the maximal hydrogen production potential was 269.9 mL, the maximal hydrogen yield was 275.1 mL/g glucose and the shortest lag time was 7.0 h. The temperature for fermentative hydrogen production by mixed cultures was optimized to be 40 °C. The expanded Ratkowsky models could be used to describe the effect of temperatures on the hydrogen production potential, maximum hydrogen production rate and the lag time during fermentative hydrogen production.     

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.     

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.     

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.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.     

海洋光合菌群利用乙酸产氢的实验研究

通过富集获得产氢海洋光合菌群,该菌群可以有效利用发酵产氢的关键副产物乙酸作为产氢碳源.温度、光照强度、起始pH和乙酸浓度都对该菌群产氢和生长有明显影响.当在30℃、4000lx光照和起始pH=8.0的条件下培养时,此光合菌群产氢量和底物转化效率较高.乙酸浓度对产氢影响巨大,低浓度乙酸的底物转化效率较高,但总产氢量不高;高浓度乙酸的底物转化效率不高,但总产氢量较高.此实验结果为海洋光合细菌与海洋发酵细菌偶联产氢提供科学参考.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

Selection and isolation of hydrogen-producing fermentative bacteria with high yield and rate and its bioaugmentation process

One of the most effective approaches is to obtain bacteria with high hydrogen-producing capacity and introduce into hydrogen production process in order to perform utmost efficiency. It was proved by analysis of 16S rDNA sequence that it, R3, possibly belongs to a novel genus. The hydrogen production varies depending on glucose concentration and pH respectively. It was found that the optimal glucose concentration is 12.0 g/l and the optimal initial pH is about 5.5. Glucose degradation rate of R3 conforms to Michaelis–Menten formula. There are three stages to actualize bioaugmentation which are cultivated hydrogen-producing fermentative bacteria with high yield and rate, actualize maximum bearing capacity and high physiological activity of hydrogen-producing bacterium added in the system for improving a special performance in the mixed system. It is very important for bioaugmentation application to the biological hydrogen-producing system in order to further enhance hydrogen-producing capacity of the system and stability of the system operation.     

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.     

Fermentative hydrogen production by the newly isolated Enterobacter asburiae SNU-1

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.     

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.     

Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill

The fermentative hydrogen production capability of the newly isolated Clostridium sp. 6A-5 bacterium was studied in a batch cultivation experiment. Various culture conditions (temperature, initial pH, and glucose concentration) were evaluated for their effects on cell growth and hydrogen production (including the yield and rate) of Clostridium sp. 6A-5. Optimal cell growth was observed at 40 °C, initial pH 7.5–8, and glucose concentration 16–26 g/L. The optimal hydrogen yield was obtained at 43 °C, initial pH 8, and glucose concentration 10–16 g/L. Hydrogen began to evolve when cell growth entered the mid-exponential phase and reached the maximum production rate at the late exponential and stationary phases. The maximum hydrogen yield, and rate were 2727 mL/L, and 269.3 mL H₂/L h, respectively. These results indicate that Clostridium sp. 6A-5 is a good candidate for mesophilic fermentative hydrogen production.     

Performance of a groove-type photobioreactor for hydrogen production by immobilized photosynthetic bacteria

The biofilm technique has been proved to be an effective cell immobilization method for wastewater biodegradation but it has had restricted use in the field of photobiological H₂ production. In the present study, a groove-type photobioreactor was developed and it was shown that a groove structure with large specific surface area was beneficial to cell immobilization and biofilm formation of the photosynthetic bacteria on photobioreactor surface as well as light penetration. A series of experiments was carried out on continuous hydrogen production in the groove-type photobioreactor illuminated by monochromatic LED lights and the performance was investigated. The effects of light wavelength, light intensity, inlet glucose concentration, flow rate and initial substrate pH were studied and the results were compared with those obtained in a flat panel photobioreactor. The experimental results show that the optimum operational conditions for hydrogen production in the groove-type photobioreactor were: inlet glucose concentration 10 g/L, flow rate 60 mL/h, light intensity 6.75 W/m², light wavelength 590 nm and initial substrate pH 7.0. The maximum hydrogen production rate, H₂ yield and light conversion efficiency in the groove-type photobioreactor were 3.816 mmol/m²/h, 0.75 molH₂/molglucose and 3.8%, respectively, which were about 75% higher than those in the flat panel photobioreactor.     

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.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

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.     

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.