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.     

Producing hydrogen from the fermentation of cheese whey and glycerol as cosubstrates in an anaerobic fluidized bed reactor

This study aimed to evaluate the effect of the organic loading rate (OLR) (60, 90, and 120 g Chemical Oxygen Demand (COD). L^?1. d^?1) on hydrogen production from cheese whey and glycerol fermentation as cosubstrates (50% cheese whey and 50% glycerol on a COD basis) in a thermophilic fluidized bed reactor (55 °C). The increase in the OLR to 90 gCOD.L^?1. d^?1 favored the hydrogen production rate (HPR) (3.9 L H₂. L^?1. d^?1) and hydrogen yield (HY) (1.7 mmol H₂. gCOD^?1_app) concomitant with the production of butyric and acetic acids. Employing 16S rRNA gene sequencing, the highest hydrogen production was related to the detection of Thermoanaerobacterium (34.9%), Pseudomonas (14.5%), and Clostridium (4.7%). Conversely, at 120 gCOD.L^?1. d^?1, HPR and HY decreased to 2.5 L H₂. L^?1. d^?1 and 0.8 mmol H₂. gCOD^?1_app, respectively, due to lactic acid production that was related to the genera Thermoanaerobacterium (50.91%) and Tumebacillus (23.56%). Cofermentation favored hydrogen production at higher OLRs than cheese whey single fermentation.     

The buffer composition impacts the hydrogen production and the microbial community composition in non-axenic cultures

Hydrogen obtained from biomass via dark fermentation is considered a sustainable and clean energy carrier. Batch fermentations with cheese whey powder were performed to assess total hydrogen production (H_max), volumetric hydrogen production rate (VHPR), maximum lactose consumption (S_max), maximum lactose consumption rate (R_max,S), hydrogen molar yield (HMY) and the bacterial species present using two mineral media formulation (A, B). The highest VHPR was 304.8 cm³ dm⁻³ h⁻¹ and the HMY was 1.8 mol mol⁻¹. Medium B yielded around twice the VHPR than the attained with medium A, but HMY only had a slight increment with the use of medium B. The values reached for S_max (17.3 g dm⁻³), H_max (4.863 dm³) and R_max,S (2.7 g dm⁻³ h⁻¹) were also enhanced with medium B. Results suggest that butyrate levels and lower pH are the reasons for diminished hydrogen production with medium A. The microbial communities were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Only one band was observed in the experiments with medium A, the sequence retrieved from this band presented a closest relative match to the sequence from Citrobacter freundii JCM (100% identity); whereas for medium B, three bands were detected. Sequences from these bands presented high homology to sequences from Clostridium perfringens W11 (95% identity), uncultured Lachnospiraceae bacterium clone MS146A1 E12 (100% identity) and Enterobacter cloacae GH1 (100% identity). From the results obtained it is clear that the formulation of culture media had a strong effect on hydrogen production, kinetics and also on the microbial diversity.     

Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation

This article describes the ability of the Chlorella vulgaris BEIJ strain G-120 to produce hydrogen (H₂) via both direct and indirect pathways without the use of nutrient starvation. Photobiological H₂ production reached a maximum rate of 12 mL H₂ L^?1 h^?1, corresponding to a light conversion efficiency (light to H₂) of 7.7% (average 3.2%, over the 8-day period) of PAR, (photosynthetically active irradiance). Cells presented a maximum in vivo hydrogenase activity of 25.5 ± 0.2 nmoles H₂ μgChl^?1 h^?1 and the calculated in vitro hydrogenase activity was 830 ± 61 nmoles H₂ μgChl^?1 h^?1. The strain is able to grow either heterotrophically or photo autotrophically. The total output of 896 mL of H₂ was attained for illuminated culture and 405 mL for dark cultures. The average H₂ production rate was 4.98 mL L^?1 h^?1 for the illuminated culture and 2.08 mL L^?1 h^?1 for the one maintained in the dark.     

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.     

Electro-hydrolysis of cheese whey solution for hydrogen gas production and chemical oxygen demand (COD) removal using photo-voltaic cells (PVC)

Diluted cheese whey (CW) solution was used for hydrogen gas production by electro-hydrolysis using photo-voltaic cells (PVC) as source of electricity. Effects of initial chemical oxygen demand (COD) concentration on the rate and yield of hydrogen gas production were investigated using a completely mixed and sealed reactor with aluminum electrodes. Cumulative hydrogen gas formation (CHF) increased with increasing initial COD concentration. The highest cumulative hydrogen gas volume (26472 mL), hydrogen gas production rate (4553 mL d⁻¹), hydrogen yield (7004 mL H₂ g⁻¹ COD), and percent COD removal (21.5%) were obtained with initial COD of 35172 mg L⁻¹. H₂ gas formation from water control was only 5365 mL. pH of the CW solution increased with decreasing conductivities during the course of experiments. Gas phase contained more than 99% H₂ at the end of experiments. The highest energy efficiency (20.4%) was also obtained with the highest COD content. Nearly pure hydrogen gas formation by electro-hydrolysis of cheese whey using PVC panels was proven to be an effective method.     

Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates

Acetone butanol ethanol (ABE) was produced from hydrolysed corn stover and switchgrass using Clostridium beijerinckii P260. A control experiment using glucose resulted in the production of 21.06 g L^?1 total ABE. In this experiment an ABE yield and productivity of 0.41 and 0.31 g L^?1 h^?1 was achieved, respectively. Fermentation of untreated corn stover hydrolysate (CSH) exhibited no growth and no ABE production; however, upon dilution with water (two fold) and wheat straw hydrolysate (WSH, ratio 1:1), 16.00 and 18.04 g L^?1 ABE was produced, respectively. These experiments resulted in ABE productivity of 0.17–0.21 g L^?1 h^?1. Inhibitors present in CSH were removed by treating the hydrolysate with Ca(OH)₂ (overliming). The culture was able to produce 26.27 g L^?1 ABE after inhibitor removal. Untreated switchgrass hydrolysate (SGH) was poorly fermented and the culture did not produce more than 1.48 g L^?1 ABE which was improved to 14.61 g L^?1. It is suggested that biomass pretreatment methods that do not generate inhibitors be investigated. Alternately, cultures resistant to inhibitors and able to produce butanol at high concentrations may be another approach to improve the current process.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

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.     

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.     

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.     

Using a food and paper-cardboard waste blend as a novel feedstock for hydrogen production: Influence of key process parameters on microbial diversity

In this study, fermentation of a thermally treated simulated organic solid waste into hydrogen (H₂) was examined using a pretreated anaerobic mixed culture. The culture was fed a steam exploded food waste plus paper-cardboard waste blend liquor with and without linoleic acid (LA). The individual and interaction effects of the initial pH, LA concentration and the initial chemical oxygen demand (COD) concentration on H₂ and methane (CH₄) production was assessed using a Box–Behnken design (BBD). The BBD model predicted a maximum H₂ yield of 87 mL g⁻¹ COD or 98 mL H₂ g⁻¹ VS with 1.6 g L⁻¹ LA, an initial pH of 5.93 and an initial COD of 9.34 g COD L⁻¹. The major microbial populations detected in cultures at pH 5.5 with and without LA included Clostridium sp., Enterococcus asini, Enterococcus faecalis, and Lactobacillus gallinarum. The dendrogram for the 16S rRNA gene T-RFs profiles showed four major groups with a similarity index of 72–75% for Clade III. The major H₂-producing populations were grouped in Clade I with a similarity index range of 55–75%.     

Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield,prolonged production

Escherichia coli wild type has the ability to utilize lactose or the mixture of lactose and glycerol producing bio-hydrogen (H₂) at different pH values. At pH 7.5 in hyaB (lacking large subunit of hydrogenase (Hyd)-1) and hybC (lacking large subunit of Hyd-2) single mutants fermenting lactose (1 g L⁻¹) H₂ yield was ∼7- and 5-fold more, respectively, compared to the wild type. During the fermentation of lactose (1 g L⁻¹) and glycerol (10 g L⁻¹) mixture H₂ yield in wild type increased ∼3-fold, compared to fermenting lactose only. H₂ generation in wild type was monitored in batch cultures during 168 h of growth when utilizing the mixture of lactose and glycerol in all combinations of different concentration. In hyaB but not in hybC mutant H₂ evolution was detected till 240 h in the mixture of 5 g L⁻¹ lactose and 10 g L⁻¹ glycerol. The highest H₂ production rate of 21.94 mL L⁻¹ h⁻¹ was detected in hyaB mutant at pH 7.5 when 1 g L⁻¹ lactose was applied. The results showed optimized H₂ production using different mutants, lactose and its mixture with glycerol. They can be applied for renewable energy, especially bio-H₂ production.     

Optimization of molecular hydrogen production by Rhodobacter sphaeroides O.U.001 in the annular photobioreactor using response surface methodology

Photofermentative H₂ production at higher rate is desired to make H₂ viable as cheap energy carrier. The process is influenced by C/N composition, pH levels, temperature, light intensity etc. In this study, Rhodobacter sphaeroides strain O.U 001 was used in the annular photobioreactor with working volume 1 L, initial pH of 6.7 ± 0.2, inoculum age 36 h, inoculum volume 10% (v/v), 250 rpm stirring and light intensity of 15 ± 1.1 W m⁻². The effect of parameters, i.e. variation in concentration of DL malic acid, L glutamic acid and temperature on the H₂ production was noted using three factor three level full factorial designs. Surface and contour plots of the regression models revealed optimum H₂ production rate of 7.97 mL H₂ L⁻¹ h⁻¹ at 32 °C with 2.012 g L⁻¹ DL malic acid and 0.297 g L⁻¹ L glutamic acid, which showed an excellent correlation (99.36%) with experimental H₂ production rate of 7.92 mL H₂ L⁻¹ h⁻¹.     

Biohydrogen production from Humulus scandens by dark fermentation: Potential evaluation and process optimization

As a perennial grass, Humulus scandens is rich in cellulose which can be fermented for bioenergy producing. Hence, the hydrogen production potential of the Humulus scandens from dark fermentation by Enterobacter aerogenes was investigated in this paper. Cellulase amount, inoculation amount, and initial pH value were evaluated. The interrelationship between these factors were studied by Response Surface Box-Behnken Experiments. Results showed that there was a significant correlation between the three factors. Through the correction of the regression equation, the optimized technological conditions were obtained. The amount of cellulase was 0.203 g g⁻¹ TS, the inoculation amount was 42.6%, the initial pH value was 6.59, the pre-estimated maximum cumulative value of hydrogen production was 65.12 mL g⁻¹ TS, and it was similar to the test mean value which was 64.08 mL g⁻¹ TS.     

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.     

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.     

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.     

Photohydrogen production from lactose and lactate by recombinant strains of Rhodobacter capsulatus: Modeling and optimization

Photofermentative hydrogen production from synthetic mixtures of lactose and lactate mimicking cheese whey was modeled and optimized using Design of Experiments and Response Surface Methodology. Five continuous parameters (light intensity, pH, lactose, lactate and glutamate concentrations) were studied as a function of buffer type (KPi or Borax) using two recombinant bacterial strains. For Rhodobacter capsulatus B10(lacZ), buffer type influenced the optimal parameter values but the optimal responses were similar in both buffers. In contrast, for R. capsulatus IR3(lacZ), responses were higher in Borax buffer than in KPi and were significantly higher than in strain B10(lacZ). Thus, the experimental optimized responses for specific volumetric H₂ production, volumetric H₂ production rate and substrate (lactose plus lactate) to H₂ conversion rate in Borax buffer, were 12,150 ml L^?1, 48.5 ml L^?1 h^?1 and 41.2%, respectively, for IR3(lacZ) compared to 6150 ml L^?1, 33.5 ml L^?1 h^?1 and 32.5%, respectively, for B10(lacZ).