Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

Co-digestion of cassava starch wastewater with buffalo dung for bio-hydrogen production

Batch and continuous modes for bio-hydrogen production by co-digesting cassava starch wastewater with buffalo dung were investigated. Response surface methodology with central composite design was used to optimize the bio-hydrogen production conditions. A hydrogen production potential of 1787 mL H₂/L was achieved under optimal conditions of 2.84 g/L of NaHCO₃, an initial pH of 6.77 and a total chemical oxygen demand (tCOD)/total nitrogen ratio of 42.36. A continuous stirred tank reactor was operated under the optimum conditions from batch mode to investigate the effects of hydraulic retention time (HRT) of 72, 60 and 48 h on hydrogen production. The highest hydrogen content, hydrogen production rate and hydrogen yield of 33%, 839 mL H₂/L.d and 16.90 mL H₂/g-COD_added, respectively, were achieved at a HRT of 60 h. The predominant hydrogen producer under the optimal conditions in batch mode was Clostridium sp. while Clostridium sp., Megasphaera sp. and Chloroflexi sp. were observed in the continuous hydrogen production mode at an optimal HRT.     

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H₂/g-substrate, hydrogen production rate of 39 mL/g h⁻¹ and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0-5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H₂/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Hydrogen production from water hyacinth through dark- and photo- fermentation

This article discusses the method of producing hydrogen from water hyacinth. Water hyacinth was pretreated with microwave heating and alkali to enhance the enzymatic hydrolysis and hydrogen production in a two-step process of dark- and photo- fermentation. Water hyacinth with various concentrations of 10–40 g/l was pretreated with four methods: (1) steam heating; (2) steam heating and microwave heating/alkali pretreatment; (3) steam heating and enzymatic hydrolysis; (4) steam heating, microwave heating/alkali pretreatment and enzymatic hydrolysis. Water hyacinth (20 g/l) pretreated with method 4 gave the maximum reducing sugar yield of 30.57 g/100 g TVS, which was 45.6% of the theoretical reducing sugar yield (67.0 g/100 g TVS). The pretreated water hyacinth was used to produce hydrogen by mixed H₂-producing bacteria in dark fermentation. The maximum hydrogen yield of 76.7 ml H₂/g TVS was obtained at 20 g/l of water hyacinth. The residual solutions from dark fermentation (mainly acetate and butyrate) were used to further produce hydrogen by immobilized Rhodopseudomonas palustris in photo fermentation. The maximum hydrogen yield of 522.6 ml H₂/g TVS was obtained at 10 g/l of water hyacinth. Through a combined process of dark- and photo- fermentation, the maximum hydrogen yield from water hyacinth was dramatically enhanced from 76.7 to 596.1 ml H₂/g TVS, which was 59.6% of the theoretical hydrogen yield.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.     

Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation

Hydrogen formation performances of different anaerobic bacteria were investigated in batch dark fermentation of waste wheat powder solution (WPS). Serum bottles containing wheat powder were inoculated with pure cultures of Clostridium acetobutylicum (CAB), Clostridium butyricum (CB), Enterobacter aerogenes (EA), heat-treated anaerobic sludge (ANS) and a mixture of those cultures (MIX). Cumulative hydrogen formation (CHF), hydrogen yield (HY) and specific hydrogen production rate (SHPR) were determined for every culture. The heat-treated anaerobic sludge was found to be the most effective culture with a cumulative hydrogen formation of 560 ml, hydrogen yield of 223 ml H₂ g⁻¹ starch and a specific hydrogen production rate of 32.1 ml H₂ g⁻¹ h⁻¹.     

Co-digestion of oil palm trunk hydrolysate with slaughterhouse wastewater for thermophilic bio-hydrogen production by Thermoanaerobacterium thermosaccharolyticm KKU19

The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H₂SO₄ method using 1.56% (w/v) H₂SO₄ and 7.50 min reaction time at 450 W. The Plackett–Burman method was used to screen the key factors that influenced the hydrogen production potential (P_s). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO₄ concentration influenced the P_s. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum P_s of 2604 ± 86 mL H₂/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO₄ concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The P_s obtained when the hydrolysate was supplemented with SHW (2604mL ± 86 mL H₂/L substrate) was comparable to the P_s obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 ± 87 mL H₂/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source.     

Enhancement effect of zero-valent iron nanoparticle and iron oxide nanoparticles on dark fermentative hydrogen production from molasses-based distillery wastewater

This study investigates the effect of two different iron compounds (zero-valent iron nanoparticle: nZVI and iron oxide nanoparticles: nIO) and pH on fermentative biohydrogen production from molasses-based distillery wastewater. The nZVI and nIO of optimum particle sizes of 50 nm and 55 nm respectively were synthesized and applied for fermentative hydrogen (H₂) production. The addition of nIO & nZVI at (0.7 g/L, pH: 6) resulted in the highest H₂ yield, H₂ production rate, H₂ content and COD reduction. Moreover, the kinetic parameters of H₂ production potential (P) and H₂ production rate (R_m) increased to 387 mL, and 22.2 mL/h, respectively for nZVI, these values were 363 mL and 21.8 mL/h for nIO. The results obtained indicated the positive effect of nZVI and nIO addition on enhanced fermentative H₂ production. The addition of nZVI & nIO resulted in 71% and 69.4% enhancement in biohydrogen production respectively.     

Ultrasonic pretreatment for an enhancement of biohydrogen production from complex food waste

The efficacy of ultrasonication pretreatment method for complex food waste prior to anaerobic digestion is evaluated for enhancement of H₂ yield (HY) and rate (R). The RSM results showed that the ultimate H₂ production increased with increasing TS content and ultrasonication time (UT). Desirability function integrated with RSM predicted an optimum condition of TS and UT as: 8% TS and 12 min, for maximization of HY and R. The highest HY, 149 mL/g VS_added, and R, 5.23 mL/h, were achieved during the verification test at optimized conditions. Furthermore, a significant decreased lag phase followed by highest molar HBu/HAc ratio (2.2) was also achieved at optimized conditions with lowest specific energy input (13,500 kJ/kg TS). The significant relative enhancement of HY, 75%, and R, 104%, implies that ultrasonically pretreated complex food waste with higher TS loading is about 1.7–2.1 times more effective for enhanced bioH₂ production compared to unsonicated food waste.     

Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).     

Biohydrogen production from cassava starch processing wastewater by thermophilic mixed cultures

Natural microbial consortia from hot spring samples were used to developed thermophilic mixed cultures for biohydrogen production from cassava starch processing wastewater (CSPW). Significant hydrogen production potentials were obtained from three thermophilic mixed cultures namely PK, SW and PR with maximum hydrogen production yields of 249.3, 180 and 124.9 mL H₂/g starch, respectively from raw cassava starch and 252.4, 224.4 and 165.4 mL H₂/g starch, respectively from gelatinized cassava starch. Acetic acid-ethanol and acetic-lactic acid type fermentation were observed in cassava starch fermentation, based on three thermophilic mixed cultures performance. The thermophilic mixed cultures PK, SW and PR exhibited the maximum hydrogen yield of 287, 264 and 232 mL H₂/g starch in CSPW, respectively corresponding to 53%, 48.7% and 42.8% of the theoretical values. Phylogenetic analysis of thermophilic mixed cultures revealed that members involved cassava starch degrading bacteria and hydrogen producers in both raw cassava starch and CSPW were phylogenetically related to the Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Anoxybacillus sp., Geobacillus sp. and Clostridium sp.     

Microbial diversity of hydrogen-producing bacteria in batch reactors fed with cellulose using leachate as inoculum

Hydrogen production using cellulosic residues offers the possibility of waste minimization with renewable energy recovery. In the present study, heat-treated biomass purified from leachate was used as inoculum in batch reactors for hydrogen production fed with different concentrations of cellulose (2.5, 5.0 and 10 g/L), in the presence and absence of exogenous cellulase. The heat-treated biomass did not degrade cellulose and hydrogen production was not detected in the absence of cellulase. In reactors with cellulase, the hydrogen yields were 1.2, 0.6 and 2.3 mol H₂/mol of hydrolyzed cellulose with substrate degradation of 41.4, 28.4 and 44.7% for 2.5, 5.0 and 10 g/L cellulose, respectively. Hydrogen production potentials (P) varied from 19.9 to 125.9 mmol H₂ and maximum hydrogen production rates (R_m) were among 0.8–2.3 mmol H₂/h. The reactor containing 10 g/L of cellulose presented the highest P and R_m among the conditions tested. The main acid produced in reactors were butyric acid, followed by acetic, isobutyric and propionic acids. Bacteria similar to Clostridium sp. (98–99%) were identified in the reactors with cellulase. The heat-treated leachate can be used as an inoculum source for hydrogen production from hydrolyzed cellulose.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Co-fermentation of sewage sludge and algae and Fe2+ addition for enhancing hydrogen production

Co-fermentation of sewage sludge and algae was performed for enhancing the hydrogen production, and the effect of Fe²⁺ on co-fermentation process was examined. Results showed that both co-fermentation process and Fe²⁺ addition promoted hydrogen production. Highest hydrogen production of 28 mL/100 mL (14.8 mL H₂/g VS_added) was obtained from the co-fermentation group with 600 mg/L Fe²⁺ addition, which was 2.15 times, 2.00 times and 1.87 times of mono-fermentation of sludge, mono-fermentation of algae, and the co-fermentation group without Fe²⁺ addition. Both volatile solids and protein degradation were stimulated by co-fermentation process. Microbial analysis showed that co-fermentation groups with Fe²⁺ addition enriched Clostridium sensu stricto 13, Clostridium tertium and Terrisporobacter, which were positively correlated with cumulative hydrogen production. This study suggested that the co-fermentation of sludge and algae in the presence of Fe²⁺ could significantly improve the hydrogen production by stimulating the hydrogen-producing metabolism.     

Effect of initial pH,nutrients and temperature on hydrogen production from palm oil mill effluent using thermotolerant consortia and corresponding microbial communities

Thermotolerant consortia were obtained by heat-shock treatment on seed sludge from palm oil mill. Effect of the initial pH (4.5–6.5) on fermentative hydrogen production palm oil mill effluent (POME) showed the optimum pH at 6.0, with the maximum hydrogen production potential of 702.52 mL/L-POME, production rate of 74.54 mL/L/h. Nutrients optimization was investigated by response surface methodology with central composite design (CCD). The optimum nutrients contained 0.25 g urea/L, 0.02 g Na₂HPO₄/L and 0.36 g FeSO₄·7H₂O/L, giving the predicted value of hydrogen production of 1075 mL/L-POME. Validation experiment revealed the actual hydrogen production of 968 mL/L-POME. Studies on the effect of temperature (25–55 °C) revealed that the maximum hydrogen production potential (985.3 mL/L-POME), hydrogen production rate (75.99 mL/L/h) and hydrogen yield (27.09 mL/g COD) were achieved at 55, 45 and 37 °C, respectively. Corresponding microbial community determined by the DGGE profile demonstrated that Clostridium spp. was the dominant species. Clostridium paraputrificum was the only dominant bacterium presented in all temperatures tested, indicating that the strain was thermotolerant.     

Enhancement of biohydrogen production from sustainable orange peel wastes using Enterobacter species isolated from domestic wastewater

Five facultative anaerobic bacterial isolates were recovered from domestic wastewater. These isolates were identified based on the 16S rRNA as Enterobacter aerogenes (one isolate), Enterobacter cloacae (two isolates), and Cronobacter sakazakii (three isolates). These isolates were examined for their potential to evolve hydrogen on a glucose medium. The most potent hydrogen‐producing isolates, E aerogenes (KY549389) and E cloacae (KY524293), were examined for their capacity to generate hydrogen, acetone, butanol, and ethanol using orange peel (OP) hydrolysate. OP powder was pretreated with n‐hexane to remove the toxicity of d ‐limonene. Different concentrations (4%, 6%, and 8% w/v) of limonene‐free OP were subjected to the boiling water (temperature of 100°C) or acid (HCl) treatments. The maximum fermentative H₂ production of 1700 and 1620 mL/L was obtained from 6% OP hydrolysate extracted with boiling water using facultative anaerobic E aerogenes (KY549389) and E cloacae (KY524293), respectively. Hydrogen production efficiency was 0.99 and 1.19 mol H₂/mol glucose for E aerogenes and E cloacae, respectively. The total fermentative acetone, butanol, and ethanol (ABE) generated by E aerogenes and E cloacae were 0.78 and 0.38 g/L including acetone (0.05 and 0.04 g/L), butanol (0.011 and 0.013 g/L), and ethanol (0.71 and 0.32 g/L), respectively. The maximum ABE productivity was 0.01 and 0.005 g/L/h generated at 60 g/L OP hydrolysate by E aerogenes and E cloacae, respectively. These strains were positive for nitrogen fixation (nitrogenase) capability estimated by the acetylene reduction assay. Application of OP hydrolysate without the addition of any nutritional components or reducing agent is considered an eco‐friendly, economical, and commercial substrate for desired biofuel production.     

Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization

Batch production of biohydrogen from cassava wastewater pretreated with (i) sonication, (ii) OPTIMASH BG^® (enzyme), and (iii) α-amylase (enzyme) were investigated using anaerobic seed sludge subjected to heat pretreatment at 105 °C for 90 min. Hydrogen yield at pH 7.0 for cassava wastewater pretreated with sonication for 45 min using anaerobic seed sludge was 0.913 mol H₂/g COD. Results from pretreatment with OPTIMASH BG^® at 0.20% and pH 7 showed a hydrogen yield of 4.24 mol H₂/g COD. Superior results were obtained when the wastewater was pretreated with α-amylase at 0.20% at pH 7 with a hydrogen yield of 5.02 mol H₂/g COD. In all cases, no methane production was observed when using heat-treated sludge as seed inoculum. Percentage COD removal was found to be highest (60%) using α-amylase as pretreatment followed by OPTIMASH BG^® at 54% and sonication (40% reduction rate). Results further suggested that cassava wastewater is one of the potential sources of renewable biomass to produce hydrogen.