Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Application of immobilized upflow anaerobic sludge blanket reactor using Clostridium LS2 for enhanced biohydrogen production and treatment efficiency of palm oil mill effluent

Polyethylene glycol (PEG) gel was used to immobilize hydrogen producing Clostridium LS2 bacteria for hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. The UASB reactor with a PEG-immobilized cell packing ratio of 10% weight to volume ratio (w/v) was optimal for dark hydrogen production. The performance of the UASB reactor fed with palm oil mill effluent (POME) as a carbon source was examined under various hydraulic retention time (HRT) and POME concentration. The best volumetric hydrogen production rate of 365 mL H₂/L/h (or 16.2 mmol/L/h) with a hydrogen yield of 0.38 L H₂/g COD_added was obtained at POME concentration of 30 g COD/L and HRT of 16 h. The average hydrogen content of biogas and COD reduction were 68% and 65%, respectively. The primary soluble metabolites were butyric acid and acetic acid with smaller quantities of other volatile fatty acid and alcohols formed during hydrogen fermentation. More importantly, the feasibility of PEG-immobilized cell UASB reactor for the enhancement of the dark-hydrogen production and treatment of wastewater is demonstrated.     

Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Electrohydrolysis of landfill leachate organics for hydrogen gas production and COD removal

Hydrogen gas production with simultaneous COD removal was realized by application of DC voltages (0.5-5.0 V) to landfill leachate. The rate and the yield of hydrogen gas production were investigated at different DC voltages by using aluminum electrodes and DC power supply. The highest cumulative hydrogen production (5000 mL), hydrogen yield (2400 mL H₂ g⁻¹ COD), daily hydrogen gas formation (1277 mL d⁻¹), and percent hydrogen (99%) in the gas phase were obtained with 4 V DC voltage. Energy conversion efficiency (H₂ energy/electrical energy) reached the highest level (80.6%) with 1 V DC voltage. Hydrogen gas production was mainly realized by electrohydrolysis of leachate organics due to negligible H₂ gas production in water and leachate control experiments. The highest COD removal (77%) was also obtained with 4 V DC voltage. Electrohydrolysis of landfill leachate was proven to be an effective method for hydrogen gas production with simultaneous COD removal.     

Control strategies for hydrogen production through co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53

This study evaluated hydrogen production by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 with different control strategies. To enhance cooperation of dark and photo-fermentation bacteria during hydrogen production process, the glucose concentration, phosphate buffer concentration and initial pH were controlled at 6 g/l, 50 mmol/l and 7.5, respectively. The maximum yield and rate of hydrogen production were 3.10 mol H₂/mol glucose and 17.2 mmol H₂/l/h, respectively. Ethanol from E. harbinense B49 in acetate medium can enhance hydrogen production by R. faecalis RLD-53 except the ratio of ethanol to acetate (R_E/A) among 0.8 to 1.0. Control of the proper phosphate buffer concentration (50 mmol/l) not only increased acetic acid production by E. harbinense B49, but also maintained stable pH of co-culture system. Therefore, the results showed that co-culture of E. harbinense B49 and immobilized R. faecalis RLD-53 was a promising way of converting glucose into hydrogen.     

Fermentative production of hydrogen and soluble metabolites from crude glycerol of biodiesel plant by the newly isolated thermotolerant Klebsiella pneumoniae TR17

A thermotolerant fermentative hydrogen-producing strain was isolated from crude glycerol contaminated soil and identified as Klebsiella pneumoniae on the basis of the 16S rRNA gene analysis as well as physiological and biochemical characteristics. The selected strain, designated as K. pneumoniae TR17, gave good hydrogen production from crude glycerol. Culture conditions influencing the hydrogen production were investigated. The strain produced hydrogen within a wide range of temperature (30–50 °C), initial pH (4.0–9.0) and crude glycerol concentration (20–100 g/L) with yeast extract as a favorable nitrogen source. In batch cultivation, the optimal conditions for hydrogen production were: cultivation temperature at 40 °C, initial pH at 8.0, 20 g/L crude glycerol and 2 g/L yeast extract. This resulted in the maximum cumulative hydrogen production of 27.7 mmol H₂/L and hydrogen yield of 0.25 mol H₂/mol glycerol. In addition, the main soluble metabolites were 1,3-propanediol, 2,3-butanediol and ethanol corresponding to the production of 3.52, 2.06 and 3.95 g/L, respectively.     

Xylose fermentation to biofuels (hydrogen and ethanol) by extreme thermophilic (70 °C) mixed culture

Combined biohydrogen and bioethanol (CHE) production from xylose was achieved by an extreme thermophilic (70 °C) mixed culture. Effect of initial pH, xylose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations on bioethanol and biohydrogen yield was investigated in batch experiments. Results obtained showed that initial pH, concentration of xylose, peptone, and FeSO₄ significantly affected biohydrogen and bioethanol production, while the concentration of NaHCO₃ was only significant for bioethanol production. By changing cultivation conditions the culture could be directed to mainly produce ethanol with maximum ethanol yield of 1.60 mol ethanol/mol-xylose corresponding to 95.8% of the theoretical ethanol yield based on degradation of xylose through ethanologenic pathway, or mainly hydrogen with maximum hydrogen yield of 1.84 mol H₂/mol-xylose corresponding to 55% of the theoretical hydrogen yield based on acetate metabolic pathway. An empirical model was established to reveal the quantitative effect of factors significant for biohydrogen (quadratic model) production and for bioethanol (linear model) production. Changes in hydrogen/ethanol yields observed were due to the shift of the metabolic pathway between ethanol or hydrogen production, rather than changes in bacterial community composition at genus level. Thermoanaerobacter related bacteria were found to be the dominant hydrogen/ethanol producers.     

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.     

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).     

Biohydrogen production from wastewater by Clostridium beijerinckii: Effect of pH and substrate concentration

An investigation of biological hydrogen production from glucose by Clostridium beijerinckii was conducted in a synthetic wastewater solution. A study examining the effect of initial pH (range 5.7–6.5) and substrate loading (range 1–3 g COD/L) on the specific conversion and hydrogen production rate has shown interaction behaviour between the two independent variables. Highest conversion of 10.3 mL H₂/(g COD/L) was achieved at pH of 6.1 and glucose concentration of 3 g COD/L, whereas the highest production rate of 71 mL H₂/(h L) was measured at pH 6.3 and substrate loading of 2.5 g COD/L. In general, there appears to be a strong trend of increasing hydrogen production rate with an increase in both substrate concentration and pH. Butyrate (14–63%), formate (10–45%) and ethanol (16–40%) were the main soluble products with other volatile fatty acids and alcohols present in smaller quantities.     

Ozonation aided mesophilic biohydrogen production from palm oil mill effluent

Ozone pretreatment of palm oil mill effluent (POME) was employed to improve sustrate biodegradability prior to biological H₂ production. The H₂ production was conducted at varing pHs from 4.0 to 6.0 to examine the impact of pH on the H₂ mesophilic production (37 °C). The optimal pH for H₂ production was 6.0 for both raw and ozonated POME. The POME concentrations were greatly influenced the yields and rates of H₂ production. At the optimal pH, the maximum H₂ production yield of 182 ± 7.2 mL.g⁻¹ COD (7.96 mmoL.g⁻¹ COD) was achieved at the ozonated POME concentration of 30,000 mg COD.L⁻¹. The maximum H₂ production rate (R_max) of 43.1 ± 2.5 mL.h⁻¹ was obtained at the ozonated POME concentration of 25,000 mg COD.L⁻¹. The highest total COD removal was 44% at of 15,000 mg COD.L⁻¹ ozonated POME. Acetic and butyric acids were dominant products during H₂ fermentation and tended to increase with the increased POME concentrations. Ozonation as a pretreatment process showed significant enhancement of the POME biodegradability , and subsequently improved the H₂ production H₂.     

Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone

Ceramic ring and pumice stone were used as a support matrix for the enhancement of biohydrogen production in immobilized cell culture systems. The reactors were continuously operated for the hydrogen fermentation using sucrose as the major carbon source at varying hydraulic retention times (HRT) as an important operational factor. In terms of volumetric hydrogen production, the best value was obtained with ceramic ring at 1.5 h HRT (2.98 l H₂/l/d), on the other hand, the pumice stone packed reactor resulted in 30% less volumetric hydrogen production (2.28 l H₂/l/d) at two fold longer retention time (HRT 3 h). It was demonstrated that volumetric hydrogen production with the immobilized bioreactor configurations was 6 fold better than the suspended culture bioreactor configuration (CSTR). Furthermore, up to 4 mol and 5 mol hydrogen yields per mole of sucrose used (which are 62.5% and 50% of the theoretical values) were achieved by pumice stone and ceramic ring packed reactors, respectively, whereas suspended culture system yielded only 0.5 mol H₂/mol sucrose.     

Ultrasonic pretreatment of palm oil mill effluent: Impact on biohydrogen production,bioelectricity generation,and underlying microbial communities

Substrate bioavailabity is one of the critical factors that determine the relative biohydrogen (bioH₂) yield in fermentative hydrogen production and bioelectricity output in a microbial fuel cell (MFC). In the present undertaking, batch bioH₂ production and MFC-based biolectricity generation from ultrasonically pretreated palm oil mill effluent (POME) were investigated using heat-pretreated anaerobic sludge as seed inoculum. Maximum bioH₂ production (0.7 mmol H₂/g COD) and COD removal (65%) was achieved at pH 7, for POME which was ultrasonically pretreated at a dose of 195 J/mL. Maximum value for bioH₂ productivity and COD removal at this sonication dose was higher by 38% and 20%, respectively, than unsonicated treatments. In batch MFC experiments, the same ultrasound dose led to reduced lag-time in bioelectricity generation with concomitant 25% increase in bioelectricity output (18.3 W/m³) and an increase of COD removal from 30% to 54%, as compared to controls. Quantitative polymerase chain reaction (qPCR) tests on sludge samples from batch bioH₂ production reflected an abundance of gene fragments coding for both clostridial and thermoanaerobacterial [FeFe]-hydrogenase. Fluorescence in situ hybridization (FISH) tests on sludge from MFC experiments showed Clostridium spp. and Thermoanaerobacterium spp. as the dominant microflora. Results suggest the potential of ultrasonicated POME as sustainable feedstock for dark fermentation-based bioH₂ production and MFC-based bioelectricity generation.     

Impacts of nano-metal oxides on hydrogen production in anaerobic digestion of palm oil mill effluent – A novel approach

In the present study, hydrogen production from palm oil mill effluent (POME) was investigated with the incorporation of nanoparticles (NPs) comprising of nickel (NiO) and cobalt oxides (CoO). The NPs of NiO and CoO were prepared using hydrothermal method and were further applied to analyse, their effect on hydrogen production. The results demonstrated that, a maxima volumetric hydrogen production rate of 21 ml H₂/L-POME/h with the hydrogen yield of 0.563 L H₂/g-COD_removed was obtained with 1.5 mg/L concentration of NiO NPs. On the other hand, the addition of CoO NPs produced maximum volumetric hydrogen production rate of 18 ml H₂/L-POME/h with a hydrogen yield of 0.487 L H₂/g-COD_removed with 1.0 mg/L of CoO NPs. Results showed that addition of optimal concentration of NiO and CoO NPs to the POME enhances the hydrogen yield by 1.51 and 1.67 fold respectively. Besides, this addition of NiO and CoO enhanced the COD removal efficiency by 15 and 10% respectively as compared to an un-additive NPs POME. The toxicity of NPs was also tested using bacterial viability test, which revealed that application of 3.0 mg/L of NiO and CoO NPs to modified Luria-Bertani (LB) medium had 63% and 83% reduction in bacterial cell growth. The results concluded that supplementation of NiO and CoO NPs under an optimal range to the wastewater can improve the hydrogen productivity.     

Fermentative hydrogen production from indigenous mesophilic strain Bacillus anthracis PUNAJAN 1 newly isolated from palm oil mill effluent

In the present study, a new mesophilic bacterial strain, identified as Bacillus anthracis strain PUNAJAN 1 was isolated from palm oil mill effluent (POME) sludge, and tested for its hydrogen production ability. Effect of physico-chemical factors such as temperature, initial pH, nitrogen source and carbon sources were investigated in order to determine the optimal conditions for hydrogen production. The maximum hydrogen yield of 2.42 mol H₂/mol mannose was obtained at 35 °C and initial pH of 6.5. Yeast and mannose were used as the main carbon and nitrogen sources respectively in the course of the hydrogen production. Apart from synthetic substrate, specific hydrogen production potentials of the strain using POME was calculated and found to be 236 ml H₂/g chemical oxygen demand (COD). The findings of this study demonstrate that the indigenous strain PUNAJAN 1 could be a potential candidate for hydrogen using POME as substrate.