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 bio-hydrogen production from cellulose by cow dung compost enriched cultures

The performance of hydrogen production from cellulose by the cow dung compost enriched continuously in defined medium containing cellulose was investigated. In the initial experiments, batch-fermentation was carried out to observe the effects of different substrate concentration conditions on the rate of cellulose-degrading, growth of bacteria and the capability of hydrogen-producing from cellulose. The result showed that the cellulose degradation decreased from 55% at 5 g/l to 22% at 30 g/l. The maximum cumulative hydrogen production and the rate of hydrogen production first increased from 828 ml/l at 5 g/l to 1251 ml/l at 10 g/l then remained constant beyond 10 g/l. The maximum hydrogen production potential, the rate of hydrogen production and the yield of hydrogen was 1525 ml/l, 33 ml/l.h, and 272 ml/g-cellulose (2.09 mol/mol-hexose) was obtained at substrate concentration 10 g/l, the hydrogen concentration in biogas was 47–50%(v/v) and there was no methane observed. During the conversion of cellulose into hydrogen, acetate and butyrate were main liquid end-products in the metabolism of hydrogen fermentation. These results proposed that cow dung compost enriched cultures were ideal microflora for hydrogen production from cellulose.     

Pretreatment of sweet sorghum bagasse for hydrogen production by Caldicellulosiruptor saccharolyticus

Pretreatment of sweet sorghum bagasse, an energy crop residue, with NaOH for the production of fermentable substrates, was investigated. Optimal conditions for the alkaline pretreatment of sweet sorghum bagasse were realized at 10% NaOH (w/w dry matter). A delignification of 46% was then observed, and improved significantly the efficiency of enzymatic hydrolysis. Under hydrolysis conditions without pH control, up to 50% and 41% of the cellulose and hemicellulose contained in NaOH-pretreated sweet sorghum bagasse were converted by 24 h enzymatic hydrolysis to soluble monomeric sugars. The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed normal growth on hydrolysates of NaOH-pretreated biomass up to a sugar concentration of 20 g/L. Besides hydrogen, the main metabolic products detected in the fermentations were acetic and lactic acid. The maximal hydrogen yield observed in batch experiments under controlled conditions was 2.6 mol/mol C6 sugar. The maximal volumetric hydrogen production rate ranged from 10.2 to 10.6 mmol/(L h). At higher substrate concentrations the production of lactic acid increased at the expense of hydrogen production.     

Enhancement of biohydrogen producing using ultrasonication

Ultrasonication was evaluated as a pretreatment for biological hydrogen production from glucose in batch studies, in comparison with heat-shock pretreatment, acid pretreatment, and base pretreatment. The optimized sonication energy for hydrogen production using anaerobic digester sludge was 79 kJ/gTS. Sonication with temperature control (less than 30 °C) increased volumetric hydrogen production by 120% over the untreated sludge, and by 40% over the heat-shock and acid pretreated sludge, with a marginal (∼10%) increase in hydrogen production rate. Upon comparing the molar hydrogen yield in sonicated sludge with and without temperature control, the deleterious effect of heat on some hydrogen producers as reflected by a 30% decrease in yield to 1.03 mol H₂/mol glucose is evident. Sonication with temperature control affected a 45% increase in molar hydrogen yield to 1.55 mol H₂/mol glucose over heat-shock pretreatment at 70 °C for 30 min and acidification to pH 3.0 for 24 h at 4 °C. Sonication with temperature control produced a biomass yield of 0.13 g VSS/g COD, as compared to 0.24 g VSS/g COD for the untreated sludge. The hydrogen yield increased linearly with the molar acetate to butyrate ratio and decreased linearly with the biomass yield.     

Biohydrogen production from apple pomace by anaerobic fermentation with river sludge

The biological hydrogen (bio-H₂) production from apple pomace (AP) by fermentation using natural mixed microorganisms in batch process was studied under various experimental conditions. The river sludge was used as a seed after being boiled for 15 min. The results show that the optimal pretreatment for AP was to soak it in the ammonia liquor of 6% for 24 h at room temperature. An optimal fermentation condition for bio-H₂ production was proposed that the pretreated AP at 37 °C, the initial pH of 7.0 and the fermentation concentration of 15 g/l could produce a maximum cumulative H₂ yield (CHYm) of 101.08 ml/g total solid (TS) with an average H₂ production rate (AHPR) of 8.08 ml/g TS/h. During the conversion of AP into H₂, acetic acid, ethanol, propionic acid and butyric acid were main liquid end-products.     

Evaluation of pretreatment methods on harvesting hydrogen producing seeds from anaerobic digested organic fraction of municipal solid waste (OFMSW)

In order to harvest high-efficient hydrogen producing seeds, five pretreatment methods (including acid, heat, sonication, aeration and freeze/thawing) were performed on anaerobic digested sludge (AS) which was collected from a batch anaerobic reactor for treating organic fraction of municipal solid waste. The hydrogen production tests were conducted in serum bottles containing 20 gVS/L (24.8 g COD/L) mixture of rice and lettuce powder at 37 °C. The experimental results showed that the heat and acid pretreatment completely repressed the methanogenic activity of AS, but acid pretreatment also partially repressed hydrogen production. Sonication, freeze/thawing and aeration did not completely suppress the methanogen activity. The highest hydrogen yields were 119.7, 42.2, 26.0, 23.0, 22.7 and 22.1 mL/gVS for heated, acidified, freeze/thawed, aerated, sonicated and control AS respectively. A pH of about 4.9 was detected at the end of hydrogen producing fermentation for all tests. The selection of an initial pH can markedly affect the hydrogen producing ability for heated and acidified AS. The higher initial pH generated higher hydrogen yield and the highest hydrogen yield was obtained with initial pH 8.9 for heated AS.     

Biological hydrogen production from steam-exploded straw by simultaneous saccharification and fermentation

Hydrogen was produced by simultaneous saccharification and fermentation from steam-exploded corn straw (SECS) using Clostridium butyricum AS1.209. Effect of various process parameters, such as solid to liquid ratio, enzyme loading and initial pH, etc., were examined with respect to maximum hydrogen productivity which was obtained by fitting the cumulative hydrogen production data to a modified Gompertz equation. Maximum specific hydrogen production rate and maximal hydrogen yield were 126 ml/g VSS d and 68 ml/g SECS, respectively. The yield of soluble metabolites was 197.7 mg/g SECS. Acetic acid accounted for 46% of the total was the most abundant product and this shows that hydrogen production from SECS was essentially acetate-type fermentation. Hydrogen production by simultaneous saccharification and fermentation of SECS has the predominance of short lag-stage and high maximum specific hydrogen production rate and it was a promising method for hydrogen production and straw biomass conversion.     

Production of hydrogen from sewage biosolids in a continuously fed bioreactor: Effect of hydraulic retention time and sparging

Hydrogen was produced from primary sewage biosolids via mesophilic anaerobic fermentation in a continuously fed bioreactor. Prior to fermentation the sewage biosolids were heated to 70 °C for 1 h to inactivate methanogens and during fermentation a cellulose degrading enzyme was added to improve substrate availability. Hydraulic retention times (HRT) of 18, 24, 36 and 48 h were evaluated for the duration of hydrogen production. Without sparging a hydraulic retention time of 24 h resulted in the longest period of hydrogen production (3 days), during which a hydrogen yield of 21.9 L H₂ kg⁻¹ VS added to the bioreactor was achieved. Methods of preventing the decline of hydrogen production during continuous fermentation were evaluated. Of the techniques evaluated using nitrogen gas to sparge the bioreactor contents proved to be more effective than flushing just the headspace of the bioreactor. Sparging at 0.06 L L min⁻¹ successfully prevented a decline in hydrogen production and resulted in a yield of 27.0  L H₂ kg⁻¹ VS added, over a period of greater than 12 days or 12 HRT. The use of sparging also delayed the build up of acetic acid in the bioreactor, suggesting that it serves to inhibit homoacetogenesis and thus maintain hydrogen production.     

Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp

Hydrogen gas production from sugar solution derived from acid hydrolysis of ground wheat starch by photo-fermentation was investigated. Three different pure strains of Rhodobacter sphaeroides (RV, NRLL and DSZM) were used in batch experiments to select the most suitable strain. The ground wheat was hydrolyzed in acid solution at pH = 3 and 90 °C in an autoclave for 15 min. The resulting sugar solution was used for hydrogen production by photo-fermentation after neutralization and nutrient addition. R. sphaeroides RV resulted in the highest cumulative hydrogen gas formation (178 ml), hydrogen yield (1.23 mol H₂ mol⁻¹ glucose) and specific hydrogen production rate (46 ml H₂ g⁻¹ biomass h⁻¹) at 5 g l⁻¹ initial total sugar concentration among the other pure cultures. Effects of initial sugar concentration on photo-fermentation performance were investigated by varying sugar concentration between 2.2 and 13 g l⁻¹ using the pure culture of R. sphaeroides RV. Cumulative hydrogen volume increased from 30 to 232 ml when total sugar concentration was increased from 2.2 to 8.5 g l⁻¹. Further increases in initial sugar concentration resulted in decreases in cumulative hydrogen formation. The highest hydrogen formation rate (3.69 ml h⁻¹) and yield (1.23 mol H₂ mol⁻¹ glucose) were obtained at a sugar concentration of 5 g l⁻¹.     

Hydrogen production from biopolymers by Caldicellulosiruptor saccharolyticus and stabilization of the system by immobilization

The biopolymers agarose and alginic acid, and hemicellulose-rich pine tree wood shavings, frequently discarded as waste, proved to be utilized as energy sources for hydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus. The addition of 0.5% (w/v) pine wood shavings to the growth medium yielded a 14-fold increase in hydrogen production over a period of 55 days relative to cultures grown in the same medium without wood shavings (average rate was about 0.45 ml H₂ ml culture⁻¹ day⁻¹). The shavings were also shown to be degraded by C. saccharolyticus in the absence of any other carbohydrate source.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

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.     

Microwave-assisted alkali pretreatment of rice straw to promote enzymatic hydrolysis and hydrogen production in dark- and photo-fermentation

Rice straw was pretreated by microwave-assisted alkali to improve saccharification in enzymatic hydrolysis and hydrogen yield in combined dark- and photo-fermentation in this paper. A maximum reducing sugar yield of 69.3 g/100 g TVS was obtained when 50 g/l rice straw was pretreated by microwave heating for 15 min at 140 °C in 0.5% NaOH solution and then enzymatically hydrolyzed for 96 h. When hydrolyzed rice straw was used for hydrogen production by anaerobic bacteria in dark-fermentation, a maximum hydrogen yield of 155 ml/g TVS was obtained. The residual solution (mainly acetate and butyrate) from dark-fermentation was reutilized for hydrogen production by immobilized photosynthetic bacteria in photo-fermentation. By combination of dark- and photo-fermentation, the maximum hydrogen yield was greatly enhanced to 463 ml/g TVS, which is 43.2% of the theoretical hydrogen yield.     

Hydrogen production by combined dark and light fermentation of ground wheat solution

Ground waste wheat was subjected to combined dark and light batch fermentation for hydrogen production. The dark to light biomass ratio (D/L) was changed between 1/2 and 1/10 in order to determine the optimum D/L ratio yielding the highest hydrogen formation rate and the yield. Hydrogen production by only dark and light fermentation bacteria was also realized along with the combined fermentations. The highest cumulative hydrogen formation (CHF = 76 ml), hydrogen yield (176 ml H₂ g⁻¹ starch) and formation rate (12.2 ml H₂ g⁻¹ biomass h⁻¹) were obtained with the D/L ratio of 1/7 while the lowest CHF was obtained with the D/L ratio of 1/2. Dark–light combined fermentation with D/L ratio of 1/7 was faster as compared to the dark and light fermentations alone yielding high hydrogen productivity and reduced fermentation time. Dark and light fermentations alone also yielded considerable cumulative hydrogen, but slower than the combined fermentation.     

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

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

Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge

An anaerobic fermentation process to produce hydrogen from cornstalk wastes was systematically investigated in this work. Batch experiments numbered series I, II and III were designed to investigate the effects of acid pretreatment, enzymatic hydrolysis (enzymatic temperature, enzymatic time and enzymatic pH) on hydrogen production by using the natural sludge as inoculant. A maximum cumulative H₂ yield of 126.22 ml g⁻¹-CS (Cornstalk, or 146.94 ml g⁻¹-TS, Total Solid) and an average H₂ production rate of 9.58 ml g⁻¹-CS h⁻¹ were obtained from fermentation cornstalk with a concentration of 20 g/L and an initial pH of 7.0 at 36 °C through an optimal pretreatment process. The optimal process was that the substrate was soaked with an HCl concentration of 0.6 wt% at 90 °C for 2 h, and subsequently enzymatic hydrolysis for 72 h at 50 °C and pH 4.8 before fermentation. The biogas consisted of only H₂ and CO₂. In addition, the fermentation system was the typical ethanol-type fermentation according to ethanol and acetate as the main liquid by-products.     

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

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

Hydrogen production from cassava wastewater using an anaerobic sequencing batch reactor: Effects of operational parameters,COD:N ratio,and organic acid composition

In this work, hydrogen production from cassava wastewater using anaerobic sequencing batch reactors (ASBR) was investigated to determine the optimum number of cycles per day, chemical oxygen demand (COD) loading rate, and COD:N ratio. The system operated at a COD loading rate of 30 kg/m³d and 6 cycles per day provided maximum hydrogen production performance in terms of specific hydrogen production rate (SHPR) (388 ml H₂/g VSS d or 3800 ml H₂/l d) and hydrogen yield (186 ml H₂/g COD removed). The effect of nitrogen supplementation was also studied by adding NH₄HCO₃ into the system at the COD:N ratios of 100:2.2, 100:3.3, and 100:4.4 under the COD loading rate of 30 kg/m³d and 6 cycles per day. The maximum SHPR and hydrogen yield of 524 ml H₂/g VSS d (5680 ml H₂/l d) and 438 ml H₂/g COD removed, respectively, were obtained at the stoichiometric COD:N ratio of 100:2.2. An excess nitrogen was found to promote the productions of higher organic acids and ethanol, resulting in lowering hydrogen production efficiency.     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: Effects of temperature and pH

Fermentative hydrogen production from cassava stillage was conducted to investigate the influences of temperature (37 °C, 60 °C, 70 °C) and initial pH (4–10) in batch experiments. Although the seed sludge was mesophilic anaerobic sludge, maximum hydrogen yield (53.8 ml H₂/gVS) was obtained under thermophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme-thermophilic (70 °C) conditions respectively. The difference was mainly due to the different VFA and ethanol distributions. Higher hydrogen production corresponded with higher ratios of butyrate/acetate and butyrate/propionate. Similar hydrogen yields of 66.3 and 67.8 ml H₂/gVS were obtained at initial pH 5 and 6 respectively under thermophilic condition. The total amount of VFA and ethanol increased from 3536 to 7899 mg/l with the increase of initial pH from 4 to 10. Initial pH 6 was considered as the optimal pH due to its 19% higher total VFA and ethanol concentration than that of pH 5. Homoacetogenesis and methonogenesis were very dependent on the initial pH and temperature even when the inoculum was heat-pretreated. Moreover, a difference between measured and theoretical hydrogen was observed in this study, which could be attributed to homoacetogenesis, methanogenesis and the degradation of protein.