Biohydrogen production from specified risk materials co-digested with cattle manure

Biohydrogen production from the anaerobic digestion of specified risk materials (SRM) co-digested with cattle manure was assessed in a 3 × 5 factorial design. Total organic loading rates (OLR) of 10, 20, and 40 g L⁻¹ volatile solids (VS) were tested using manure:SRM (wt/wt) mixtures of 100:0 (control), 90:10, 80:20, 60:40, and 50:50 using five 2 L continuously stirred biodigesters operating at 55 °C. Gas samples were taken daily to determine hydrogen production, and slurry samples were analyzed daily for volatile fatty acid (VFA) concentration, total ammonia nitrogen (TAN), and VS degradation. Hydrogen production (mL g⁻¹ VS fed) varied quadratically according to OLR (P < 0.01), with maximum production at OLR20, while production decreased linearly (P < 0.0001) as SRM concentration increased. Reduced hydrogen production associated with SRM inclusion at >10% VS may be attributed to a rapid increase in TAN (r = −0.55) or other inhibitors such as long chain fatty acids. Reduced hydrogen production (P < 0.01) at OLR40 versus OLR20 may be related to increased rate of VFA accumulation and final VFA concentration (P < 0.001), as well as inhibition due to hydrogen accumulation (P < 0.001). Biohydrogen production from SRM co-digested with cattle manure may not be feasible on an industrial scale due to reduced hydrogen production with increasing levels of SRM.     

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.     

High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant

A mesophilic high hydrogen producing strain DMHC-10 was isolated from a lab scale anaerobic reactor being operated on distillery wastewater for hydrogen production. DMHC-10 was identified as Clostridium sp. on the basis of 16S rRNA gene sequencing. Various medium components (carbon and nitrogen sources) and environmental factors (initial pH, temperature of incubation) were optimized for hydrogen production by Clostridium sp. DMHC-10. The strain, in late exponential growth phase, showed maximum hydrogen production (3.35 mol-H₂ mol⁻¹ glucose utilized) at 37 °C, pH 5.0 in a medium supplemented with organic nitrogen source. Butyric acid to acetic acid ratio was ca. 2.3. Hydrogen production declined when organic nitrogen was replaced with inorganic nitrogen.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

Effect of temperature and iron concentration on the growth and hydrogen production of mixed bacteria

Anaerobic mixed culture acclimated with sucrose was used as inoculum in batch experiments to investigate the effects of various parameters on biological hydrogen production from sucrose. In particular, the effect of the culture temperature has been investigated in detail. The optimum of the iron concentration in the external environment on hydrogen production was also studied at different temperatures. Experimental results show that the hydrogen production ability of the anaerobic bacteria was deeply affected by both culture temperature and iron concentration. Increasing the culture temperature favored the production of hydrogen when it was in the range of 25–40 °C, and high sucrose conversion efficiencies (ca. 98%) were consistently obtained with the mixed bacteria at the same time. While the temperature went on increasing to 45 °C, the hydrogen production was almost inhibited. The optimum concentrations of iron for hydrogen production decreased obviously along with increasing the reactor's temperature. For 25, 35, and 40 °C, the maximum production yield of hydrogen were 356.0, 371.7, and 351.1 ml obtained at the iron concentration of 800, 200, and 25 mg FeSO₄l^-1${\mathrm{l}}^{-1}$, respectively.     

Fermentative hydrogen production by diverse microflora

In this study, hydrogen production with activated sludge, a diverse bacterial source has been investigated and compared to microflora from anaerobic digester sludge, which is less diverse. Batch experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The hydrogen production yields with activated sludge at 37 °C and 55 °C were 0.56 and 1.32 mol H₂/mol glucose consumed, respectively. While with anaerobically digested sludge hydrogen yield was 2.18 mol H₂/mol glucose consumed at 37 °C and 1.25 mol H₂/mol glucose consumed at 55 °C. The results of repeated batch experiments for 615 h resulted in average yields of 1.21 ± 0.62 and 1.40 ± 0.16 mol H₂/mol glucose consumed for activated sludge and anaerobic sludge, respectively. The hydrogen production with activated sludge was not stable during the repeated batches and the fluctuation in hydrogen production was attributed to formation of lactic acid as the predominant metabolite in some batches. The presence of lactic acid bacteria in microflora was confirmed by PCR-DGGE.     

Effect of temperature on fermentative hydrogen production by mixed cultures

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

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.     

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.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

Hydrogen gas production by electrohydrolysis of volatile fatty acid (VFA) containing dark fermentation effluent

Dark fermentation effluents of wheat powder (WP) solution containing different concentrations of volatile fatty acids (VFAs) were subjected to low voltage (1–3 V) DC current to produce hydrogen gas. Graphite and copper electrodes were tested and the copper electrode was found to be more effective due to higher electrical conductivity. The effects of solution pH (2–7), applied voltage (1–3 V) and the total VFA (TVFA) concentration (1–5 g L⁻¹) on hydrogen gas production were investigated. Hydrogen production increased with decreasing pH and became maximum at pH = 2. Increases in applied voltage and the TVFA concentration also increased the cumulative hydrogen formation. The most suitable conditions for the highest cumulative hydrogen production was pH = 2, with 3 V applied voltage and 5 g TVFA L⁻¹. Up to 110 ml hydrogen gas was obtained with 5 g L⁻¹ TVFA at pH = 5.8 and 2 V applied voltage within 37.5 h. The highest energy efficiency (56%) was obtained with the 2 V applied voltage and 10.85 g L⁻¹ TVFA. Hydrogen production by electrolysis of water in control experiments was negligible for pH > 4. Hydrogen production by electrohydrolysis of VFA containing anaerobic treatment effluents was found to be an effective method with high energy efficiency.     

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.     

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.     

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.     

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.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

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.     

Biohydrogen production from starch wastewater and application in fuel cell

Biohydrogen was produced from starch in wastewater by anaerobic fermentation. The effects of parameters, such as pH, starch concentration were investigated and optimum operating conditions were determined. The optimal pH and starch concentration for hydrogen production at 37 °C were 6.5 and 5 g/L, respectively with a maximum hydrogen yield of 186 ml/g-starch. The produced biogas contains 99% of hydrogen after passing through KOH solution to remove CO₂. The anaerobic fermentation installation was integrated with a proton-exchange-membrane fuel cell (PEMFC) system for on-line electricity generation. This combination system of biohydrogen and fuel cell achieved a power output of 0.428 W at 0.65 V per cell.     

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.     

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.