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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 H 2 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 H 2 /g volatile solid (VS) (4.1 mmolH 2 /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 H 2 /L-d and 0.30 L H 2 /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 H 2 /g VS (without treatment) was increased to 94.6 mL H 2 /g VS and 82.6 mL H 2 /g VS (without treatment) was increased to 85.7 mL H 2 /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.

A pilot-scale high-rate dark fermentative hydrogen production plant has been established in the campus of Feng Chia University to develop biohydrogen production pilot-plant technology. This pilot-plant system is composed of two feedstock storage tanks (0.75 m3 each), a nutrient storage tank (0.75 m3), a mixing tank (0.6 m3), an agitated granular sludge bed fermentor (working volume 0.4 m3), a gas–liquid–solid separator (0.4 m3) and a control panel. The seed mixed microflora was obtained from a lab-scale agitated granular sludge bed bioreactor. This pilot-scale fermentor was operated for 67 days at 35 °C, an organic loading rate (OLR) of 40–240 kg COD/m3/d, and the influent sucrose concentration of 20 and 40 kg COD/m3. Both biogas and hydrogen production rates increased with increasing OLR. However, the biomass concentration (volatile suspended solids, VSS) only increased with an increasing OLR at an OLR range of 40–120 kg COD/m3/d, whereas it decreased when OLR was too high (i.e., 240 kg COD/m3/d). The biogas consisted mainly of H2 and CO2 with a H2 content range of 23.2–37.8%. At an OLR of 240 kg COD/m3/d, the hydrogen content in biogas reached its maximum value of 37% with a hydrogen production rate (HPR) of 15.59 m3/m3/d and a hydrogen yield of 1.04 mol H2/mol sucrose. This HPR value is much higher than 5.26 m3/m3/d (fermented molasses substrate) and 1.56 m3/m3/d (glucose substrate) reported by other pilot-scale systems. Moreover, HPR was also greatly affected by pH. At an optimal pH of 5.5, the bacterial community became simple, while the efficient hydrogen producer Clostridium pasteurianum was dominant. The factors of energy output compared with the energy input (Ef) ranged from 13.65 to 28.68 on biohydrogen, which is higher than the Ef value on corn ethanol, biodiesel and sugarcane ethanol but in the similar range of cellulosic ethanol.

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp. A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H 2 /L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum -like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.

The hydraulic retention time (HRT) effects on fermentative hydrogen production from sucrose at high sulfate concentration of 3 g- SO 4 2 - / l were studied using enriched mixed-microflora in a continuously fed reactor. The tested HRTs and organic loading rate ranged from 10 to 2 h and 48 to 240 g-COD/l-day, respectively, and the operating pH was 5.5. The experimental results indicate that hydrogen production could not be inhibited under high sulfate concentration and the efficiency was HRT-dependent with a short HRT of 4 h efficiently enhanced hydrogen production. At this HRT the biogas production rate and hydrogen gas content peaked with the hydrogen yield, hydrogen production rate and specific hydrogen production rate of 4.70 mol-H2/mol-sucrose, 874 mmol-H2/l-day and 432 mmol-H2/g-VSS-day, respectively. These values were 50%, 80% and 300%, respectively, higher than those reported for 12 h HRT at the same sulfate concentration. The metabolite concentration fractions were butyrate 77.3%, acetate 15.6%, ethanol 4.4% and propionate 2.0% and changed to 55%, 27.3%, 11.2% and 6.5%, respectively, at HRT 2 h. Therefore, intimate HRT control is important to obtain efficient hydrogen production. Based on a biological growth comparison, pH 5.5 was considered to be the optimal value for operating a hydrogen-producing fermenter fed on sulfate-rich substrate.

An anaerobic continuous-flow fermentation system with condensed molasses fermentation soluble (CMS) from monosodium glutamate production as substrate was developed for the biohydrogen production. The production rate of the system was increased, while hydraulic retention time (HRT) was varied from 12 to 3 h and the optimal hydrogen production rate was obtained at HRT 3 h. The major bacterial composition of each HRT was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Acidaminococcus sp. that could evolve hydrogen from glutamate was the only hydrogen producer and could be also detected from substrate. Hydrogenase mRNA-targeted reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that except HRT 3 h, two clusters of clostridial hydrogenase gene sequences were obtained. RT-PCR-DGGE analysis was employed to illustrate the active clostridial strains confirming that clostridial strains were not present at HRT 3 h. Acidaminococcus sp. existing in substrate might be the major strain that contributes to hydrogen production at HRT 3 h.