Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria

Leaves are one of the main by-products of forestry. In this study, batch experiments were carried out to convert poplar leaves pretreated by different methods into hydrogen using anaerobic mixed bacteria at 35 °C. The effects of acid (HCl), alkaline (NaOH) and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, β-glucanase, hemicellulase and xylanase) pretreatments on the saccharification of poplar leaves were studied. Furthermore, the effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves was achieved from substrate pretreated with 2% Vicozyme L, which was approximately 3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective method for enhancing the hydrogen yield from poplar leaves.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

Isolation of hydrogen generating microflora from cow dung for seeding anaerobic digester

The effectiveness of using cow dung as a source for isolating hydrogen generating microflora was investigated under varying isolating conditions based on viz.: pH adjustment and pH adjustment coupled with heat treatment. The viability of the isolated microflora was tested in an anaerobic jar with respect to biogas generation, hydrogen content and pH. The results showed that for pH adjusted microflora isolated from cow dung with solids content at 10% resulted in a cumulative biogas generation of 1494, 2404 and 3327 ml, whereas the corresponding cumulative hydrogen generation was found to be 424, 701 and 47 ml during the anaerobic fermentation for 120 h at a pH of 4, 5 and 6, respectively. The biogas was free from methane when operated at pH 4 and 5, whereas at pH 6 methane generation was observed. In the case of microflora isolated from cow dung with 10% solids, by subjecting to pH adjustment coupled with heat treatment resulted in biogas free from methane content during the fermentation at pH 4, 5 and 6, respectively. At the end of 120 h of fermentation for a reactor pH at 4, 5 and 6 the cumulative biogas generation was 1685, 2610 and 2353 ml, whereas the cumulative hydrogen generation was 509, 1198 and 1165 ml, respectively. A maximum of 41% and 62% hydrogen was obtained at pH 5 for microflora isolated based on pH adjustment and pH adjustment coupled with heat treatment. The effect of initial solids content of the cow dung on the isolating efficiency of hydrogen generating microflora was also investigated at pH 5 and 6 coupled with heat treatment. The results revealed that with the increase in initial solids content of the cow dung the optimum heat treatment period also increased as the pH increased from 5 to 6.     

Impact of low lignin containing brown midrib sorghum mutants to harness biohydrogen production using mixed anaerobic consortia

Three low lignin containing bmr 3 derivatives, namely DRT 07K1, DRT 07K6 and DRT 07K15 developed through backcrossing were used along with the parent, bmr 3 source mutant (IS 21888) for evaluation of biohydrogen production. Results demonstrated that biohydrogen production varied amongst bmr derivatives under similar fermentation conditions. Significant negative correlation was observed between lignin content and fermentative biohydrogen production. All bmr derivatives with lower lignin content produced higher levels of biohydrogen compared to source bmr 3 (IS 21888) which has more lignin content. The maximum and a minimum biohydrogen production observed was 72 and 50 ml/g Total Volatile Solids (TVS) for the DRT 07K6 bmr3 derivative and bmr 3 (IS 21888) respectively. Acetate and butyrate were accounted >85% of volatile fatty acids, indicating acid type fermentations. Statistical analysis revealed that all bmr mutant derivatives with respect to source differ significantly in cumulative biohydrogen production, plant height, grain yield and lignin content. Biohydrogen production from biomass associated at least two different levels, one at lignin entanglement another at the polymeric nature of cellulose and hemicellulose. Further studies are necessary to determine the effect of biomass structure associated with different bmr traits on the microbial growth and biohydrogen production rate.     

Effect of pH and sulfate concentration on hydrogen production using anaerobic mixed microflora

The effects of varying sulfate concentrations with pH on continuous fermentative hydrogen production were studied using anaerobic mixed cultures growing on a glucose substrate in a chemostat reactor. The maximum hydrogen production rate was 2.8 L/day at pH 5.5 and sulfate concentration of 3000 mg/L. Hydrogen production and residual sulfate level decreased with increasing the pH from 5.5 to 6.2. The volatile fatty acids (VFAs) and ethanol fractions in the effluent were in the order of butyric acid (HBu) > acetic acid (HAc) > ethanol > propionic acid (HPr). Fluorescence In Situ Hybridization (FISH) analysis revealed the presence of hydrogen producing bacteria (HPB) under all pH ranges while sulfate reducing bacteria (SRB) were present at pH 5.8 and 6.2. The inhibition in hydrogen production by SRB at pH 6.2 diminished entirely by lowering to pH 5.5, at which activity of SRB is substantially suppressed.     

Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities

Hydrogen (H₂) production from simulated cheese processing wastewater via anaerobic fermentation was conducted using mixed microbial communities under mesophilic conditions. In batch H₂ fermentation experiments H₂ yields of 8 and 10 mM/g COD fed were achieved at food-to-microorganism (F/M)$(F/M)$ ratios of 1.0 and 1.5, respectively. Butyric, acetic, propionic, and valeric acids were the major volatile fatty acids (VFA) produced in the fermentation process. Continuous H₂ fermentation experiments were also performed using a completely mixed reactor (CSTR). The pH of the bioreactor was controlled in a range of 4.0–5.0 by addition of carbonate in the feed material. Maximum H₂ yields were between 1.8 and 2.3 mM/g COD fed for the loading rates (LRs) tested with a hydraulic retention time (HRT) of 24 h. Occasionally CH₄ was produced in the biogas with concurrent reductions in H₂ production; however, continuous H₂ production was achieved for over 3 weeks at each LR. The 16S rDNA analysis of DNA extracted from the bioreactors during periods of high H₂ production revealed that more than 50% of the bacteria present were members of the genus Lactobacillus and about 5% were Clostridia. When H₂ production in the bioreactors decreased concurrent reductions in the genus Lactobacillus were also observed. Therefore, the microbial populations in the bioreactors were closely related to the conditions and performance of the bioreactors.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

Microbial community analysis of mixed anaerobic microflora in suspended sludge of ASBR producing hydrogen from palm oil mill effluent

This study investigated the microbial community of an anaerobic sequencing batch reactor (ASBR) operating at mesophilic temperature under varying hydraulic retention times (HRTs) for evaluating optimal hydrogen production conditions, using palm oil mill effluent (POME) as substrate. POME sludge enriched by heat treatment with hydrogen-producing bacteria was used as inoculum and acclimated with the POME. The microbial community was determined by first isolating cultivable bacteria at each operating HRT and then using polymerase chain reaction (PCR). The PCR products were sequenced and sequence identification was performed using the BLAST algorithm and Genbank database. The findings revealed that about 50% of the isolates present were members of the genus Streptococcus, about 30% were Lactobacillus species and around 20% were identified as species of genus Clostridium. Scanning electron microscopy (SEM) analysis also confirmed the presence of spherical and rod-shaped microbial morphologies in the sludge samples of bioreactor during prolonged cultivation.     

Fermentative biohydrogen production by mixed anaerobic consortia: Impact of glucose to xylose ratio

Glucose and xylose are the dominant monomeric carbohydrates present in agricultural materials which can be used as potential building blocks for various biotechnological products including biofuels production. Hence, the imperative role of glucose to xylose ratio on fermentative biohydrogen production by mixed anaerobic consortia was investigated. Microbial catabolic H₂ and VFA production studies revealed that xylose is a preferred carbon source compared to glucose when used individually. A maximum of 1550 and 1650 ml of cumulative H₂ production was observed with supplementation of glucose and xylose at a concentration of 5.5 and 5.0 g L⁻¹, respectively. A triphasic pattern of H₂ production was observed only with studied xylose concentration range. pH impact data revealed effective H₂ production at pH 6.0 and 6.5 with xylose and glucose as carbon sources, respectively. Co-substrate related biohydrogen fermentation studies indicated that glucose to xylose ratio influence H₂ and as well as VFA production. An optimum cumulative H₂ production of 1900 ml for 5 g L⁻¹ substrate was noticed with fermentation medium supplemented with glucose to xylose ratio of 2:3 at pH 6. Overall, biohydrogen producing microbial consortia developed from buffalo dung could be more effective for H₂ production from lignocellulosic hydrolysates however; maintenance of glucose to xylose ratio, inoculum concentration and medium pH would be essential requirements.     

Fermentative hydrogen production from macro-algae Laminaria japonica using anaerobic mixed bacteria

In this study, one macro-alga (Laminaria japonica) was used for fermentative hydrogen production by anaerobic mixed bacteria. The saccharification efficiency and hydrogen production by L. japonica with four different pretreatment methods, including heat, acid, alkaline and ultrasonic treatment, were investigated. The results showed that the saccharification efficiency from L. japonica that was pretreated with acid was the highest among the four methods. The saccharification efficiency for the total reducing sugars in the acid-pretreated L. japonica was 350.54 ± 19.89 mg/g (mean ± S.E.). The cumulative hydrogen production was 66.68 ± 5.68 mL/g from the heat-pretreated L. japonica, whereas that of L. japonica that was subjected to acid, alkaline, and ultrasonic pretreatment and the control was 43.65 ± 6.87 mL/g, 15.00 ± 3.89 mL/g, 23.56 ± 4.56 mL/g and 10.00 ± 1.21 mL/g, respectively. In addition, the effects of substrate concentration and initial pH on hydrogen production from heat-pretreated L. japonica were also analyzed. The results showed that the maximum hydrogen production was 83.45 ± 6.96 mL/g with a hydrogen concentration of approximately 28.4% from heat-pretreated L. japonica when the initial pH and substrate concentration were determined to be 6.0 and 2%, respectively. Heat pretreatment was the most effective method for increasing fermentative hydrogen production when L. japonica was used as the only substrate.     

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.     

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH₂) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH₂ production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L⁻¹ bromoethanesulphonic acid, 24 h) on bioH₂ production from starch powder (1% w/v) showed highest yield (323.4 mL g⁻¹ starch) in heat-treatment and peak production rate (144.5 mL L⁻¹ h⁻¹) in acid-treatment. Acetate (1.07 g L⁻¹) and butyrate (1.21 g L⁻¹) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH₂ production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H₂ yielding conditions. Peak H₂ yield and production rate were 412.6 mL g⁻¹ starch and 78.6 mL L⁻¹ h⁻¹, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH₂ yield of 126.5 mL g⁻¹ COD and 456 mL g⁻¹ starch. The net energy was calculated to be +2.97 kJ g⁻¹ COD and +0.57 kJ g⁻¹ COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 10¹³ kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH₂ production with low energy input.     

A pilot-scale high-rate biohydrogen production system with mixed microflora

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 (E_f) ranged from 13.65 to 28.68 on biohydrogen, which is higher than the E_f value on corn ethanol, biodiesel and sugarcane ethanol but in the similar range of cellulosic ethanol.     

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.