Bioconversion of corncob to hydrogen using anaerobic mixed microflora

Biohydrogen production from corncob using natural anaerobic microflora was reported for the first time. The optimum pretreatment condition for the corncob was determined to be 100 °C, 30 min, and 1% HCl (w/w). The maximum hydrogen yield of 107.9 ml/g-TVS and hydrogen production rate of 4.20 ml/g-TVS h⁻¹ was obtained under the condition of 10 g/l substrate concentration and initial pH 8.0. Butyrate and acetate were the dominant metabolic by-products of hydrogen fermentation. Chemical composition analysis, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to study the mechanism of degrading corncob for hydrogen production. The amorphous domains of cellulose and hemicellulose were hydrolyzed into fermentable saccharides through acid pretreatment and the microorganisms had a devastating effect on the crystallinity of the cellulose. The hydrogen yield from pretreated corncob was much higher than from raw corncob. Therefore, the acid pretreatment played a crucial role on hydrogen production from corncob.     

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.     

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.     

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.     

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.     

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.     

Fermentative biohydrogen production: Evaluation of net energy gain

Most dark fermentation (DF) studies had resorted to above-ambient temperatures to maximize hydrogen yield, without due consideration of the net energy gain. In this study, literature data on fermentative hydrogen production from glucose, sucrose, and organic wastes were compiled to evaluate the benefit of higher fermentation temperatures in terms of net energy gain. This evaluation showed that the improvement in hydrogen yield at higher temperatures is not justified as the net energy gain not only declined with increase of temperature, but also was mostly negative when the fermentation temperature exceeded 25 °C. To maximize the net energy gain of DF, the following two options for recovering additional energy from the end products and to determine the optimal fermentation temperature were evaluated: methane production via anaerobic digestion (AD); and direct electricity production via microbial fuel cells (MFC). Based on net energy gain, it is concluded that DF has to be operated at near-ambient temperatures for the net energy gain to be positive; and DF + MFC can result in higher net energy gain at any temperature than DF or DF + AD.     

Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures

Batch tests were conducted to evaluate the enhancement of hydrogen/ethanol (EtOH) productivity using cow dung microflora to ferment α-cellulose and saccharification products (glucose and xylose). Hydrogen/ethanol production was evaluated based on hydrogen/ethanol yields (HY/EY) under 55 °C at various initial pH conditions (5.5–9.0). Our test results indicate that cow dung sludge is a good mixed natural-microflora seed source for producing biohydrogen/ethanol from cellulose and xylose. The heat-pretreatment, commonly used to produce hydrogen more efficiently from hexose, applied to mixed anaerobic cultures did not help cow dung culture convert cellulose and xylose into hydrogen/ethanol. Instead of heat-pretreatment, the mixed culture received enrichments cultivated at 55 °C for 4 days. Positive results were observed: hydrogen/ethanol production from fermenting cellulose and xylose was effectively enhanced at increases of 4.8 (ethanol) to 8 (hydrogen) and 2.4 (ethanol) to 15.6 (hydrogen) folds, respectively. In which, the ethanol concentration produced from xylose reached 4–4.4 g/L, an output comparable to that of using heat-treated sewage sludge and better than that (1.25–3 g/L) using pure cultures. Our test results show that for the enriched cultures the initial cultivation pH can affect hydrogen/ethanol production including HY, EY and liquid fermentation product concentration and distribution. These results were also concurred using a denaturing gradient gel electrophoresis analysis saying that both cultivation pH and substrate can affect the enriched cow dung culture microbial communities. The enriched cow dung culture had an optimal initial cultivation pH range of 7.6–8.0 with peak HY/EY values of 2.8 mmol-H₂/g-cellulose, 5.8 mmol-EtOH/g-cellulose, 0.3 mol-H₂/mol-xylose and 1 mol-EtOH/mol-xylose. However, a pH change of 0.5 units from the optimal values reduced hydrogen/ethanol production efficiency by 20%. Strategies based on the experimental results for optimal hydrogen/ethanol production from cellulose and xylose using cow dung microflora are proposed.     

Fermentative hydrogen production from wastewaters: A review and prognosis

Biohydrogen is a promising candidate which can replace a part of our fossil fuels need in day-to-day life due its perceived environmental benefits and availability through dark fermentation of organic substrates. Moreover, advances in biohydrogen production technologies based on organic wastewater conversion could solve the issues related to food security, climate change, energy security and clean development in the future. An evaluation of studies reported on biohydrogen production from different wastewaters will be of immense importance in economizing production technologies. Here we have reviewed biohydrogen production yields and rates from different wastewaters using sludges and microbial consortiums and evaluated the feasibility of biohydrogen production from unexplored wastewaters and development of integrated bioenergy process. Biohydrogen production has been observed in the range of substrate concentration 0.25–160 g COD/L, pH 4–8, temperature 23–60 °C, HRT 0.5–72 h with various types of reactor configuration. The most efficient hydrogen production has been obtained at an organic loading rate (OLR) 320 g COD/L/d, substrate concentration 40 g COD/L, HRT 3 h, pH 5.5–6.0, temperature 35 °C in a continuously-stirred tank reactor system using mixed cultures and fed with condensed molasses fermentation soluble wastewater. The net energy efficiency analysis showed vinasse wastewater has the highest positive net energy gain followed by glycerin wastewater and domestic sewage as 140.39, 68.65, 51.84 kJ/g COD feedstock with the hydrogen yield (HY) of 10 mmol/g COD respectively.     

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.     

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.     

Simultaneous hydrogen and ethanol production from a mixture of glucose and xylose using extreme thermophiles II: Effect of hydraulic retention time

In this study, biofuels (hydrogen and ethanol) fermentation from glucose and xylose by extreme thermophiles in an Up-flow Anaerobic Sludge Bed (UASB) reactor was successfully demonstrated. Autoclaved methanogenic granules were used as carriers for the extreme thermophiles. High yields of hydrogen and ethanol were achieved at various HRTs from 24 h to 6 h. The highest hydrogen production rate of 121 ± 23 mL/(L h) and highest ethanol production rate of 6.7 ± 1.2 mmol/(L h) were observed at HRT = 12 h. The highest simultaneous hydrogen and ethanol yields were 0.58 ± 0.11 mol H₂/(mol hexose) and 0.72 ± 0.13 mol ethanol/(mol hexose), reaching a total energy yield of 1151 kJ/mol hexose. The substrate conversion efficiency was maintained over 90% at three HRTs (24, 18, and 12 h).     

Sequential production of hydrogen and methane using hemicellulose hydrolysate from diluted acid pretreatment of sugarcane straw

Sugarcane straw is not effectively used in the industry currently. However, sugarcane straw hemicellulose hydrolyzate (HH) application as raw material for H₂ and CH₄ production is a promising alternative. In this work, the two-stage anaerobic digestion (TS-AD) approach led to 37.86 mL_H2/L.h and 40.12 mL_CH4/L.d, while the single-stage anaerobic digestion (SS-AD) generated 46.11 mL_CH4/L.d. Hence, the two-stage process was energetically favorable than the single-stage by approximately 33%. Additionally, the comparison with standard medium (composed of glucose, xylose, and arabinose) applied as raw material indicated that although hydroxymethylfurfural and furfural from HH were not responsible for the decrease in H₂ production, they extended the adaptive phase of methanogenic archaea during the methanogenesis. Hemicellulose hydrolysate is an attractive raw material for two-stage anaerobic digestion.     

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.     

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.