Feasibility study on the application of rhizosphere microflora of rice for the biohydrogen production from wasted bread

We performed an experiment of continuous anaerobic hydrogen fermentation as a pilot-plant-scale test, in which waste from a bread factory was fermented by microflora of rice rhizosphere origin. The community structure of microflora during anaerobic hydrogen fermentation was analyzed using PCR-DGGE, FISH, and quinone profiles. The relation of those results to hydrogen generation was discussed. Results show that a suitable condition was a reactor temperature of 35 °C, with HRT 12–36 h, volume load of 30–70 kg-COD_Cr/m³ day, and maximum hydrogen production rate of 1.30 mol-H₂/mol-hexose. Regarding characteristics of microflora during fermentation, PCR-DGGE results show specific 16S rDNA band patterns; Megasphaera elsdenii and Clostridium sp. of the hydrogen-producing bacteria were identified. M. elsdenii was detected throughout the fermentation period, while Clostridium sp. of hydrogen-producing bacteria was detected on the 46th day. Furthermore, FISH revealed large amounts of Clostridium spp. in the sample. The quinone profile showed that the dominant molecular species of quinone is MK-7. Because Clostridium spp. belong to MK-7, results suggest that the quinone profile result agrees with the results of PCR-DGGE and FISH. Microflora in the rhizosphere of rice plants can be a possible resource for effective bacteria of biohydrogen production.     

Microbial characterization of hydrogen-producing bacteria in fermented food waste at different pH values

An anaerobic fermentation of food waste was conducted in a 0.5 L bioreactor incubated at a thermophilic temperature of 55 °C to evaluate the effects of different controlled pH values (5.0, 5.5 and 6.0) on biohydrogen production. Effective biohydrogen production was found at controlled pH 5.5 and 6.0 corresponding to lower lactic acid production compared to pH 5.0. It was demonstrated that biohydrogen production from food waste was pH-dependent with hydrogen yields of 79, 76 and 23 mmol H₂/L-media/d for pH 5.5, 6.0 and 5.0, respectively. Specific microbial determination for Clostridium sp. and total bacteria quantification were carried out by the fluorescent in-situ hybridization (FISH) technique. The number of Clostridium sp. for acclimatized sludge, fermentation broth at pH 5.0, 5.5 and 6.0 were 2.9 × 10⁸, 3.6 × 10⁸, 7.8 × 10⁸ and 5.4 × 10⁸ cells/ml, respectively. The quantification analysis showed that 92% of the total bacteria belonged to Clostridium sp. from clusters I and XI from the sample at controlled pH 5.5. The denaturing gradient gel electrophoresis (DGGE) bands of the sample after heat-treatment, acclimatization and during fermentation indicated the presence of Bacteroidetes, Caloromator australicus sp. and Clostridium sp.     

The factors influencing direct photohydrogen production and anaerobic fermentation hydrogen production combination bioreactors

A system that combines fermentative and photosynthetic bacteria makes combined organic wastewater treatment and energy recycling possible. This study introduces a method to separates useful byproduct, such as volatile fatty acids, to serve as the substrate for a serial photobioreactor from the outlet of anaerobic hydrogen fermentation reactor using a ceramic membrane system. Batch experiments were applied to investigate the metabolic characteristics of Rhodopseudomonas palustris WP3-5 under various conditions during photobioreactor operation. The results indicate that a higher temperature (35 °C) improved poly-β-hydroxybutyric acid (PHB) content and hydrogen accumulation. Maximal hydrogen accumulation and reduced PHB synthesis occurred at lower temperature (25 °C) at an initial pH of 6.8.. Glutamate and peptone were more suitable nitrogen sources for hydrogen production than glutamine and NH₄Cl. Photohydrogen production results, using various anaerobic fermentation effluents with a dilution ratio of 0.4X, illustrated that an increased photohydrogen production rate (HPR) could be obtained using an anaerobic fermentation influent with an initial C/N ratio of 4. The result obtained when an anaerobic hydrogen fermentation reactor was connected to the serial photobioreactor showed that when the anaerobic hydrogen fermentation reactor outlet dilution increased from 2.5- to 5-fold, HPR increased. The HPR of the three bioreactors in the serial photobioreactor were 269.2, 345.6 and 256.7 mL-H₂/L-reactor/day. A UV-sterilizer, which was placed between the fermentation reactor and photobioreactor, resulted in the longer (336 h) operating duration.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

Fermentative hydrogen production from lipid-extracted microalgal biomass residues

The conversion of lipid-extracted microalgal biomass residues (LMBRs) into hydrogen plays the dual role in renewable energy production and sustainable development of microalgal biodiesel industry. An anaerobic fermentation process to covert LMBRs into hydrogen was investigated in this work. Using batch experiments, the effects of pretreatment of inoculum (by acid, base, heat, and chloroform, respectively), initial pH (5.0–7.0), inoculum concentrations at 0.59–2.94 g VSS/l (volatile suspended solids, VSS) and substrate concentrations at 4.5–45 g VS/l (volatile solids, VS) were investigated, respectively. The results showed that the most effective hydrogen production was obtained from fermentation of LMBRs with a concentration of 36 g VS/l at the initial pH 6.0–6.5 using the heat-treated anaerobic digested sludge as inoculum. Acetate, propionate and butyrate were the main fermentation byproducts in the conversion of LMBRs into hydrogen.     

Optimization of key variables for the enhanced production of hydrogen by Ethanoligenens harbinense W1 using response surface methodology

The optimization of process conditions for the production of hydrogen by Ethanoligenens harbinense W1 was investigated using response surface methodology (RSM). Three parameters namely inoculum to substrate ratio, initial pH value and temperature were chosen as variables. The adequately high R² value (99.4%) indicated the statistical significance of the model. The optimum process conditions for hydrogen production rate were determined by analyzing the response surface three-dimension surface plot and contour plot and by solving the regression model equation with Design Expert software. The central composite design (CCD) was used to optimize the process conditions, which showed that an inoculum to substrate ratio of 14%, initial pH value of 4.32 and the experimental temperature of 34.97 °C were the best conditions. Under the optimized conditions, the maximum specific hydrogen production rate (SHPR) was 35.74 mL/g-CDW.h based on cell dry weight. The results were further verified by triplicate experiments. The batch reactors were operated under an optimized condition of the inoculum to substrate ratio of 14%, initial pH value of 4.3 and the experimental temperature of 35 °C. The maximum SHPR was estimated at 35.57 mL/g-CDW.h, which further verified the practicability of this optimum strategy.     

Enhanced bio-hydrogen production by anaerobic fermentation of apple pomace with enzyme hydrolysis

A series of batch experiments were conducted to investigate the effects of the HCl-pretreated concentrations, enzyme hydrolysis time and temperature, the cellulase dosage, the ultrasonic time and the fermentation substrate concentration on hydrogen (H₂) production from the anaerobic fermentation of apple pomace (AP). The natural mixed microorganisms from river sludge was used as the seed after being boiled for 15 min. A maximum cumulative H₂ yield (CHYm) of 134.04 ml/g total solid (TS) and an average H₂ production rate (AHPR) of 12.00 ml/g TS/h were obtained from the fermentation of the enzyme-hydrolyzed AAP (a AP soaked with 6% ammonia liquor for 24 h) at the substrate concentration of 15 g/L. The optimal enzyme hydrolysis conditions were proposed as follows: AAP with a cellulase dosage of 12.5 mg/g TS at the hydrolysis substrate concentration of 20 g/L after the ultrasonic irradiation for 20 min was hydrolyzed by the enzyme catalysis at 45 °C and initial pH 5.0 for 48 h.     

Biohydrogen and biogas production from mashed and powdered vegetable residues by an enriched microflora in dark fermentation

This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35^?C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H₂ with hydrogen yield and hydrogen concentration of 151.67 mL H₂/g VS_added and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.     

Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

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 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.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 °C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57–70%, 41–55% and 37–67%.     

Development of a simple bio-hydrogen production system through dark fermentation by using unique microflora

In order to ensure efficient functioning of hydrogen fermentation systems that use Clostridium as the dominant hydrogen producer, energy-intensive process such as heat pretreatment of inoculum and/or substrate, continuous injection, and control of anaerobic conditions are required. Here, we describe a simple hydrogen fermentation system designed using microflora from leaf-litter cattle-waste compost. Hydrogen and volatile fatty acid production was measured at various hydraulic retention times, and bacterial genera were determined by PCR amplification and sequencing. Although hydrogen fermentation yield was approximately one-third of values reported in previous studies, this system requires no additional treatment and thus may be advantageous in terms of cost and operational control. Interestingly, Clostridium was absent from this system. Instead, Megasphaera elsdenii was the dominant hydrogen-producing bacterium, and lactic acid-producing bacteria (LAB) were prevalent. This study is the first to characterize M. elsdenii as a useful hydrogen producer in hydrogen fermentation systems. These results demonstrate that pretreatment is not necessary for stable hydrogen fermentation using food waste.     

Biohydrogen using leachate from an industrial waste landfill as inoculum

Since hydrogen is a renewable energy source, biohydrogen has been researched in recent years. However, there is little data on hydrogen fermentation by a leachate from a waste landfill as inoculum. We investigated hydrogen production using a leachate from an industrial waste landfill in Kanagawa prefecture. The results showed no methane gas production and the leachate was a suitable inoculum for hydrogen fermentation. The maximum H₂ yield was 2.67 mol of H₂ per mol of carbohydrate added, obtained at 30 °C and initial pH 7. The acetate and butyrate production was significant when the H₂ yield was higher. The oxidation–reduction potential analysis of the culture suggested that hydrogen-producing bacteria in the leachate were facultatively anaerobic. Scanning electron microscope observations revealed hydrogen-producing bacteria comprised bacilli of about 2 μm in length.     

Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H₂ yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H₂ generation potential (171.3 NL H₂/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.     

Effect of temperature on anaerobic fermentative hydrogen gas production from feedlot cattle manure using mixed microflora

Hydrogen production from the anaerobic fermentation of feedlot cattle manure was examined in batch cultures over a temperature range from 36 to 60 °C at a pH of 5.2. The amount of hydrogen produced increased with temperature to a maximum of 65 L H₂ kg TS⁻¹ at 52 °C. At temperatures > 52 °C, acetate was the main volatile fatty acid (VFA) accumulated, while at <52 °C butyrate accumulated the most. Formate was detected in the 56 and 60 °C treatments but was absent in all others. Thermophilic conditions resulted in the highest hydrogen production rates, with maximum hydrogen production occurring 52 °C. Changing incubation temperature by small (4 °C) increments up or down from 52 °C resulted in changes in the metabolic flux (conversion of substrate to VFA and gaseous products) of the anaerobic digestion system. These findings indicate that the hydrogen production potential of anaerobic systems utilizing heat treated cattle manure as inoculum is affected greatly by incubation temperature.     

Effect of system optimizing conditions on biohydrogen production from herbal wastewater by slaughterhouse sludge

The study evaluates the biohydrogen production from herbal wastewater as the substrate by the enriched mixed slaughterhouse sludge as the seed source. In the following experiments, batch-fermentations are carried out with the optimum substrate concentrations, fermentation pH and fermentation temperature to observe the effects of H₂ production, hydrogen yield and other fermentation end products at different conditions. The hydrogen production is increased as substrate concentration increased up to 8 g COD/L WW, but drastically decreased at 10 g COD/L WW. When the pH of fermentation is controlled to 6.5, a maximum amount of hydrogen yield could be obtained. The hydrogen production is maximum at 50 °C (930 ± 30 mL/L WW) compared to 30 °C (436 ± 16 mL/L WW). Acid-forming pathway with butyric acid as a major metabolite dominated the metabolic flow during the hydrogen production. The experimental results indicated that effective hydrogen production from the herbal wastewater could be obtained by thermophilic acidogenesis at proper operational conditions.     

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.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.