Fermentative hydrogen production by microbial consortium

Heat pre-treatment of the inoculum associated to the pH control was applied to select hydrogen-producing bacteria and endospores-forming bacteria. The source of inoculum to the heat pre-treatment was from a UASB reactor used in the slaughterhouse waste treatment. The molecular biology analyses indicated that the microbial consortium presented microorganisms affiliated with Enterobacter cloacae (97% and 98%), Clostridium sp. (98%) and Clostridium acetobutyricum (96%), recognized as H₂ and volatile acids' producers. The following assays were carried out in batch reactors in order to verify the efficiencies of sucrose conversion to H₂ by the microbial consortium: (1) 630.0 mg sucrose/L, (2) 1184.0 mg sucrose/L, (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L. The subsequent yields were obtained as follows: 15% (1.2 mol H₂/mol sucrose), 20% (1.6 mol H₂/mol sucrose), 15% (1.2 mol H₂/mol sucrose) and 4% (0.3 mol H₂/mol sucrose), respectively. The intermediary products were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors.     

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%.     

Bio-hydrogen production by mixed culture of photo- and dark-fermentation bacteria

Clostridium butyricum and Rhodopseudomonas faecalis RLD-53 were employed to produce hydrogen in mixed culture with glucose as sole substrate. Due to the great difference on growth rate and acid-resistant capacity between photo-fermentative bacteria and dark-fermentative bacteria, directly mixed culture of the two kinds of bacteria in different ratio was studied in this work. Hydrogen yield, volatile acids, pH and biomass in different periods were evaluated. Acetic acid and butyric acid produced by C. butyricum were dominant terminal fermentation products, and they were effective substrates for photo-fermentative bacteria. The cooperation was formed in a way like food chain. But compared to the production rate of volatile acids produced by C. butyricum, the utilization rate by photo-fermentative bacteria was far slower. The results demonstrated that the growth of photo-fermentative bacteria was limited when pH decreased sharply. The best ratio of C. butyricum to R. faecalis RLD-53 was 1:600. The maximum yield of hydrogen reached 122.4 ml-H₂/vessel and hydrogen production rate was 0.5 ml-H₂/ml-culture/day.     

Effect of changing temperature on anaerobic hydrogen production and microbial community composition in an open-mixed culture bioreactor

The temperature effect (37–65 °C) on H₂ production from glucose in an open-mixed culture bioreactor using an enrichment culture from a hot spring was studied. The dynamics of microbial communities was investigated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). At 45 and 60 °C the H₂ production was the highest i.e. 1.71 and 0.85 mol H₂/mol glucose, respectively. No H₂ was produced at temperatures 50 and 55 °C. At 37–45 °C, H₂ production was produced by butyrate type fermentation while fermentation mechanism changed to ethanol type at 60 °C. Clostridium species were dominant at 37–45 °C while at 50–55 °C and 60 °C the culture was dominated by Bacillus coagulans and Thermoanaerobacterium, respectively. In the presence of B. Coagulans the metabolism was directed to lactate production. The results show that the mixed culture had two optima for H₂ production and that the microbial communities and metabolic patterns promptly changed according to changing temperatures.     

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H₂ generation was utilized to study the production of H₂ from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H₂ production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H₂ from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.     

Dark fermentative hydrogen production by defined mixed microbial cultures immobilized on ligno-cellulosic waste materials

Mixed microbial cultures (MMCs) based on 11 isolates belonging to Bacillus spp. (Firmicutes), Bordetella avium, Enterobacter aerogenes and Proteus mirabilis (Proteobacteria) were employed to produce hydrogen (H₂) under dark fermentative conditions. Under daily fed culture conditions (hydraulic retention time of 2 days), MMC6 and MMC4, immobilized on ligno-cellulosic wastes – banana leaves and coconut coir evolved 300–330 mL H₂/day. Here, H₂ constituted 58–62% of the total biogas evolved. It amounted to a H₂ yield of 1.54–1.65 mol/mol glucose utilized over a period of 60 days of fermentation. The involvement of various Bacillus spp. – Bacillus sp., Bacillus cereus, Bacillus megaterium, Bacillus pumilus and Bacillus thuringiensis as components of the defined MMCs for H₂ production has been reported here for the first time.     

Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production

Volatile fatty acids (VFA) from dark fermentation hydrogen production were tested as carbon sources for the culture of oleaginous yeast Cryptococcus curvatus, which is a promising feedstock for biofuel production. The optimal acetate concentration and pH were investigated when potassium acetate was used as the sole carbon source. Comparisons were then made when hydrogen production effluent (HPE) from synthetic wastewater was tested as feedstock. A pH-stat culture fed with acetic acid ultimately produced 168 g/L biomass, with a lipid content of 75.0%. No inhibitor to yeast growth was produced in the hydrogen production process. However, inhibition occurred in culture with HPE from food waste (FW), indicating that inhibitors may be present in the original raw food waste. This inhibition could be avoided by a process that uses glucose as the initial carbon source and then is continuously fed with FW-HPE. The biomass productivity in this continuous culture process reached 0.34 g/L/h, but the lipid content was only 13.5%. These results suggest that FW-HPE alone is not an optimal feedstock, but HPE derived from nitrogen-deficient waste streams could be good feedstocks. This study provides preliminary evidence for the feasibility of using organic waste for the co-production of hydrogen and lipid.     

Effect of organic loading rate and solids retention time on microbial population during bio-hydrogen production by dark fermentation in large lab-scale

The experimental investigation aimed at the study of the microbial population during the continuous operation of a complete mixed reactor in large lab-scale (30 L) by variation of the Organic Loading Rate (OLR) and Sludge Retention Time (SRT) ranging from 10 g sucrose/(L?d) to 30 g/(L?d) and from 12 h to 48 h respectively. H₂ yield reached to 1.72 mol H₂/mol hexose for HRT = 1.6 d and OLR = 20 g sucrose/(L?d). In each phase the dominant microbial genera were identified by sequencing after a Polymerase Chain Reaction (PCR) with universal primers for the domains of Archaea and Eubacteria and specific for Clostridium species and genetic material isolation by Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The phylogenetic analyses showed that hydrogen producing Clostridium species could be affiliated in all experimental phases. Other dominant genera were affiliated mainly to Ethanoligenes harbinense and uncultured Prevotella and Selonomonas species. Bio-hydrogen production was associated to a mixed butyric/ethanol type fermentation facilitated mainly by Clostridium tyrobutyricum and E. harbinense in the presence of lactate as intermediate metabolic product.     

Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a photobioreactor

Rhodobacter sphaeroides O.U.001 is a photosynthetic non-sulfur bacterium which produces hydrogen from organic compounds under anaerobic conditions. Halobacterium salinarum is an archaeon and lives under extremely halophilic conditions (4 M NaCl). H. salinarum contains a retinal protein bacteriorhodopsin in its purple membrane which acts as a light-driven proton pump. In this study the Rhodobacter sphaeroides O.U.001 culture was combined with different amounts of packed cells of H. salinarum S9 or isolated purple membrane fragments in order to increase the photofermentative hydrogen gas production. The packed cells of H. salinarum have the ability to pump protons upon illumination due to the presence of bacteriorhodopsin. The proton gradient produced may be used for the formation of ATP or protons may be used for H₂ production by R. sphaeroides. Similar to intact cells purple membrane fragments may also form vesicles around certain ions and may act like closed systems.     

Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond

A new hydrogen-producing photosynthetic bacterium, designated as Rhodovulum sulfidophilum P5, was enriched and isolated from the sludge of a marine shrimp cultivation farm. During fermentation, hydrogen was mainly produced in the late exponential and stationary phases. The optimum culture conditions of strain P5 for hydrogen production were NaCl concentration of 20 g/L, initial pH of 8, temperature of 30 °C, and light intensity of 100 μmol photons/m² s. The maximum hydrogen yield and rate were 2.56 ± 0.18 mol/mol acetate and 19.4 ± 1.6 mL/L h, respectively. Under optimum culture conditions, the hydrogen conversion efficiencies of P5 from acetate, propionate, and butyrate were (64.62 ± 5.05)%, (17.95 ± 0.72)%, and (41.83 ± 2.68)%, respectively. Taken together, these results suggest that this strain has a high salt tolerance and the potential to be used for biohydrogen production and biological treatment of marine organic wastewater.     

Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation

Chlamydomonas reinhardtii UTEX 90 accumulated 1.45 g dry cell weight and 0.77 g starch/L during photosynthetic growth using TAP media at 25 ^°C${}^{°}\mathrm{C}$ in presence of 2% _CO2${\mathrm{CO}}_2$ for 3 days. C. reinhardtii biomass was concentrated and then converted into hydrogen and organic acids by anaerobic fermentation with Clostridium butyricum. Organic acids in the fermentate of algal biomass were consecutively photo-dissimilated to hydrogen by Rhodobacter sphaeroides KD131. In the concentrated algal biomass 52% of the starch was hydrolyzed to 37.1 mmol _H2${\mathrm{H}}_2$/L-concentrated algal biomass and 13.6, 25.5, 7.4 and 493 mM of formate, acetate, propionate, and butyrate, respectively by C. butyricum. R. sphaeroides KD131 evolved 5.72 mmol _H2${\mathrm{H}}_2$ per ml-fermentate of algal biomass under illumination of 8 klux at 30 ^°C${}^{°}\mathrm{C}$. Only 80% of the organic acids, mainly butyrate, were hydrolyzed during photo-incubation. During anaerobic conversion, 2.58 mol _H2/mol${\mathrm{H}}_2/\mathrm{mol}$ starch–glucose was evolved using C. butyricum and then 5.72 mol _H2/L${\mathrm{H}}_2/\mathrm{L}$-anaerobic fermentate was produced by R. sphaeroides KD131. Thus, the two-step conversion process produced 8.30 mol _H2${\mathrm{H}}_2$ from 1 mol starch–glucose equivalent algal biomass via organic acids.     

Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures

Dark fermentation using mixed cultures is an attractive biological process for producing hydrogen (H₂) from lignocellulosic biomass at a low cost. Physicochemical pretreatment is generally used to convert lignocellulosic materials into monosaccharides. However, the processes also involved release degradation byproducts which can, in turn, inhibit microbial growth and metabolism and, hence, impact substrate conversion. In this study, the impact on H₂ production of lignocellulose-derived compounds (i.e. furan derivatives, phenolic compounds and lignins) was assessed along with their effect on bacterial communities and metabolisms. Batch tests were carried out using xylose as model substrate (1.67 mol_H2 mol_xylose⁻¹ in the control test). All the putative inhibitory compounds showed a significant negative impact on H₂ production performance (ranging from 0.34 to 1.39 mol_H2 mol_xylose⁻¹). The H₂ yields were impacted more strongly by furan derivatives (0.40–0.51 mol_H2 mol_xylose⁻¹) than by phenolic compounds (1.28–1.39 mol_H2 mol_xylose⁻¹). Except for the batch tests supplemented with lignins, the lag phase was shorter for inhibitors having the highest molecular weight (8 days versus 22 days for the lowest MW). Variability of the lag phase was clearly related to a shift in bacterial community structure, as shown by multivariate ordination statistics. The decrease in H₂ yield was associated with a decrease in the relative abundance of several H₂-producing clostridial species. Interestingly, Clostridium beijerinkii was found to be more resistant to the inhibitors, making this bacterium an ideal candidate for H₂ production from hydrolyzates of lignocellulosic biomass.     

Effect of microwave pretreatment of mixed culture on biohydrogen production from waste of sweet produced from Benincasa hispida

To enhance the production of biohydrogen from biomass, various pretreatment methods play important role. In this study, effect of microwave irradiation on the culture was studied on biohydrogen production from Benincasa hispida (Petha) solid waste at different powers for a fixed interval of time. The highest power studied was 800 W with a frequency of 2450 MHz. The amount of soluble sugars found in the waste was 13.9 mg/L having the chemical oxygen demand (COD) of 3000 mg/L. Studies have been performed in batch reactors using mixed consortia and results were also compared with the reactor operated at the normal conditions i.e. without any inoculum pretreatment. Maximum hydrogen produced was 14 mmol H₂ per mol of soluble sugar consumed in the reactor in which the inoculum was exposed to 320 W of microwave for 5 min. SEM analysis of this microwave pretreated culture was done.     

Hydrogen production by Chlamydomonas reinhardtii in a two-stage process with and without illumination at alkaline pH

This work presents the results of a two-stage (carbon fixation and hydrogen production) experimental study for hydrogen production from microalgae using optical fiber as an internal light source. Effect of absence and presence of light on Chlamydomonas reinhardtii culture’s pH shift is also evaluated. The culture pH value is a function of light intensity; the pH in the alkaline range changes from 7.5 to 9.5 in the presence and absence of optical fiber respectively. The maximum rate of hydrogen production in the presence of exogenic glucose and optical fiber is 6 mL/L_cult/hour, which is higher than other reported values. This study has also revealed that the presence of light reduces the lag time for hydrogen production from 12 to 5 h.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

A viable antibiotic strategy against microbial contamination in biotechnological production of polyhydroxyalkanoates from surplus whey

The study demonstrates the successful application of vancomycin for inhibiting growth of the contaminant Bacillus cereus in a polyhydroxyalkanoate (PHA) production process with Hydrogenophaga pseudoflava on whey. The minimum inhibiting concentration (MIC) of vancomycin for both strains was calculated in minimal medium H3 as well as in H3 medium supplemented with yeast extract. MIC for B. cereus was 0.57 ??g mL⁻¹ and 1.63 ??g mL⁻¹ in H3 and H3 plus yeast extract respectively. For H. pseudoflava, MIC amounted to 2.8 ??g mL⁻¹ in H3 medium and 6.88 ??g mL⁻¹ in H3 plus yeast extract. The effect of vancomycin on PHA production was minimal or negligible up to a vancomycin concentration of 1 ??g mL⁻¹ in both media. The specific PHA production rates of H. pseudoflava decreased with increasing antibiotics in a minimal medium but in media supplemented with yeast extract the specific PHB production rates increased as the antibiotics concentrations increased.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using a novel strategy — shaking and extra-light supplementation approach

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H₂ production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H₂ production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H₂/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H₂ production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H₂ production efficiency.