Hydrogen production from sulfate- and ferrous-enriched wastewater

The quantitative relationship between sulfate reducing bacteria (SRB) and hydrogen (H₂) production from sulfate (SO₄²⁻) and ferrous [Fe(II)] enriched wastewater was investigated. Both Fe(II) (0–11,600 mg/L) and SO₄²⁻ (0–20,000 mg/L) improved the H₂ production efficiency from wastewater. The H₂ yields were increased up to 1.9 mol H₂/mol glucose in 580–1750 mg Fe(II)/L and 1000–3000 mg SO₄²⁻/L enriched wastewater at pH 5.8–6.2. Quantitative Fluorescence In Situ Hybridization (FISH) analyses revealed that the specific sulfate reducing activities (SSRA) were increased from 0.08 and 0.06 to 0.16 and 0.21 g TS/g SRB h in response to variations in sulfate concentration from 300–20,000 mg/L at pH 5.8 and 6.2, respectively. H₂ production was not influenced by low SSRA (≤0.1 g TS/g SRB h), which was independent of pH variation. The results demonstrated that the SSRA and Fe(II) concentration can significantly influence on the biological H₂ production from SO₄²⁻ and Fe(II) containing wastewater.     

Sulfate- and pH-driven metabolic flexibility in sugarcane vinasse dark fermentation stimulates biohydrogen evolution,sulfidogenesis or homoacetogenesis

Dark fermentation of sugarcane vinasse can be used as a “cleaning” step to remove sulfate prior to methanogenesis because sulfidogenic conditions can be successfully established in parallel with biohydrogen production. Using a 2² central composite rotational design (CCRD) and response surface methodology (RSM), this study assessed the impacts of bicarbonate and sulfate availability on the establishment of sulfidogenesis in the thermophilic (55 °C) fermentation of vinasse in batch reactors, equally assessing the impacts on biohydrogen evolution. CCRD-RSM results indicated the favoring of biohydrogen production at the lowest sulfate and bicarbonate concentrations, whilst the opposite was observed for sulfidogenesis. Glycerol, lactate, and hydrogen were the preferential electron donors utilized by sulfate-reducing bacteria (SRB), whilst ethanol was markedly consumed only at high sulfate concentrations. SRB were inhibited by sodium when dosing excess NaHCO₃ and Na₂SO₄. Complementary tests revealed maximum biohydrogen production (2.40 mmol) out of the CCRD, at pH exceeding 7.5 with no interference of sulfidogenesis. Non-efficient biohydrogen production was observed at low pH (<5.0; ～1.90 mmol) because the uptake of lactate was inhibited. Meanwhile, homoacetogenesis was established under intermediate pH range (5.5–6.5), as revealed by the accumulation of acetate (up to 2.5 g L^?1). 16S rRNA gene amplicon sequencing further revealed the genera Thermoanaerobacterium/Pseudoclostridium, Desulfotomaculum/Desulfohalotomaculum and Sporomusaceae/Moorella as the main biohydrogen-producing, sulfate-removing and biohydrogen-consuming (homoacetogens) microbial groups, respectively. Hence, using a single inoculum source, vinasse may provide a butyrate-rich (along with biohydrogen-rich biogas) or a sulfate-free and acetate-rich fermented effluent, depending mainly on proper pH control.     

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.     

Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.     

Silage as source of bacteria and electrons for dark fermentative hydrogen production

In this study, grass silage was used both as a source of bacteria and as a substrate for dark fermentative hydrogen production. Silage is produced by lactic acid fermentation controlled by end point pH (<4.0). In this study, the fermentation of silage was successfully continued and directed to hydrogen production by neutralizing the pH. Highest hydrogen yield of 37.8 ± 5.8 mL H₂/g silage was obtained at 25 g/L of silage. The main soluble metabolites were acetate and butyrate with the final concentrations of 1.5 ± 0.2 and 0.5 ± 0.0 g/L, respectively. Bacteria present (at 25 g silage/L) included Ruminobacillus xylanolyticum, Acetanaerobacterium elongatum and Clostridium populeti and were involved in silage fermentation to hydrogen. In summary, this work demonstrates that grass silage becomes amenable to hydrogen fermentation by indigenous silage bacteria through pH neutralization.     

Modeling and statistical analysis of heat-shocked sulfate-reducers and methanogens rich consortiums for hydrogen and methane production in a bio-electrochemical cell

This study was carried to investigate the effect of thermal pre-treatment at 55 °C, 65 °C, 75 °C, and 85 °C on sulfate-reducing bacteria (SRB) and methanogens to check their potential in a Bio-electrochemical system (BES) for hydrogen and methane production. The experimental results indicate that SRB culture pre-treated at 55 °C could be used efficiently in hydrogen production up to 2.814 ± 0.091 M/M of glucose. A significant drop in methane production from Phase-I to Phase-III with an increase in pre-treatment temperature was observed. Volatile fatty acids production was found highest in Phase-III, with butyric acid in major concentration at the pre-treatment temperature range of 55 °C–65 °C in SRB-based BES. The higher hydrogen production conditions were tested with various models i.e. the Gompertz model, Richard model, and Logistic model to confirm its validity and hydrogen production prediction. Richard's model was found best fitted for cumulative hydrogen production.     

Enhancement of batch biohydrogen production from prehydrolysate of acid treated oil palm empty fruit bunch

Carbohydrates from hydrolyzed biomass has been a potential feedstock for fermentative hydrogen production. In this study, oil palm empty fruit bunch (OPEFB) was treated by sulfuric acid in different concentrations at 120 °C for 15 min in the autoclave. The optimal condition for pretreatment was obtained when OPEFB was hydrolyzing at 6% (w/v) sulfuric acid concentration, which gave the highest total sugar of 26.89 g/L and 78.51% of sugar production yield. However, the best conversion efficiency of OPEFB pretreatment was 39.47 at sulfuric acid concentration of 4%. A series of batch fermentation were performed to determine the effect of pH in fermentation media and the potential of this prehydrolysate was used as a substrate for fermentative hydrogen production under optimum pretreatment conditions. The prehydrolysate of OPEFB was efficiently converted to hydrogen via fermentation by acclimatized mixed consortia. The maximum hydrogen production was 690 mL H₂ L⁻¹ medium, which corresponded to the yield of 1.98 molH₂/mol_xylose achieved at pH 5.5 with initial total sugar concentration of 5 g/L. Therefore, the results implied that OPEFB prehydrolysate is prospective substrate for efficient fermentative hydrogen conducted at low controlled pH. No methane gas was detected throughout the fermentation.     

Utilization of palm kernel cake as a renewable feedstock for fermentative hydrogen production

Fermentative hydrogen generation was studied using palm kernel cake (PKC) as sustainable cellulosic biomass. PKC was subjected to an acid hydrolysis approach using dilute H₂SO₄ (7% v/v). PKC hydrolysate obtained was then diluted (70%) and used as a substrate for hydrogen generation. Chemical analysis showed that the main fermentable sugars in diluted PKC hydrolysate were glucose, xylose and mannose with the concentrations of 2.75 g/L, 2.60 g/L and 27.75 g/L, respectively. Hydrogen production was carried out by the cultivation of Clostridium acetobutylicum YM1 on PKC hydrolysate. The effect of incubation temperature, the initial pH of culture medium and microbial inoculum size on hydrogen production was studied using a statistical model. The analysis of the model generated showed that the initial pH value of the culture medium and inoculum size had significant effects on the hydrogen production. The study showed that the optimum conditions for the biohydrogen production were 30.57 °C temperature, pH 5.5 and 20% inoculum size. A verification experiment was performed in the optimum conditions determined. Experimental results of the verification test showed that a cumulative hydrogen volume of 1575 ml/L was generated with consuming 2.75 g/L glucose, 2.20 g/L xylose and 16.31 g/L mannose.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

A newly isolated Rhodobacter sphaeroides HY01 with high hydrogen production performance

A newly isolated strain, HY01, was identified to be Rhodobacter sphaeroides by phylogenetic analysis. Some DNA sequence data indicate that it is highly similar to R. sphaeroides ATCC 17029. The effects of initial pH values, nitrogen sources and carbon sources on hydrogen production were studied. The results showed that pH 7.25 is optimum for its hydrogen production. Among the nitrogen sources of glutamic acid, yeast extract, glycine, ethanolamine and l-aspartic acid, the maximum hydrogen production rate of 98.0 ± 6.2 mL/(Lh) using 10 mM glutamic acid and maximum hydrogen yield of 7012.5 ± 150 mL/L using 5 mM glutamic acid was obtained. Maximum hydrogen production rates of 148.7 ± 4.6 mL/(Lh) and 94.8 mL/(Lh) were obtained under 10 klux and 5 klux light intensity using 7 mM glutamic acid as nitrogen source, respectively. Compared with the reported data, it shows high hydrogen production performance and is a good candidate for further study.     

Kinetics of cotton cellulose hydrolysis using concentrated acid and fermentative hydrogen production from hydrolysate

The kinetics of cotton cellulose hydrolysis using concentrated sulfuric acid and the performance of fermentative hydrogen production from the hydrolysate in the batch system was carried out in this study. Effects of sulfuric acid concentrations, cotton cellulose concentrations and operating temperatures on the cotton cellulose hydrolysis were investigated. It was found that cotton cellulose can dissolve completely in sulfuric acid concentration above 55% (by volume) at room temperature. The reduced sugar yields were varied from 64.3 to 73.9% (g R-sugar/g cotton cellulose) with the initial cotton cellulose concentrations of 30-70 g/L at a temperature of 40 °C.The reduced sugar concentrations and the initial pH of biohydrogen production were investigated at 37 °C. It was found that the optimal values of the hydrogen yield and substrate utilization were 0.95 mol H₂/mol R-sugar and 98% with an initial pH of 8.2, when substrate concentration was fixed at 20 g R-sugar/L. The maximum hydrogen yield was 0.99 mol H₂/mol R-sugar at a substrate concentration of 15 g R-sugar/L. Using the Gompertz Equation Model simulation, the maximum hydrogen production rate was 253 mL H₂/h/L at a substrate of 30 g/L and initial pH of 8.4.     

Gamma irradiation as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge

Gamma irradiation was used as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The experimental results demonstrated that 5.0 kGy was optimal dose among the different doses (0.5–10 kGy) applied in this study. The maximum cumulative hydrogen production, hydrogen yield, hydrogen production rate and substrate degradation efficiency of the sludge irradiated at such dose were 529.4 mL, 267.7 mL/g glucose, 37.25 mL/h and 98.9%, respectively when the fermentation conditions were as follows: at 36 °C, initial pH 7.0 and 10 g/L glucose as substrate. In comparison with the conventional pretreatment methods, such as heat-shock, acid, base, aeration and chloroform, gamma irradiation was more powerful pretreatment method for enriching hydrogen-producing bacteria. The effect of Gamma irradiation on the microbial community structure of the pretreated sludge needs further study.     

Biohydrogen production,sludge granulation,and microbial community in an anaerobic inner cycle biohydrogen production (AICHP) reactor at different hydraulic retention times

This study investigated the continuous biohydrogen production in an anaerobic inner cycle biohydrogen production (AICHP) reactor fed with synthetic molasses wastewater as the model substrate under mesophilic conditions (37 ± 1 °C). The hydraulic retention times (HRTs) were set as 6.12, 4.90, 4.08, 3.50, and 3.06 h. Both maximum hydrogen production rate (HPR) (8.08 ± 0.48 L/L/d) and maximum granule formation were achieved at the HRT of 3.50 h. Acetic acid and butyric acid were the dominant metabolites in all tested HRTs throughout the experiment. Microbial community analysis showed that shortening the HRT promoted hydrogen production. This was mainly achieved by enhancing the growth of acetogenic bacteria in the AICHP reactor, rather than the growth of hydrogen-producing bacteria.     

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 sulfate on hydrogen production from the organic fraction of municipal solid waste using co-culture of E. coli and Enterobacter aerogenes

In the present study, the effect of sulfate on the hydrogen production from the organic fraction of municipal solid (OFMSW) waste using co-culture of Enterobacter aerogenes and E. coli has been studied under varying pH conditions. The presence of sulfate in the feedstock declines hydrogen production efficiency. To evaluate the effect of sulfate on hydrogen production from OFMSW, COD/sulfate ratio of 17.5, 15.0, 12.5, 10.0, 7.5, 5.0 and 2.5 were applied at different pH conditions (i.e. pH 5.5, 6.0 and 6.5). The hydrogen production continuously declined with the decreasing COD/sulfate ratio and increase in pH. The cumulative hydrogen production decreased from 220.8 ± 10.5 mL in control to a minimum of 98.3 ± 10.5 mL, 74.4 ± 10.4 mL, and 44.6 ± 2.6 mL at pH 5.5, 6.0 and 6.5 respectively. The major content of gaseous composition included hydrogen and CO₂ at higher COD/sulfate ratio and low pH, while H₂S formation started with the decrease in COD/sulfate ratio and increase in the pH. Similarly, sulfate removal efficiency was found to be influenced by COD/sulfate ratio and pH condition. Soluble metabolite analysis revealed that total volatile fatty acid concentration was not affected by sulfate addition. Thus, Sulfate removal is essential prior to fermentation in order to improve hydrogen yield.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

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.     

Hydrogen production from acidogenic food waste fermentation using untreated inoculum: Effect of substrate concentrations

The effect of substrate concentrations (0, 7.5, 15, 22.5, 30, and 37.5 g-VS/L) on hydrogen production from heat-treated and fresh food waste (FW) using untreated inoculums was investigated in this work. The highest hydrogen yield (75.3 mL/g-VS) was obtained with heat-treated FW at 15 g-VS/L. Lower substrate content could not provide enough organic matter for hydrogen fermentation, while higher substrate concentrations shifted the metabolic pathways from hydrogen fermentation to lactic acid fermentation by enriching the lactic acid bacteria (LAB), which lowered the slurry pH and decreased enzyme activity, resulting in a lower chemical oxygen demand (COD), volatile solid (VS), carbohydrate removal rate, and hydrogen yield. Compared with fresh FW, heat-treated FW is preferred for biohydrogen process with acetate as the main organic product. Additionally, at the optimal concentration (15 g-VS/L) using fresh FW, lactic acid is first accumulated and then degraded to produce hydrogen with butyrate as the main metabolite.     

Effect of the initial total solids concentration and initial pH on the bio-hydrogen production from cafeteria food waste

In this paper, the influence of the initial pH and the total solids (TS) concentration on hydrogen production from the organic fraction of cafeteria food waste at mesophilic conditions in batch reactors was determined. It was found that the yield and specific hydrogen production rate were influenced by the initial pH and the initial total solids concentration. The highest hydrogen production rate, 2.90 mmolH₂/d, was obtained at 90 gTS/L and a pH of 5.5. Under this condition, the TS and chemical oxygen demand (COD) removal were the lowest (10% as TS and 14% as COD). However, considering the specific values, the highest specific degradation rate (192.2 mLH₂/gVS_removed/d) was obtained with the lowest TS concentration and an initial pH of 7.0. It was found that the influence of the TS concentration on hydrogen production was more significant than that of the initial pH for this type of residues.     

Catalytic activity and characterization of Fe–Zn–Mg–Al hydrotalcites in biohydrogen production

Four different M²⁺–Mg–Al hydrotalcite (HT) materials were investigated for their effect on biohydrogen enhancement, where M²⁺ is Fe and/or Zn. HTs were synthesized by the coprecipitation method and characterized by infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). The effect of Fe–Zn–Mg–Al HTs dose (0–833 mg/L) on hydrogen production was investigated in batch tests using sucrose-fed anaerobic mixed culture at 37 °C. The best catalytic activity was observed on Mg–Al HT at 167 mg/L with the maximum hydrogen yield of 2.30 ± 0.37 mol H₂/mol sucrose, which was 44% higher than the control. The major metabolites detected in the test were acetic acid (3.6 g/L), butyric acid (4.1 g/L), and lactic acid (0.5 g/L). The basic properties of the different catalysts played an important role in stimulating or inhibiting the activity of hydrogen producing bacteria. Calcined Mg–Al HT did not promote biohydrogen production, suggesting that the catalytic enhancement was related to immobilization of bacteria in the electrostatically charged HT interlayers.