Synergic effect of various amino acids and ferric oxide on hydrogen production

To overcome nitrogen and iron deficiency in the organic fraction of municipal solid waste, amino acids and ferric oxide were separately added in the feedstock to evaluate their effect on hydrogen production. Furthermore, synergic effect of amino acids and ferric oxide on hydrogen production was evaluated. The co-culture of E. coli and Enterobacter aerogenes was used in the present study. The amino acids were applied in the concentration range of 1.0, 2.5, 5.0, 7.5 and 10.0 g/L while ferric oxide was used in the concentration range of 10, 20, 30, 40, 50, 100, 150, 200 and 500 mg/L. Modified Gompertz model was used to analyze cumulative hydrogen production (P), maximum hydrogen production rate (Rmax) and lag phases (λ). The results exhibited that the hydrogen production was positively affected by each amino acid at every concentration applied. Application of alanine resulted in the highest cumulative and volumetric hydrogen production of 685.4 ± 10.1 mL and 1.9561L_H2/L_substrate respectively which increased to 872.5 ± 10.1 mL and 2.492L_H2/L_substrate for ferric oxide addition along with alanine. COD removal and VFA generation were positively affected by the synergic effect of amino acid and ferric oxide.     

Effects of pH and ferrous iron on the coproduction of butanol and hydrogen by Clostridium beijerinckii IB4

Butanol and hydrogen gas are main products in butanol production by solventogenic Clostridia. Effects of pH and ferrous iron on hydrogen and butanol production by Clostridium beijerinckii IB4 were investigated in this work. With the increasing of the pH value, the hydrogen yields increased during acidogenic phase and decreased during solventogenic phase. Compared with the process without pH control, butanol and hydrogen increased by 15.43% and 11.77%, respectively, when pH was controlled at 5.2. Under the control of pH at 5.2 and supplementation of 250 mg/L of FeSO₄·7H₂O, the maximum hydrogen quantity of 8.24 L/L was obtained, and hydrogen productivity achieved 187 mL/L/h with yield of 145 mL/g glucose, which increased by 65%, 46% and 37%, respectively. However, butanol yield was reduced slightly by 4.8%. The reducing power was enhanced in solventogenic phase with supplementation of 250 mg/L FeSO₄·7H₂O, and the energy content of fuel produced in this process also remained stable. These results indicated that hydrogen production was enhanced by pH control and ferrous iron regulation, and butanol production performance was also maintained, which was favorable for coproduction of butanol and hydrogen in ABE fermentation.     

Anaerobic solid-state fermentation of bio-hydrogen from microalgal Chlorella sp. biomass

A considerable amount of volatile solids (VS) contained in the biomass of microalgae makes it promising for use as feedstock in fermentation processes. In this study, a biomass of microalga Chlorella sp. was used as a sole substrate for hydrogen production in an anaerobic solid-state fermentation (ASSF). Optimization of the process was investigated on the selected critical variables, i.e., total solid (TS) content, initial pH, and feed to inoculum (F/I) ratio (on a VS basis) using response surface methodology (RSM) with central composite design (CCD). TS content and F/I ratio were found to have statistically significant effects on hydrogen production. Maximal hydrogen production of 165 ± 12 mL H₂, equivalent to 18.58 mL H₂/g VS and 0.28 L H₂/L reactor·d, was achieved under the optimal conditions of 38.83% TS, pH 6.03, and an F/I ratio of 4.33. Acetic and butyric acids were found to be main soluble microbial products (SMPs) in the fermented biomass. Based on the compositions of the biomass, an equation for theoretical bioconversion of Chlorella sp. biomass to hydrogen was proposed.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Overexpressing atpXF enhanced photo-fermentative hydrogen production performance of Rhodobacter sphaeroides

The effects of overexpressing atpXF genes encoding b₂ subunits of F₀F₁-ATPase in Rhodobacter sphaeroides HY01 on ATPase, nitrogenase and photo-fermentative hydrogen production activities were studied. The results showed that the expression levels of atpXF and f₁ operon in ZTHBB (F₀P⁺atpXF⁺) enhanced by 1870% and 40% while its expression levels of f₀ operon and nif decreased by 20% and 50% compared with those of ZTH, respectively; and the expression levels of b₂ subunits, f₀ operon, f₁ operon and nif in ZTHCB (pucP⁺atpXF⁺) enhanced by 370%, 60%, 130% and 160% compared with those of ZTH, respectively. The ATP contents of ZTHBB and ZTHCB increased by 41.5% and 52.8% compared with that of ZTH, and their nitrogenase activities were increased by 30.0% and 41.0% compared with that of ZTH, respectively. When 25 mM acetic acid and 34 mM butyric acid were employed as carbon source and 6 mM l-glutamate was employed as nitrogen source, their hydrogen yields were 7677 ± 176 mL/L (ZTHBB) and 8458 ± 129 mL/L (ZTHCB), which increased by 16.0% and 29.1% compared with that of ZTH, and their maximum hydrogen production rates were 98.0 ± 4.3 mL/(L·h) (ZTHBB) and 100.1 ± 5.1 mL/(L·h) (ZTHCB) under the same conditions, which increased by 14.6% and 17.1% compared with that of ZTH, respectively. The results suggest that overexpressing atpXF could enhance the expression level of F₀F₁-ATPase and hydrogen production performance of purple non-sulfur bacteria.     

Photofermentation of tequila vinasses by Rhodopseudomonas pseudopalustris to produce hydrogen

Hydrogen production by photofermentation of tequila vinasses (VT) was studied using Rhodopseudomonas pseudopalustris DSM 123. To the best of our knowledge this is the first report on hydrogen production by photofermentation on VT. Hydrogen production was doubled on VT (260 m$props_value{literPattern}/L) as compared to a synthetic medium. Storing VT changed its chemical composition; however, in photofermentations of a sixty-day old stock, growth and hydrogen production were similar to fresh VT. The periodic displacement of hydrogen with nitrogen resulted in a three-fold increase in both hydrogen and growth of R. pseudopalustris (860 mL H₂/L and 4.5 g/L respectively) as compared to non-displaced headspace. Hydrogen almost doubled at a light intensity of 270 W/m² (2249 mL H₂) as compared to 68 W/m². In dark-light cycles, biomass and hydrogen production were highest with continuous illumination. The potential of VT to produce hydrogen in high amounts using R. pseudopalustris has been demonstrated.     

Effect of bioaugmentation on hydrogen production from organic fraction of municipal solid waste

In the present study, the effect of bioaugmentation with three bacterial species (i.e. E. coli, Bacillus subtilis and Enterobacter aerogenes) on the hydrogen production from organic fraction of municipal solid waste was evaluated at different bacteria/sludge ratios (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40). Cumulative hydrogen production, lag phases, and maximum hydrogen production rates were analyzed using modified Gompertz model. The highest cumulative and volumetric hydrogen production of 564.4 ± 10.9 mL and 1.61L_H2/L_substrate respectively was achieved for bioaugmentation with Bacillus subtilis at bacteria/sludge ratio of 0.25. The corresponding highest hydrogen yield was 43.68 mLH₂/gCarbo. For bioaugmentation with E. coli and Enterobacter aerogenes, the maximum cumulative hydrogen production of 423.4 ± 10.6 mL and 486.3 ± 10.6 mL respectively was obtained from bacteria/sludge ratio of 0.20. Corresponding highest hydrogen yields were 32.9 mLH₂/gCarbo and 37.1 mLH₂/gCarbo respectively. Bioaugmentation shortened the lag phases and improved COD removal. Volatile fatty acid generation was also improved with the bioaugmentation.     

Continuous biogenic hydrogen production from dilute acid pretreated algal hydrolysate using hybrid immobilized mixed consortia

This study investigated the bioconversion of dilute acid (2% H₂SO₄) pretreated red algae (Gelidium amansii) hydrolysate into H₂ by anaerobic fermentation in a continuous stirred tank reactor under mesophilic conditions using hybrid immobilized cells as microbial catalyst. Two different hydraulic retention times (HRT) of 24 h and 16 h with a feed concentration of 15 g/L hexose equivalent have been investigated over 85 days of operation to evaluate H₂ production performance and stability of the continuous system. The highest hydrogen production rate (HPR) and hydrogen yield (HY) of 2.7 L/L/d and 1.3 mol/mol substrate hexose_added was achieved at 24 h HRT, while further operation at 16 h HRT led to a significant drop in the hydrogen production with a HPR and HY values of 1.8 L/L/d and 0.7 mol/mol substrate hexose_added, respectively. The bacterial community analysis characterized by 454 pyrosequencing revealed that the changes in HRT significantly influence the composition of the dominant microflora. At longer HRT (24 h), the phyla Firmicutes was abundant over 98%, whereas at shorter HRT (16 h), Proteobacteria being the dominant populations with 84%. These outcomes suggested that controlling appropriate HRT is prerequisite for efficient hydrogen production.     

Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor

The production of hydrogen from glucose by using Enterobacter aerogenes ATCC 13048 (E. aerogenes) in an immobilized cell reactor (ICR) was investigated. The effect of several factors, such as the glucose concentration, feed flow rate, and fermentation time were examined. The highest amount of hydrogen (9.44 mmol H₂/g glucose) was obtained at a glucose concentration of 8 g/L, flow rate of 0.5 mL/min, retention time of 24 h and at a temperature of 30 °C. Meanwhile, the highest amount of carbon dioxide (1.68 mmol CO₂/g glucose) was obtained at a glucose concentration of 10 g/L, flow rate of 0.7 mL/min, hydraulic retention time of 24 h and at a temperature of 30 °C. The hydrogen and carbon dioxide production were affected by glucose concentration, hydraulic retention time (HRT) and fermentation time. This study showed that the ICR was a very efficient method for the production of hydrogen and carbon dioxide gases.     

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.     

Enhancement on hydrogen production performance of Rhodobacter sphaeroides HY01 by overexpressing fdxN

Ferredoxin I (FdI), encoded by fdxN gene, is proved to be the main electron donor of nitrogenase for hydrogen production. In this work, fdxN gene overexpression was implemented in a mutant MHY01, which was constructed by inserting fdxN gene into the hupSL region in Rhodobacter sphaeroides HY01 genome. Its photo-fermentative H₂ production performance was studied. The results showed that the expression level of fdxN and nitrogenase activity in MHY01 (hupSL::fdxN) were enhanced by 177% and 61.7% respectively compared with that of wild type HY01. Using 25 mM acetate and 34 mM butyrate as carbon source and 6 mM l-glutamate as nitrogen source, the maximum H₂ production rate was 156.1 mL/(L·h), which was increased by 50.7% compared with that of HY01. The maximum H₂ production rates of MHY01 were enhanced by 30.0%, 52.5% and 50.7% compared with those obtained from HY01 at the inoculation size of 5%, 10% and 15% respectively. The results suggested that overexpressing fdxN could enhance the nitrogenase activity and H₂ production performance of purple non-sulfur bacteria. The abundancy of ferredoxin I might limit the efficiency of electron transfer flux associated with the biohydrogen production process.     

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h^?1 and the average pseudo-steady state hydrogen production rate was 36 mL H₂ L^?1 medium h^?1. The cumulative hydrogen production was 430 mL H₂ L^?1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H₂ L^?1 h^?1 and the cumulative hydrogen production was 517 mL H₂ L^?1 medium. Reducing the feed rate to less than 48 mL h^?1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.     

Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H₂/mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations.     

Effect of substrate concentration on the competition between Clostridium and Lactobacillus during biohydrogen production

To evaluate the effect of substrate concentration on competition between hydrogen producing bacteria and lactic acid bacteria during hydrogen fermentation, a series of batch tests was conducted using galactose or glucose as a carbon source. Clostridium butyricum, Lactobacillus casei, and mixture of the two species were used for inoculum, respectively. The batch tests based on single species revealed that C. butyricum utilized galactose faster than L. casei, regardless of substrate concentration (0.2–5.7 g/L), while L. casei utilized glucose faster than C. butyricum at low levels of glucose (<1 g/L). These trends were also observed in the batch test based on a mixture of the two species. L. casei outcompeted C. butyricum only when glucose was provided at low concentration. Taken together, the results suggest that failure of biohydrogen production can occur at low glucose concentration via outcompeting of lactic acid bacteria over hydrogen producing bacteria.     

The photosynthetic hydrogen production performance of a newly isolated Rhodobacter capsulatus JL1 with various carbon sources

Rhodobacter capsulatus is a photosynthetic bacterium with the ability to produce H₂ under photosynthetic condition. In this study, a new strain JL1 isolated from lake water was identified as Rhodobacter capsulatus by phylogenetic analysis of 16S ribosomal DNA (rDNA) sequence. Initial medium pH and l-glutamate (nitrogen source) concentration were optimized. At optimum pH 7.0 and 7 mmol/L l-glutamate, R. capsulatus JL1 could grow and produce hydrogen on the carbon sources of acetate, butyrate, glucose, xylose and fructose with the maximum substrate to H₂ conversion efficiencies of 67.5%, 26.6%, 46.1%, 46.2% and 46.6%, respectively. The maximum H₂ production rate, 124 ± 0.6 mL/(L·h), was obtained using 20 mmol-glucose/L as the carbon source. The addition of appropriate acetic acid to the tests with low concentration of glucose was able to improve the H₂ yield. Under the optimum operation parameters, the maximum H₂ yield and H₂ production rate of R. capsulatus JL1 from 16.4 g-corn straw/L-culture were 2966.5 ± 43.2 mL/L and 71.1 ± 4.5 mL/(L·h), while the chemical oxygen demand (COD) removal rate was up to 49.6%. This study indicates that R. capsulatus JL1 can serve as good candidate strain for H₂ production with organic waste water as well as effluent of dark-fermentation.     

Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization

This work evaluated the effects of individual alkaline, sodium carbonate (Na₂CO₃ denoted as; NaC), sodium sulfide (Na₂SO₃ denoted as; NaS) and combination of NaC + NaS pretreatment for the saccharification of sugarcane bagasse (SCB). The effects of different pretreatments on chemical composition and structural complexity of SCB in relation with its saccharification were investigated. For enzymatic hydrolysis of pretreated SCB we have utilized the produced crude enzymes by Streptomyces sp. MDS to make the process more cost effective. A enzyme dose of 30 filter paperase (FPU) produced a maximum reducing sugar (RS) 592 mg/g with 80.2% hydrolysis yield from NaC + NaS pretreated SCB under optimized conditions. The resulted enzymatic hydrolysates of each pretreated SCB were applied for hydrogen production using Clostridium beijerinckii KCTC1785. NaC + NaS pretreated SCB hydrolysates exhibited maximum H₂ production relative to other pretreatment methods. Effects of temperature, initial pH of culture media and increasing NaC + NaS pretreated SCB enzymatic hydrolysates concentration (2.5–15 g/L) on bioH₂ production were investigated. Under the optimized conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1485 mL/L, 61.87 mL/L/h and 1.24 mmol H₂/mol of RS (0.733 mmol H₂/g of SCB), respectively. The efficient conversion of the SCB hydrolysate to H₂ without detoxification proves the viability of process for cost-effective hydrogen production.     

Optimizing hydrogen production from organic wastewater treatment in batch reactors through experimental and kinetic analysis

Anaerobic hydrogen production from organic wastewater, an emerging biotechnology to generate clean energy resources from wastewater treatment, is critical for environmental and energy sustainability. In this study, hydrogen production, biomass growth and organic substrate degradation were comprehensively examined at different levels of two critical parameters (chemical oxygen demand (COD) and pH). Hydrogen yields had a reverse correlation with COD concentrations. The highest specific hydrogen yield (SHY) of 2.1 mole H₂/mole glucose was achieved at the lowest COD of 1 g/L and decreased to 0.7 mole H₂/mole glucose at the highest COD of 20 g/L. The pH of 5.5–6.0 was optimal for hydrogen production with the SHY of 1.6 mole H₂/mole glucose, whereas the acidic pH (4.5) and neutral pH (6.0–7.0) lowered the hydrogen yields. Under all operational conditions, acetate and butyrate were the main components in the liquid fermentation products. Additionally, a comprehensive kinetic analysis of biomass growth, substrate degradation and hydrogen production was performed. The maximum rates of microbial growth (μ_m) and substrate utilization (R_su) were 0.03 g biomass/g biomass/day and 0.25 g glucose/g biomass/day, respectively. The optimum pH for the rate of hydrogen production (RH₂$R_{{\text{H}}_2}$) and SHY were 5.89 and 5.74 respectively. Based on the kinetic analysis, the highest RH₂$R_{{\text{H}}_2}$ and SHY for batch-mode anaerobic hydrogen production systems were projected to be 13.7 mL/h and 2.32 mole H₂/mole glucose.     

Repeated batch fermentation for photo-hydrogen and lipid production from wastewater of a sugar manufacturing plant

Hydrogen and lipid production from sugar manufacturing plant wastewater (SMW) by Rhodobacter sp. KKU-PS1 were investigated. Aji-L (i.e., a waste from the process of crystallizing monosodium glutamate) was used as nitrogen source. Batch fermentation was conducted in 300 mL serum bottles with the working volume of 180 mL to investigate the optimal inoculum size by varying the initial inoculum concentration from 0.23 to 0.92 g_CDW/L. The photo-fermentation was conducted at an initial pH 7.0 and 25.6 °C with continuously light illumination at 7500 lux. The optimal inoculum size of 0.77 g_CDW/L gave the hydrogen production rate (R_m) and lipid production of 5.24 mL H₂/L.h and 407 mg lipid/L, respectively. The hydrogen production from SMW was further examined in 1.7-L photo-bioreactor with the working volume of 1.2-L using the optimal condition from batch experiment. A photo-bioreactor yielded 1.73 times higher R_m than that obtained from the fermentation in serum bottles with a greater lipid production of 424 mg lipid/L. Hydrogen production from SMW in the repeated-batch fermentation was further studied by varying the medium replacement ratios of 25, 50–75%. A maximum biomass and lipid concentration of 2.83 g_CDW/L and 685 mg lipid/L, respectively were achieved at a medium replacement ratio of 75%. C18:1 (51.2%), C18:0 (24.9%) and C16:0 (9.1%) were found as the major free fatty acid. Lactic acid followed by propionic, acetic and butyric acids containing in SMW were the suitable carbon source for biomass production of KKU-PS1.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.