Fermentative production of hydrogen and soluble metabolites from crude glycerol of biodiesel plant by the newly isolated thermotolerant Klebsiella pneumoniae TR17

A thermotolerant fermentative hydrogen-producing strain was isolated from crude glycerol contaminated soil and identified as Klebsiella pneumoniae on the basis of the 16S rRNA gene analysis as well as physiological and biochemical characteristics. The selected strain, designated as K. pneumoniae TR17, gave good hydrogen production from crude glycerol. Culture conditions influencing the hydrogen production were investigated. The strain produced hydrogen within a wide range of temperature (30–50 °C), initial pH (4.0–9.0) and crude glycerol concentration (20–100 g/L) with yeast extract as a favorable nitrogen source. In batch cultivation, the optimal conditions for hydrogen production were: cultivation temperature at 40 °C, initial pH at 8.0, 20 g/L crude glycerol and 2 g/L yeast extract. This resulted in the maximum cumulative hydrogen production of 27.7 mmol H₂/L and hydrogen yield of 0.25 mol H₂/mol glycerol. In addition, the main soluble metabolites were 1,3-propanediol, 2,3-butanediol and ethanol corresponding to the production of 3.52, 2.06 and 3.95 g/L, respectively.     

Simultaneous production of hydrogen and ethanol from waste glycerol by Enterobacter aerogenes KKU-S1

Factors affecting simultaneous hydrogen and ethanol production from waste glycerol by a newly isolated bacterium Enterobacter aerogenes KKU-S1 were investigated employing response surface methodology (RSM) with central composite design (CCD). The Plackett-Burman design was first used to screen the factors influencing simultaneous hydrogen and ethanol production, i.e., initial pH, temperature, amount of vitamin solution, yeast extract (YE) concentration and glycerol concentration. Results indicated that initial pH, temperature, YE concentration, and glycerol concentration had a statistically significant effect (p ≤ 0.05) on hydrogen production rate (HPR) and ethanol production. The significant factors were further optimized using CCD. Optimum conditions for simultaneously maximizing HPR and ethanol production were YE concentration of 1.00 g/L, glycerol concentration of 37 g/L, initial pH of 8.14, and temperature of 37 °C in which a maximum HPR and ethanol production of 0.24 mmol H₂/L h and 120 mmol/L were achieved.     

Statistical optimization of medium components affecting simultaneous fermentative hydrogen and ethanol production from crude glycerol by thermotolerant Klebsiella sp. TR17

A thermotolerant Klebsiella sp. TR17 for production of hydrogen from crude glycerol was investigated. Results from Plackett–Burman design indicated that the significant variables, which influenced hydrogen production, were KH₂PO₄ and NH₄Cl (for buffer capacity and nitrogen source). Subsequently, the two selected variables and crude glycerol were optimized by the Central Composite design for achieving maximum hydrogen and ethanol yield. The concentration of crude glycerol, KH₂PO₄, and NH₄Cl had an individual effect on both hydrogen and ethanol yield (p < 0.05), while KH₂PO₄ and NH₄Cl had an interactive effect on ethanol yield (p < 0.05). The optimum medium components were 11.14 g/L crude glycerol, 2.47 g/L KH₂PO₄, and 6.03 g/L NH₄Cl. The predicted maximum simultaneous hydrogen and ethanol yield were 0.27 mol H₂/mol glycerol and 0.63 mol EtOH/mol glycerol, respectively. Validation of the predicted optimal conditions exhibited similar hydrogen and ethanol yield of 0.26 mol H₂/mol glycerol and 0.58 mol EtOH/mol glycerol, respectively.     

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was operated under non-sterile conditions at 40^○C and initial pH 8.0 at different hydraulic retention times (HRTs) (2–12 h) and glycerol concentrations (10–30 g/L). Decreasing the HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The highest HPR of 242.15 mmol H₂/L/d and HY of 44.27 mmol H₂/g glycerol consumed were achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively.     

Surfactant mediated enhanced glycerol uptake and hydrogen production from biodiesel waste using co-culture of Enterobacter aerogenes and Clostridium butyricum

In the present study, Tween 80, a non-ionic surfactant, has been used for enhanced hydrogen production by crude glycerol bioconversion using co-culture of Enterobacter aerogenes and Clostridium butyricum. The purpose of introducing the surfactant was to decrease the crude glycerol viscosity, so that apparent solubility and bioavailability of glycerol could be improved at the expenses of pretreatment steps. Experiments were planned using central composite design (CCD); crude glycerol and Tween 80 concentrations were optimized whereas, hydrogen production, glycerol utilization and viscosity of the media were considered as responses. The response surface for quadratic model showed, Tween 80 concentration had significant effect (p < 0.05) on all the three responses. Using the optimized conditions at 17.5 g/L crude glycerol and 15 mg/L Tween 80, hydrogen production reached a maximum of 32.1 ± 0.03 mmol/L of medium. The increase in hydrogen production was around 1.25-fold in presence of Tween 80 in comparison to its absence with 25.56 ± 0.91 mmol/L production. Selected optimum conditions were also validated against absence of crude glycerol (4.69 ± 0.76), with pretreated crude glycerol (20.06 ± 0.51) and across mono-culture system (15.43 ± 0.79 to 22.14 ± 0.94). Introduction of Tween 80 to the fermentation medium improved the glycerol utilization rate, resulting in increased hydrogen production and eliminated pretreatment steps.     

Media optimization for biohydrogen production from waste glycerol by anaerobic thermophilic mixed cultures

Media compositions affecting thermophilic biohydrogen production from waste glycerol were optimized using response surface methodology (RSM) with central composite design (CCD). Investigated parameters used were waste glycerol concentration, urea concentration, the amount of Endo-nutrient addition and disodium hydrogen phosphate (Na₂HPO₄) concentration. Waste glycerol concentration and the amount of Endo-nutrient addition had a significant individual effect on the cumulative hydrogen production (HP) (p ≤ 0.05). The interactive effect on HP was found between waste glycerol and urea concentration as well as waste glycerol concentration and the amount of Endo-nutrient addition (p ≤ 0.05). The optimal media compositions were 20.33 g/L of waste glycerol, 0.16 g/L of urea, 3.97 g/L of Na₂HPO₄ and 0.20 mL/L of the amount of Endo-nutrient addition which gave the maximum HP of 1470.19 mL H₂/L. The difference between observed HP (1502.84 mL H₂/L) and predicted HP was 2.22%. The metabolic products from the fermentation process were 1,3-propanediol (1,3-PD), ethanol, acetic, formic, lactic, butyric, and propionic acids. Results from polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis indicated that the hydrogen producers present in the fermentation broth was Thermoanaerobacterium sp.     

Biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures

Key factors affecting biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures were optimized using response surface methodology (RSM) with central composite design (CCD). Investigated parameters were waste glycerol concentration, sludge concentration, and the amount of Endo–nutrient addition. Concentrations of waste glycerol and sludge had a significant individual effect on hydrogen production rate (HPR) (p ≤ 0.05). The interactive effect on HPR (p ≤ 0.05) was found between waste glycerol concentration and sludge concentration. The optimal conditions for the maximum HPR were: waste glycerol concentration 22.19 g/L, sludge concentration 7.16 g-total solid (TS/L), and the amount of Endo–nutrient addition 2.89 mL/L in which the maximum HPR of 1.37 mmol H₂/L h was achieved. Using the optimal conditions, HPR from a co-digestion of waste glycerol and sludge (1.37 mmol H₂/L h) was two times greater than the control (waste glycerol without addition of sludge) (0.76 mmol H₂/L h), indicating a significant enhancement of HPR by sludge. Major metabolites of the fermentation process were ethanol, 1,3-propanediol (1,3-PD), lactate, and formate.     

Enrichment of activated sludge for enhanced hydrogen production from crude glycerol

Enriched activated sludge that can effectively convert crude glycerol into bio-hydrogen was selected by an eco-biotechnological approach, in very strict conditions, using biodiesel-derived glycerol as the only carbon source. The thus obtained functional consortium was characterized by the genera Klebsiella, Escherichia/Shigella and Cupriavidus. During enrichment, the dominant metabolic end-product shifted from a 1,3 propanediol to ethanol, with a concomitant increase of the hydrogen yield from 0.18 ± 0.003 to 0.66 ± 0.06 mol/mol and an almost five-fold increase of the hydrogen production. Glycerol degradation efficiency showed an increase of around 50%. In optimized and upscaled conditions it was possible to obtain a hydrogen production rate of 2960 mL H₂/L/day ± 185 at a near stoichiometric yield (of 0.90 mol/mol ± 0.01), with a carbon recovery of almost 90%, both in sterile and non-sterile conditions. Glycerol was almost totally degraded (degradation efficiency of 97.42% ± 0.98), independently of the glycerol type used.     

Bioconversion of crude glycerol from biodiesel production to hydrogen

This study evaluates the potential of bioconversion of crude glycerol, discharged from biodiesel production plant, to hydrogen (H₂) by an enriched microbial community. Microbial community was enriched from activated sludge in a medium amended with 2.5 g/L of crude glycerol. Optimal cultivation parameters for H₂ production such as initial pH, cultivation temperature and substrate concentration were investigated. H₂ yields from raw glycerol at optimal conditions (pH 6.5; 40 °C and 1 g/L raw glycerol) were 1.1 ± 0.1 mol-H₂/mol-glycerol_consumed. H₂ production was associated with acetate-butyrate type fermentation, along with ethanol as one of the end products. Kinetic experiments on H₂ production from pure and crude glycerol indicated the absence of any inhibitory effects from the impurities present in crude glycerol. The community analysis revealed that the enriched microbial consortium was dominated mainly by Clostridium species.     

Statistical optimization of biohydrogen and ethanol production from crude glycerol by microbial mixed culture

Design of Experiments (DoE) was applied to improve the ability of enriched activity sludge to efficiently convert crude glycerol from biodiesel industry into hydrogen and ethanol, using a very simple synthetic medium. Based on Plackett–Burman screening design, glycerol concentration, temperature and initial pH were identified as significant variables. Box–Behnken design and Response Surface Method (RSM) were then used for optimization. The maximum hydrogen yield of 0.96 mol H₂/mol glycerol was estimated at the temperature of 37.0 °C, initial pH of 7.9 and glycerol concentration of 15.0 g/L. Maximum hydrogen production rate of 2191 mL/L/d was estimated at the temperature of 37.3 °C, initial pH of 8.0 and glycerol concentration of 15.2 g/L. Finally maximum ethanol production of 7.92 g/L was estimated at an initial pH of 8.0 and glycerol concentration of 15.0 g/L (temperature had no significant effect). These results show that it is possible to obtain both, high yield and production of hydrogen and ethanol together, using a very simple synthetic medium, without trace element- and vitamin solution, tryptone or yeast extract.     

Effects of increasing the NAD(H) pool on hydrogen production and metabolic flux distribution in Enterobacter aerogenes mutants

The effects of combining two strategies, recycling NAD and improving the availability of NADH, on hydrogen production in Enterobacter aerogenes were investigated. The NAD synthetase encoded by nadE gene was homologously overexpressed in AB91002-O, which had been obtained previously, to increase the intracellular concentration of the NAD(H/⁺) pool. This overexpression was duplicated in mutant strains in which the phosphoenolpyruvate carboxylase (PEPC) gene (ppc) and hybO gene were knocked out, yielding AB91102-OP (ΔhybO/Δppc), AB91102-ON (ΔhybO/nadE), and AB91102-OP/N (ΔhybO/Δppc/nadE). Chemostat experiments showed that the total NAD(H) pool size in AB91102-ON increased 2-fold compared with the control strain AB91102-OC, but the NADH/NAD⁺ ratio decreased by 24%. Metabolic analysis of batch experiments indicated that a larger NAD(H/⁺) pool and inactivation of PEPC led to a significant shift in metabolic patterns, whereas a smaller NADH/NAD⁺ ratio improved glucose uptake. Thus, compared with the control strain, the hydrogen yields per glucose of the mutant strains AB91102-OP, AB91102-ON, and AB91102-OP/N were enhanced by 36.2%, 66.0%, and 149%, respectively, and the total volumes of hydrogen production increased by 27%, 165%, and 301%, respectively. The maximum hydrogen production of 5.1 L/L was achieved by AB91102-OP/N, suggesting that the double modification strategy exhibits markedly positive synergistic effects on hydrogen production.     

Optimization of selected salts concentration for improved biohydrogen production from biodiesel-based glycerol using Enterobacter aerogenes

Enterobacter aerogenes have a known ability to convert glycerol (GL) in a fermentative process to yield hydrogen and ethanol as the main by-products. The concentration of some media constituents was optimized to maximize biohydrogen yield and rate of production. E. aerogenes were cultured in aerobic conditions, and then transferred into anaerobic conditions before being cultured in a minimum mineral synthetic media (MMSM) containing 15 g/L GL. The concentration of selected salts were optimized in the following ranges: 0–300 mg/L MgSO₄, 0–14 g/L Na₂EDTA, 0–10 mg/L CaCL₂, 0–10 g/L Na₂HPO₄, and 0–9.7 g/L KH₂PO₄. The results of the full factorial design indicated that the production of biohydrogen required a minimal concentration of 3.5 mg/L EDTA, 200 mg/L MgSO₄.7H₂O and no CaCl₂.2H₂O. A significant interaction between EDTA and MgSO₄ was also observed. Results from the phosphate salts optimization showed that Na₂HPO₄ gave better results than KH₂PO₄. The optimal conditions determined using pure glycerol (commercial grade glycerol), were successfully applied to the fermentation of crude glycerol from biodiesel production. The results indicated promising yields of 0.79 and 0.84 mol/mol of glycerol for bioethanol and biohydrogen, respectively, and this at a faster rate than reported previously for E. aerogenes.     

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.     

Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria

Glycerol is an inevitable by-product from biodiesel synthesis process and could be a promising feedstock for fermentative hydrogen production. In this study, the feasibility of using crude glycerol from biodiesel industry for biohydrogen production was evaluated using seven isolated hydrogen-producing bacterial strains (Clostridium butyricum, Clostridium pasteurianum, and Klebsiella sp.). Among the strains examined, C. pasteurianum CH4 exhibited the best biohydrogen-producing performance under the optimal conditions of: temperature, 35 °C; initial pH, 7.0; agitation rate, 200 rpm; glycerol concentration, 10 g/l. When using pure glycerol as carbon source for continuous hydrogen fermentation, the average H₂ production rate and H₂ yield were 103.1 ± 8.1 ml/h/l and 0.50 ± 0.02 mol H₂/mol glycerol, respectively. In contrast, when using crude glycerol as the carbon source, the H₂ production rate and H₂ yield was improved to 166.0 ± 8.7 ml/h/l and 0.77 ± 0.05 mol H₂/mol glycerol, respectively. This work demonstrated the high potential of using biodiesel by-product, glycerol, for cost-effective biohydrogen production.     

A sequential process of anaerobic solid-state fermentation followed by dark fermentation for bio-hydrogen production from Chlorella sp.

Microalgal biomass has recently been one of the most widely studied feedstocks for bio-hydrogen production, owing to its richness in fermentable components, e.g. polysaccharides and proteins, and high biomass productivity. In this study, biomass of microalga Chlorella sp. TISTR 8411 was converted to hydrogen through a sequential process consisting of an anaerobic solid-state fermentation (ASSF) followed by a dark fermentation. The microalga was grown photoautothrophically in 80-L rectangular glass tanks and then scaled-up to a 240-L open pond for the production of biomass. The highest biomass concentration attained was 4.45 g L⁻¹. The biomass was harvested with over 90% flocculation efficiency at pH 11.5 and a biomass concentration of 2.6 g/L. The sequential process gave a total hydrogen yield (HY) of 16.2 mL/g-volatile-solid (VS), of which 11.6 mL/g-VS was from ASSF. The high HY obtained from the ASSF indicated that it was effective and could be integrated with a conventional hydrogen production process to improve energy recovery from biomass.     

Bioenergy production from glycerol in hydrogen producing bioreactors (HPBs) and microbial fuel cells (MFCs)

The supply of glycerol has increased substantially in recent years as a by-product of biodiesel production. To explore the value of glycerol for further application, the conversion of glycerol to bioenergy (hydrogen and electricity) was investigated using Hydrogen Producing Bioreactors (HPBs) and Microbial Fuel Cells (MFCs). Pure-glycerol and the glycerol from biodiesel waste stream were compared as the substrates for bioenergy production. In terms of hydrogen production, the yields of hydrogen and 1,3-propanediol at a pure-glycerol concentration of 3 g/L were 0.20 mol/mol glycerol and 0.46 mol/glycerol, respectively. With glucose as the co-metabolism substrate at the ratio of 3:1 (glycerol:glucose), the yields of hydrogen and 1,3-propanediol from glycerol significantly increased to 0.37 mol/mol glycerol and 0.65 mol/glycerol, respectively. The glycerol from biodiesel waste stream had good hydrogen yields (0.17-0.18 mol H₂/mole glycerol), which was comparable with the pure-glycerol. In terms of power generation in MFCs, pure-glycerol was examined at concentrations of 0.5-5 g/L with the highest power density of 4579 mW/m³ obtained at a concentration of 2 g/L. The power densities from the biodiesel waste glycerol were 1614-2324 mW/m³, which were likely caused by the adverse effects of impurities on electrode materials. An economic analysis indicates that with the annual waste stream of 70 million gallons of glycerol, the expected values generated from HPBs and MFCs were $311 and $98 million, respectively.     

Glycerol fermentation to hydrogen by Thermotoga maritima: Proposed pathway and bioenergetic considerations

The production of biohydrogen from glycerol, by the hyperthermophilic bacterium Thermotoga maritima DSM 3109, was investigated in batch and chemostat systems. T. maritima converted glycerol to mainly acetate, CO₂ and H₂. Maximal hydrogen yields of 2.84 and 2.41 hydrogen per glycerol were observed for batch and chemostat cultivations, respectively. For batch cultivations: i) hydrogen production rates decreased with increasing initial glycerol concentration, ii) growth and hydrogen production was optimal in the pH range of 7–7.5, and iii) a yeast extract concentration of 2 g/l led to optimal hydrogen production. Stable growth could be maintained in a chemostat, however, when dilution rates exceeded 0.025 h⁻¹ glycerol conversion was incomplete. A detailed overview of the catabolic pathway involved in glycerol fermentation to hydrogen by T. maritima is given. Based on comparative genomics the ability to grow on glycerol can be considered as a general trait of Thermotoga species. The exceptional bioenergetics of hydrogen formation from glycerol is discussed.     

Hydrogen production by glycerol steam reforming with in situ hydrogen separation: A thermodynamic investigation

Thermodynamic features of hydrogen production by glycerol steam reforming with in situ hydrogen extraction have been studied with the method of Gibbs free energy minimization. The effects of pressure (1–5 atm), temperature (600–1000 K), water to glycerol ratio (WGR, 3–12) and fraction of H₂ removal (f, 0–1) on the reforming reactions and carbon formation were investigated. The results suggest separation of hydrogen in situ can substantially enhance hydrogen production from glycerol steam reforming, as 7 mol (stoichiometric value) of hydrogen can be obtained even at 600 K due to the hydrogen extraction. It is demonstrated that atmospheric pressure and a WGR of 9 are suitable for hydrogen production and the optimum temperature for glycerol steam reforming with in situ hydrogen removal is between 825 and 875 K, 100 K lower than that achieved typically without hydrogen separation. Furthermore, the detrimental influence of increasing pressure in terms of hydrogen production becomes marginal above 800 K with a high fraction of H₂ removal (i.e., f = 0.99). High temperature and WGR are favorable to inhibit carbon production.     

Photocatalytic production of hydrogen and methane from glycerol reforming over Pt/TiO2–Nb2O5

In this study, platinized mixed oxides (TiO₂–Nb₂O₅) were tested on photocatalytic hydrogen production from a glycerol solution under UV light. Different samples with different Ti:Nb ratios were prepared by using a simple method that simultaneously combined a physical mixture and a platinum photochemical reduction. This method led to improved physicochemical properties such as low band gap, better Pt nanoparticle distribution on the surface, and the formation of different Pt species. Niobia content was also found to be an important factor in determining the overall efficiency of the Pt–TiO₂–Nb₂O₅ photocatalyst in the glycerol reforming reaction. The photocatalytic results showed that Pt on TiO₂–Nb₂O₅ enhanced hydrogen production from the aqueous glycerol solution at a 5 wt% initial glycerol concentration. The influence of different operating conditions such as the catalyst dosage and initial glycerol concentration was also evaluated. The results indicated that the best hydrogen and methane production was equal to 6657 μmol/L and 194 μmol/L, respectively after 4 h of UV radiation using Pt/Ti:Nb (1:2) sample and with 3 g/L of catalyst dosage. Moreover, the role of water in photocatalytic hydrogen production was studied through photocatalytic activity tests in the presence of D₂O. The obtained results confirmed the role of water molecules on the photocatalytic production of hydrogen in an aqueous glycerol solution.     

A robust and efficient bioprocess of hydrogen production from crude glycerol by Clostridium beijerinckii G117

Clostridia can produce hydrogen (a renewable-biofuel) from crude glycerol (CG). Reportedly, the indigenous CG-impurities (eg.methanol, soap and salt) interfere with clostridial-process, and reduces hydrogen production. Thus, it is important to develop a robust bioprocess to directly utilize native-CG for hydrogen production. In this study, among four Clostridia tested, Clostridium beijerinckii G117 is selected based on its high hydrogen (>290 mL/g) production from CG. Furthermore, optimization of process-parameters enhances hydrogen production from strain G117 by ～5 fold from 1195 ± 45 to 5893 ± 25 mL/L with 2.16 mol/mol yield. The order of impact of process-parameters (optimum value in parenthesis) on hydrogen production is: CG concentration (12.5 g/L)>inoculum size (10%v/v)>inoculum age (12 h)>temperature (39 °C)>initial medium-pH (6.4)>L-cysteine (1 g/L)>operational volume (30 mL)>agitation rate (150 rpm). Notably, this optimized bioprocess records >90% efficiency by determining total mass and electron balances. Interestingly, this optimized bioprocess retains adequate robustness to yield 1.94–2.02 mol/mol hydrogen with elevated CG-impurities including alcohols/salts/surfactants.