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.     

Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients. After being used for growing mushroom for one to two weeks this bag becomes mushroom cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced annually in Taiwan and are usually burned or discarded. The cellulosic materials and nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen fermentation. This study aims to select the inoculum from various waste sludges (sewage sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature 55 °C and an initial cultivation pH of 8. The results show that extra nutrient addition inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in case of using sewage sludge inoculum and without inoculum. This related to the inhibition caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak HY of 0.73 mmol H₂/g TVS was obtained with no inoculum and nutrient addition. However, peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H₂/L/d and 2.02 mmol H₂/g VSS/d, respectively, were obtained by using cow dung inoculum without any extra nutrient addition.     

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.     

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.     

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H₂/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.     

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.     

Effect of acid-pretreatment on hydrogen fermentation of food waste: Microbial community analysis by next generation sequencing

This work presents the effect of acid-pretreatment on H₂ fermentation of food waste with detailed microbial information by next generation sequencing. The pretreated food waste at pH 1.0–4.0 was cultivated under mesophilic conditions without external inoculum addition. From the food waste acid-pretreated at pH 1–3, H₂ yields in the range of 1.37–1.74 mol H₂/mol hexose_added were achieved, attaining the highest value at pH 2. Clostridium sp. such as Clostridium acetobutylicum ATCC 824 and Clostridium perfringens occupied more than 70% of total number of sequences at pH 1–3. On the other hand, in the control (no pretreatment) and at pH 4, lactic acid bacteria such as Lactobacillus and Streptococcus were found to be the dominant genus (>90% of total number of sequences), resulting in a low H₂ yield. In addition, the effect of substrate concentration on H₂ fermentation was investigated, and the maximum H₂ productivity was estimated to be 27.2 L H₂/L/d by Andrew's model.     

Aspect ratio effect of bioreactor on fermentative hydrogen production with immobilized sludge

The phenomenon of bacterial wash-out frequently occurs in the traditional continuous stirred tank reactor (CSTR) systems at low hydraulic retention time (HRT). In this study, the effect of different aspect ratios, height (H) to diameter (D) of 1:1, 3:1 and 5:1, of a CSTR with immobilized anaerobic sludge on hydrogen (H₂) production were investigated. The pH, volatile suspended solids (VSS) and total solids (TS) concentrations of the seed sludge were 6.8, 33.3 and 65.1 g/L, respectively. Thermally treated sludge was immobilized by silicone gel entrapment approach. The entrapped-sludge system operated stably at a low HRT without suffering from cell wash-out. Hence, the hydrogen production rate (HPR) was enhanced by increasing organic loading rates. The immobilized sludge CSTRs were operated at 40 °C with sucrose (10, 20, 30 and 40 g COD/L) and Endo nutrient medium at different HRTs (4, 2, 1 and 0.5 h). It was found that the granule formation enhanced HPR. The maximum HPR and the H₂ yield were found to be 15.36H₂ L/h/L and 3.16 mol H₂/mol sucrose, respectively, with the H₂ content in the biogas above 44% for all tests runs.     

Bioconversion of de-oiled Jatropha Waste (DJW) to hydrogen and methane gas by anaerobic fermentation: Influence of substrate concentration,temperature and pH

Cellulosic materials-based de-oiled Jatropha Waste (DJW) was fermented to H₂ and CH₄ using sewage sludge inoculum. Batch assays were performed at various substrate concentrations (40–240 g/L), temperatures (25–65 °C) and pHs (5.5–7.5). The peak hydrogen production rate (HPR) and hydrogen yield (HY) of 744.0 ± 11.3 mL H₂/L-d and 10.6 ± 0.2 mL H₂/g VS obtained when the optimal substrate concentration, pH, temperature were 200 g/L, 6.5, 55 °C, respectively. The peak methane production rate (MPR) of 178.4 ± 5.6 mL CH₄/L-d obtained while DJW concentration, pH, temperature were 200 g/L, 7.0, 45 °C, however, peak methane yield (MY) of 23.3 ± 0.1 mL CH₄/g VS obtained at 40 g/L, 7.0 and 55 °C, respectively. Effect of substrate concentration on HPR and MPR was elucidated using Monod model. Butyrate and acetate were the main soluble metabolic products. Maximal carbohydrate removal and COD reduction were achieved as 51.7 ± 0.7% and 68.3 ± 1.6%, respectively.     

Non-sterile process for biohydrogen and 1,3-propanediol production from raw glycerol

Raw glycerol is a tempting substrate for fermentations, but contains impurities that can be inhibitory for organisms. In this study, raw glycerol tolerance and contamination risk of pure bacterial culture at hypersaline process conditions were evaluated. The inhibitory effect of raw glycerol was similar on a halophilic (Halanaerobium saccharolyticum) and a non-halophilic (Clostridium butyricum) bacterium implying the inhibition originating from methanol or other impurities rather than salt. The hypersaline process conditions decreased efficiently contaminations and no growth of contaminants was observed at and above 125 g/l NaCl. Halophilic H₂ and 1,3-PD production from raw glycerol were studied separately as 1-stage processes and jointly as 2-stage process in non-sterile conditions. Non-sterile conditions were successfully applied and the highest production yields obtained were 3.0 mol H₂/mol glycerol and 0.66 mol 1,3-PD/mol glycerol (1-stage processes), whereas the highest cumulative production was 74 mmol H₂/l culture and 31 mmol 1,3-PD/l culture (2-stage process).     

Producing hydrogen from the fermentation of cheese whey and glycerol as cosubstrates in an anaerobic fluidized bed reactor

This study aimed to evaluate the effect of the organic loading rate (OLR) (60, 90, and 120 g Chemical Oxygen Demand (COD). L^?1. d^?1) on hydrogen production from cheese whey and glycerol fermentation as cosubstrates (50% cheese whey and 50% glycerol on a COD basis) in a thermophilic fluidized bed reactor (55 °C). The increase in the OLR to 90 gCOD.L^?1. d^?1 favored the hydrogen production rate (HPR) (3.9 L H₂. L^?1. d^?1) and hydrogen yield (HY) (1.7 mmol H₂. gCOD^?1_app) concomitant with the production of butyric and acetic acids. Employing 16S rRNA gene sequencing, the highest hydrogen production was related to the detection of Thermoanaerobacterium (34.9%), Pseudomonas (14.5%), and Clostridium (4.7%). Conversely, at 120 gCOD.L^?1. d^?1, HPR and HY decreased to 2.5 L H₂. L^?1. d^?1 and 0.8 mmol H₂. gCOD^?1_app, respectively, due to lactic acid production that was related to the genera Thermoanaerobacterium (50.91%) and Tumebacillus (23.56%). Cofermentation favored hydrogen production at higher OLRs than cheese whey single fermentation.     

Influence of alkaline peroxide assisted and hydrothermal pretreatment on biodegradability and bio-hydrogen formation from citrus peel waste

The effect of pretreatments by hydrothermolysis (180 °C; 15 min) and alkaline delignification (NaOH 5M; H₂O₂ 1%; 24 h) in citrus peel waste (CPW) was evaluated, as well as the effect on H₂, organic acids and alcohols production, in addition to characterization of the microbial community involved in fermentation. Batch reactors at 37 °C were operated with 3 gTVS/L of CPW with allochthonous consortium (UASB reactor sludge; 2 gTVS/L) and autochthonous of CPW (1.5 gTVS/L) as inocula. H₂ production was higher in reactors with in natura CPW (13.31 mmol/L) compared to hydrothermolysis (8.19 mmol/L) and alkaline delignification (7.27 mmol/L). The acetogenic pathway was predominant in the in natura CPW (4,355 mg/L acetic acid). The most abundant genera in the in natura CPW and after hydrothermolysis were Clostridium (18.97 and 12.90%, respectively) and Ruminiclostridium (16.65 and 1.04%, respectively) commonly related to cellulolytic bacteria and/or H₂ production.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

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.     

Isolation,characterization and optimization of photo-hydrogen production conditions by newly isolated Rhodobacter sphaeroides KKU-PS5

A purple non-sulfur (PNS) photosynthetic bacterium was isolated from an upflow anaerobic sludge blanket (UASB) bioreactor for methane production and was identified as Rhodobacter sphaeroides KKU-PS5 (GenBank Accession no. KC481702) by 16s rRNA gene sequence analysis. Strain KKU-PS5 could utilize glucose, xylose, fructose, arabinose, malate, succinate, acetate, butyrate, lactate and D-mannitol for growth and hydrogen production. Malate was a preferred carbon source while glutamate and Aji-L (waste from the process of crystallizing monosodium glutamate) were the preferred nitrogen sources. The ability to utilize Aji-L as a low-cost nitrogen supplement for photo-biohydrogen production by the strain KKU-PS5 is considered as its desirable characteristic. The threshold substrate concentration of malate was 30 mmol/L. The optimum conditions for hydrogen production from malate were an initial pH of 7.0, FeSO₄ concentration of 4 mg/L, temperature of 30 °C and light intensity of 6 klux. Under the optimum conditions, the maximum hydrogen production, the hydrogen yield (HY) and the hydrogen production rate (HPR) of 1330 mL-H₂/L, 3.80 mol-H₂/mol-malate, and 11.08 mL-H₂/L h, respectively, were achieved. Hydrogen production under a dark/light cycle led to a decreased HY and HPR in comparison to continuous illumination.     

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.     

Evaluation of different supplementary nutrients for enhanced biohydrogen production by Enterobacter aerogenes NRRL B 407 using waste derived crude glycerol

Crude glycerol (CG) has several advantages over a range of conventional substrates used for biohydrogen production by Enterobacter aerogenes NRRL B 407. Meanwhile, high process cost due to requirement of expensive supplementary media component is a concern. Therefore, different less expensive (or wastes) materials have been evaluated as supplementary nutrient for H₂ production by CG (meat processing and restaurant waste based biodiesel derived) bioconversion. Among the materials selected, slaughterhouse liquid waste (SL), brewery waste biomass (BWB) and urea was found to improve the production by 18.81 ± 3.56, 27.30 ± 3.54 and 38.57 ± 3.66%, respectively. Further, in the case of urea (10 mg/L), cumulative production as high as 116.41 ± 3.72 mmol H₂/L media has been achieved; which is comparable to other reports available on CG bioconversion. Thus, present study demonstrates successful replacement of large amount (∼5–6 g/L) of expensive nutrients/buffering agents by negligible amount (∼10 mg/L) of different waste materials, without compromising the cumulative H₂ yield. Further, the strain used in the present study was found to grow at an acidic pH as low as 3.3, indicating its prospective application for dark fermentative H₂ production.     

Biohydrogen production from palm oil mill effluent (POME) by two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of carbon sources in POME

The production of biohydrogen through dark fermentation of palm oil mill effluent (POME) was evaluated in two-stages of biohydrogen in an anaerobic sequencing batch reactor (ASBR) system using enriched mixed culture for the first time. This study attempts to examine the effect of HRT and its interaction behavior with the solid retention time (SRT), and the sugar consumption. The effluent after discharged from the thermophilic reactor contained 7.61 g/L TC and 22.87 g/L TSS was fed to the secondary mesophilic reactor system. Results indicated that the overall sugar consumption reached 88.62% at the optimum HRT of 12 h with the SRT set to 20 h. The optimum hydrogen yield and HPR in the thermophilic stage were 2.99 mol H₂/mol-sugar and 8.54 mmol H₂/L·h respectively, while for the mesophilic stage were 1.19 mol H₂/mol-sugar and 1.47 mmolH₂/L·h respectively. The overall HPR showed an improvement and increase from 8.54 mmol H₂/L·h to 10.34 mmol H₂/L.h. Microbial community analysis of mixed culture in the two-stage thermophilic (55.0 °C) and mesophilic (37.0 °C) ASBR reactor was dominated by Thermoanaerobacterium sp. based on the PCR-DGGE technique.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

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.