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.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate

Anaerobic sequencing batch reactor (ASBR) process offers great potential for H₂ production from wastewaters. In this study, an ASBR was used at first time for enhanced continuous H₂ production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times (HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at 65 mmol sugars L⁻¹. Results showed that increasing the HRT from 6 to 12 h led to the H₂ production rate increased from 6.7 to the maximum of 9.6 mmol H₂ L⁻¹ h⁻¹ and the substrate conversion reached 90.3%, although the H₂ yield remained at the same level of 1.7 mol H₂ mol⁻¹ substrate. Taking into account both H₂ production and substrate utilization efficiencies, the optimum HRT for continuous H₂ production via an ASBR was determined at 12 h. Compared with other continuous H₂ production processes, ASBR yield higher H₂ production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H₂ production from lignocellulosic biomass.     

Continuous thermophilic hydrogen production and microbial community analysis from anaerobic digestion of diluted sugar cane stillage

The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 °C using a mixture of sugar cane stillage and glucose at approximately 5000–5300 mg COD L⁻¹. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H₂ yields of 5.73 mmol g COD_added⁻¹ were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m⁻³ d⁻¹). The maximum volumetric H₂ production of 0.78 L H₂ h⁻¹ L⁻¹ was achieved using stillage as carbon source. In all operational phases, the H₂ average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacterium sp. and Clostridium sp. were identified.     

Sequencing batch reactor enhances bacterial hydrolysis of starch promoting continuous bio-hydrogen production from starch feedstock

Bio-hydrogen production from starch was carried out using a two-stage process combining thermophillic starch hydrolysis and dark H₂ fermentation. In the first stage, starch was hydrolyzed by Caldimonas taiwanensis On1 using sequencing batch reactor (SBR). In the second stage, Clostridium butyricum CGS2 was used to produce H₂ from hydrolyzed starch via continuous dark hydrogen fermentation. Starch hydrolysis with C. taiwanensis On1 was operated in SBR under pH 7.0 and 55 °C. With a 90% discharge volume, the reducing sugar (RS) production from SBR reactor reached 13.94 g RS/L, while the reducing sugar production rate and starch hydrolysis rate was 0.92 g RS/h/L and 1.86 g starch/h/L, respectively, which are higher than using other discharge volumes. For continuous H₂ production with the starch hydrolysate, the highest H₂ production rate and yield was 0.52 L/h/L and 13.2 mmol H₂/g total sugar, respectively, under a hydraulic retention time (HRT) of 12 h. The best feeding nitrogen source (NH₄HCO₃) concentration was 2.62 g/L, attaining a good H₂ production efficiency along with a low residual ammonia concentration (0.14 g/L), which would be favorable to follow-up photo H₂ fermentation while using dark fermentation effluents as the substrate.     

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).     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Overcoming propionic acid inhibition of hydrogen fermentation by temperature shift strategy

A novel temperature shift strategy has been proposed to overcome an inhibition on hydrogen fermentation of beverage industry wastewater (BW) due to the accumulation of propionic acid (HPr) during continuous reactor operation. The continuous performance at constant pH 5.5, temperature 37 °C and hydraulic retention time (HRT) 8 h with BW concentration of 20 g/L_{hexose-equivalent} in a stirred tank reactor (2 L) showed an accumulation of HPr to 2.36 g/L leading to a drop in hydrogen production rate (HPR) from 10 to 8.5 L L⁻¹ d⁻¹. To overcome the HPr inhibition, a temperature shift (from 37 °C) to 45 °C for 8 h was applied. This significantly improved the inhibited HPR and HY to 13.6 L L⁻¹ d⁻¹ and 1.68 mol-H₂ mol⁻¹ hexose, respectively, with a simultaneous reduction in the HPr concentration to 0.7 g/L. Microbial community analysis based on PCR-DGGE after temperature shift revealed the non-dominance of Selenomonas lacticifex and Bifidobacterium catenulatum (involved in HPr formation), and dominance of hydrogen producing bacteria namely Clostridium butyricum, Clostridium perfringenes, Clostridium acetobutylicum, and Ethanoligenens harbinense. This study demonstrated that temperature shift strategy could overcome the HPr inhibition and significantly improve the hydrogen fermentation of an industrial wastewater.     

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.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

Continuous fermentative hydrogen and methane production from Laminaria japonica using a two-stage fermentation system with recycling of methane fermented effluent

In this study, a two-stage fermentation system to produce H₂ and CH₄ from Laminaria japonica was developed. In the first stage (dark fermentative H₂ production, DFHP), response surface methodology (RSM) with a Box-Behnken design (BBD) was applied for optimization of operational parameters, including cycle-frequency, HRT, and substrate concentration, using an intermittent-continuously stirred tank reactor (i-CSTR). Overall performance revealed that the degree of importance of the three variables in terms of H₂ yield is as follows: cycle-frequency > substrate concentration > HRT. In the confirmation test, H₂ yield of 113.1 mL H₂/g dry cell weight (dcw) was recorded, corresponding with 96.3% of the predicted response value under desirable operational conditions (cycle-frequency of 17 hr, HRT of 2.7 days, and substrate concentration of 31.1 g COD/L). In the second stage, an anaerobic sequencing batch reactor (ASBR) and an up-flow anaerobic sludge blanket reactor (UASBr) were employed for CH₄ production from H₂ fermented solid state (HFSS) and H₂ fermented liquid state (HFLS), respectively. The CH₄ producing ASBR and UASBr showed a stable CH₄ yield and COD removal until a HRT of 12 days and OLR of 3.5 g COD/L/d, respectively. Subsequently, for recycling of CH₄ fermented effluent from the UASBr (MFE_UASBr) as diluting water in DFHP, the tap water and MFE_UASBr mixing ratio (T/M ratio) was optimized (a T/M ratio of 5:5) in a batch test using heat pretreated MFE_UASBr at 90 °C for 20 min, resulting in the best performance. Although slight decreases of H₂ yield (7.6%) and H₂ production rate (3.5%) were recorded, 100% reduction of alkali addition was possible, indicating potential to maximize economic benefits. However, a drastic decrease of H₂ productivity and a change of liquid-state metabolites were observed with the use of non-heat pretreated MFE_UASBr. These results coincided with those of the microbial analysis, where non-H₂ producing bacteria, such as Selenomonas sp., were detected. The results indicate that pretreatment of MFE_UASBr may be required in order to recycle it in DFHP.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

Effects of alginate immobilization on dynamic membrane formation and H2 fermentation from galactose

A dynamic membrane (DM) is a biofilm that forms on a support material, acting as a filter to retain high-density biomass. This study aims to explain the effects of alginate immobilization on DM formation during dark-H₂ fermentation. Galactose is used as a model substrate. Heat-treated anaerobic sludge, with and without immobilization, is used for an inoculum for two identical lab-scale DM bioreactors (DMBR). The DMBRs are continuously operated for more than 40 days by changing the hydraulic retention time (HRT) from 12 to 3 h. Biomass retention and H₂ production performance are significantly improved at an HRT of 3 h with immobilization. The alginate-added bioreactor shows higher extracellular polymeric substance content both in the mixed liquor and the DM. At an HRT of 3 h with immobilization, the fraction of Sporolactobacillus spp. and Lactobacillus spp. increases, possibly contributing to DM formation. However, lactic-acid concentration does not increase, implying it can be further consumed by the dominant bacteria, Clostridium butyricum.     

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.     

Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter

Hydrogen was produced in a biotrickling filter (BF) packed with perlite and fed with oat straw acid hydrolysate at 30 °C. Inlet chemical oxygen demand (COD) from 1.2 to 35 g/L and hydraulic retention time (HRT) between 24 h and 6 h were assayed. With increasing inlet COD or decreasing HRT, H₂ production rate (HPR) increased but H₂ production yield (HY) decreased. Maximum HPR of 81.4 mL H₂/L_reactor h (3.3 mmol H₂/L_reactor h) and HY of 2.9 mol H₂/mol_{hexose consumed} were found at an inlet COD of 0.05 g_COD/L h (HRT 24 h) and 2.9 g_COD/L h (HRT 12 h), respectively. Maximum hydrogen composition in gas was 45 ± 4% (v/v) with CO₂ as balance. Methane was not detected. Maximum HPR and inlet COD used in this work were higher than others reported for reactors with suspended or fixed biomass. However, implementation of strategies for biomass control to avoid reactor clogging is needed.     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium 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.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.