Biohydrogen production in anaerobic fluidized bed reactors: Effect of support material and hydraulic retention time

This study evaluated two different support materials (polystyrene and expanded clay) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBRs contained either polystyrene (R1) or expanded clay (R2) as support materials were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C and a pH of approximately 5.5. The AFBRs were operated with a range of hydraulic retention times (HRTs) between 1 and 8 h. For R1 with an HRT of 2 h, the maximum hydrogen yield (HY) was 1.90 mol H₂ mol⁻¹ glucose, with 0.805 mg of biomass (as total volatile solids, or TVS) attached to each g of polystyrene. For R2 operated at an HRT of 2 h, the maximum HY was 2.59 mol H₂ mol⁻¹ glucose, with 1.100 mg of attached biomass (as TVS) g⁻¹ expanded clay. The highest hydrogen production rates (HPR) were 0.95 and 1.21 L h⁻¹ L⁻¹ for R1 and R2, respectively, using an HRT of 1 h. The H₂ content increased from 16–47% for R1 and from 22–51% for R2. No methane was detected in the biogas produced throughout the period of AFBR operation. These results show that the values of HY, HPR, H₂ content, and g of attached biomass g⁻¹ support material were all higher for AFBRs containing expanded clay than for reactors containing polystyrene.     

Increased performance of continuous stirred tank reactor with calcium supplementation

Continuous biohydrogen production with calcium supplementation at low hydraulic retention time (HRT) in a continuous stirred tank reactor (CSTR) was studied to maximize the hydrogen productivity of anaerobic mixed cultures. After stable operations at HRT of 8–4 h, the bioreactor became unstable when the HRT was lowered to 2 h. Supplementation of 100 mg/L calcium at HRT 2 h improved the operation stability through enhancement of cell retention with almost two-fold increase in cell density than that without calcium addition. Hydrogen production rate and hydrogen yield reached 24.5 L/d/L and 3.74 mol H₂/mol sucrose, respectively, both of which were the highest values our group have ever achieved. The results showed that calcium supplementation can be an effective way to improve the performance of CSTR at low HRT.     

Effects of pH value and substrate concentration on hydrogen production from the anaerobic fermentation of glucose

A series of batch experiments were conducted to investigate the effects of pH and glucose concentrations on biological hydrogen production by using the natural sludge obtained from the bed of a local river as inoculant. Batch experiments numbered series I and II were designed at an initial and constant pH of 5.0–7.0 with 1.0 increment and four different glucose concentrations (5.0, 7.5, 10 and 20 g glucose/L). The results showed that the optimal condition for anaerobic fermentative hydrogen production is 7.5 g glucose/L and constant pH 6.0 with a maximum H₂ production rate of 0.22 mol H₂ mol⁻¹ glucose h⁻¹, a cumulative H₂ yield of 1.83 mol H₂ mol⁻¹ glucose and a H₂ percentage of 63 in biogas.     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor

The objective of this study was to evaluate the production of hydrogen in a two-stage CSTR system – both reactors having the same volume – and compare its performance with a conventional one-stage process. The lab-scale two-stage and one-stage systems were operated at five pHs and five hydraulic retention time (HRTs). The maximum volumetric hydrogen productivity and yield obtained with the two-stage system were 5.8 mmol L⁻¹ h⁻¹ and 2.7 mol H₂ mol glucose⁻¹, respectively, at an HRT of 12 h and pH 5.5. Overall, the two-stage system showed, at steady state, a better performance that the one-stage system for all the evaluated pHs. However, a comparison between the one-stage system, operating at 6 h of HRT, and the first reactor of the two-stage system at the same HRT did not show any significant difference, highlighting the positive impact of having a two-stage process. The determination of the ratio between the experimental measured H₂ in the gas phase and the theoretical H₂ generated in the liquid phase (discrepancy factor) indicated that an important part of the hydrogen produced in the first reactor was transferred into the second reactor instead of being desorbed in the headspace. Therefore, the improving of hydrogen production in the two-stage system is rather attributed to the increased transfer of hydrogen from liquid to gas than an actual total hydrogen production increase.     

Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone

Ceramic ring and pumice stone were used as a support matrix for the enhancement of biohydrogen production in immobilized cell culture systems. The reactors were continuously operated for the hydrogen fermentation using sucrose as the major carbon source at varying hydraulic retention times (HRT) as an important operational factor. In terms of volumetric hydrogen production, the best value was obtained with ceramic ring at 1.5 h HRT (2.98 l H₂/l/d), on the other hand, the pumice stone packed reactor resulted in 30% less volumetric hydrogen production (2.28 l H₂/l/d) at two fold longer retention time (HRT 3 h). It was demonstrated that volumetric hydrogen production with the immobilized bioreactor configurations was 6 fold better than the suspended culture bioreactor configuration (CSTR). Furthermore, up to 4 mol and 5 mol hydrogen yields per mole of sucrose used (which are 62.5% and 50% of the theoretical values) were achieved by pumice stone and ceramic ring packed reactors, respectively, whereas suspended culture system yielded only 0.5 mol H₂/mol sucrose.     

An online-monitored thermophilic hydrogen production UASB reactor for long-term stable operation

In this study, the H₂ production and chemical oxygen demand (COD) removal performances of a thermophilic upflow anaerobic sludge blanket (UASB) reactor with online monitoring system were investigated. The online monitoring system enabled a rapid monitoring and timely control of the process. As a consequence, high operating stability was achieved despite of the varied hydraulic retention time (HRT) during 310-day operation. The COD efficiency remained at above 98%, and the hydrogen yield fluctuated slightly within the range of 2.42–3.06 mol H₂ mol⁻¹ sucrose. Thermophilic H₂-producing granules were successfully cultivated in this reactor, which showed better physical and microbial properties than floc sludge and higher H₂ production rate than mesophilic granules. An analysis of the microbial growth kinetics further demonstrated a possibly higher synthesis and metabolism activity of microbes in the thermophilic granule state.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

Biohydrogen production from purified terephthalic acid (PTA) processing wastewater by anaerobic fermentation using mixed microbial communities

Purified terephthalic acid (PTA) processing wastewater was evaluated as a fermentable substrate for hydrogen (H₂) production with simultaneous wastewater treatment by dark-fermentation process in a continuous stirred-tank reactor (CSTR) with selectively enriched acidogenic mixed consortia under continuous flow condition in this paper. The inoculated sludge used in the reactor was excess sludge taken from a second settling tank in a local wastewater treatment plant. Under the conditions of the inoculants not less than 6.3 gVSS/L, the organic loading rate (OLR) of 16 kgCOD/m³ d, hydraulic retention time (HRT) of 6 h and temperature of (35 ± 1) °C, when the pH value, alkalinity and oxidation–reduction potential (ORP) of the effluent ranged from 4.2 to 4.4, 280 to 350 mg CaCO₃/L, and −220 to −250 mV respectively, soluble metabolites were predominated by acetate and ethanol, with smaller quantities of propionate, butyrate and valerate. Stable ethanol-type fermentation was formed with the sum of ethanol and acetate concentration ratio of 70.31% to the total liquid products after 25 days operation. The H₂ volume content was estimated to be 48–53% of the total biogas and the biogas was free of methane throughout the study. The average biomass concentration was estimated to be 10.82 gVSS/L, which favored H₂ production efficiently. The rate of chemical oxygen demand (COD) removal reached at about 45% and a specific H₂ production rate achieved 0.073 L/gMLVSS d in the study. This CSTR system showed a promising high-efficient bioprocess for H₂ production from high-strength chemical wastewater.     

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 biohydrogen production using cheese whey: Improving the hydrogen production rate

Due to the renewed interest in finding sustainable fuels or energy carriers, biohydrogen (Bio-H₂) from biomass is a promising alternative. Fermentative Bio-H₂ production was studied in a continuous stirred tank reactor (CSTR) operated during 65.6 d with cheese whey (CW) as substrate. Three hydraulic retention times (HRTs) were tested (10, 6 and 4 h) and the highest volumetric hydrogen production rate (VHPR) was attained with HRT of 6 h. Therefore, four organic loading rates (OLRs) at a fixed HRT of 6 h were tested thereafter, being: 92.4, 115.5, 138.6 and 184.4 g lactose/L/d. The highest VHPR (46.61 mmol H₂/L/h) and hydrogen molar yield (HMY) of 2.8 mol H₂/mol lactose were found at an OLR of 138.6 g lactose/L/d; a sharp fall in VHPR occurred at an OLR of 184.4 g lactose/L/d. Butyric, propionic and acetic acids were the main soluble metabolites found, with butyric-to-acetic ratios ranging from 1.0 to 2.4. Bacterial community was identified by partial sequence analysis of the 16S rRNA and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). The results showed that at HRT of 10 h and 6 h were dominated by the Clostridium genus. The VHPR attained in this study is the highest reported value for a CSTR system using CW as substrate with anaerobic sludge as inoculum and represents a 33-fold increase compared to a previous study. Thus, it was demonstrated that continuous fermentative Bio-H₂ production from CW can be significantly enhanced by an appropriate selection of parameters such as HRT and OLR. Enhancements in VHPR are significant because it is a critical parameter to determine the full-scale practical application of fermentation technologies that will be used for sustainable and clean energy generation.     

Comparison of the use of sucrose and glucose as a substrate for hydrogen production in an upflow anaerobic fixed-bed reactor

This study evaluated the use of two types of substrates, glucose and sucrose, feeding an anaerobic fixed-bed bioreactor. The biogas produced was composed of H₂ and CO₂, without methane. Maximum hydrogen yields were 3.22 mol H₂ mol⁻¹ sucrose_converted and 1.51 mol H₂ mol⁻¹ glucose_converted. The main intermediates were acetic acid, butyric acid, and ethanol. The greatest difference, however, was in the stability of the process. The operation of the reactor with sucrose exhibited a drop in biogas production, whereas operation with glucose was stable after a slight decrease in biogas production. This decrease may have been caused by the differential growth of microbial populations in each reactor, namely, the growth of organisms that use the Wood–Ljungdahl metabolic pathway.     

Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp

Hydrogen gas production from sugar solution derived from acid hydrolysis of ground wheat starch by photo-fermentation was investigated. Three different pure strains of Rhodobacter sphaeroides (RV, NRLL and DSZM) were used in batch experiments to select the most suitable strain. The ground wheat was hydrolyzed in acid solution at pH = 3 and 90 °C in an autoclave for 15 min. The resulting sugar solution was used for hydrogen production by photo-fermentation after neutralization and nutrient addition. R. sphaeroides RV resulted in the highest cumulative hydrogen gas formation (178 ml), hydrogen yield (1.23 mol H₂ mol⁻¹ glucose) and specific hydrogen production rate (46 ml H₂ g⁻¹ biomass h⁻¹) at 5 g l⁻¹ initial total sugar concentration among the other pure cultures. Effects of initial sugar concentration on photo-fermentation performance were investigated by varying sugar concentration between 2.2 and 13 g l⁻¹ using the pure culture of R. sphaeroides RV. Cumulative hydrogen volume increased from 30 to 232 ml when total sugar concentration was increased from 2.2 to 8.5 g l⁻¹. Further increases in initial sugar concentration resulted in decreases in cumulative hydrogen formation. The highest hydrogen formation rate (3.69 ml h⁻¹) and yield (1.23 mol H₂ mol⁻¹ glucose) were obtained at a sugar concentration of 5 g l⁻¹.     

Developing a statistical model to predict hydrogen production by a mixed anaerobic mesophilic culture

Optimizing the hydrogen (H₂) yield at several initial pH conditions in a mixed batch anaerobic mesophilic culture fed with glucose and linoleic acid (LA) was performed using a three factor three level Box–Benkhen design (BBD). Based on the BBD approach, a statistical model was developed to predict the H₂ yield. The variables considered for the experimental design were the LA concentration, the initial pH and the number of times glucose was added to the culture. The D-optimality method predicted a maximum H₂ yield of 3.49 mol H₂ mol glucose⁻¹ for cultures fed 1.9 g l⁻¹ LA, maintained at an initial pH of 5.15 and received 1.79 glucose additions. The response outcome (H₂ yield of 3.38 ± 0.22 mol mol glucose⁻¹) at the nearest setting of the experimental factors (2.0 g l⁻¹ LA, an initial pH of 5.0 and two glucose additions) was 3.3% less than the predicted maximum value. The model provides a useful approach for predicting H₂ production when H₂ consumers are inhibited in mixed batch anaerobic cultures.     

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.     

Biological hydrogen production in continuous stirred tank reactor systems with suspended and attached microbial growth

Fermentative H₂ production in continuous stirred tank reactor (CSTR) system with bacteria attached onto granular activated carbon (GAC) was designed to produce H₂ continuously. The H₂ production performances of CSTR with suspended and attached-sludge from molasses were examined and compared at various organic loading rates (8–40 g COD/L/d) at hydraulic retention time of 6 h under mesophilic conditions (35 °C). Both reactor systems achieved ethanol-type fermentation in the pH ranges 4.5–4.8 and 3.8–4.4, respectively, while ORP ranges from −450 to −470 mV and from −330 to −350 mV, respectively. The hydrogen production rate in the attached system was higher compared to that of the suspended system (9.72 and 6.65 L/d/L, respectively) while specific hydrogen production rate of 5.13 L/g VSS/d was higher in the suspended system. The attached-sludge CSTR is more stable than the suspended-sludge CSTR with regard to hydrogen production, pH, substrate utilization efficiency and metabolic products (e.g., volatile fatty acids and ethanol) during the whole test.     

Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures

Dark fermentation using mixed cultures is an attractive biological process for producing hydrogen (H₂) from lignocellulosic biomass at a low cost. Physicochemical pretreatment is generally used to convert lignocellulosic materials into monosaccharides. However, the processes also involved release degradation byproducts which can, in turn, inhibit microbial growth and metabolism and, hence, impact substrate conversion. In this study, the impact on H₂ production of lignocellulose-derived compounds (i.e. furan derivatives, phenolic compounds and lignins) was assessed along with their effect on bacterial communities and metabolisms. Batch tests were carried out using xylose as model substrate (1.67 mol_H2 mol_xylose⁻¹ in the control test). All the putative inhibitory compounds showed a significant negative impact on H₂ production performance (ranging from 0.34 to 1.39 mol_H2 mol_xylose⁻¹). The H₂ yields were impacted more strongly by furan derivatives (0.40–0.51 mol_H2 mol_xylose⁻¹) than by phenolic compounds (1.28–1.39 mol_H2 mol_xylose⁻¹). Except for the batch tests supplemented with lignins, the lag phase was shorter for inhibitors having the highest molecular weight (8 days versus 22 days for the lowest MW). Variability of the lag phase was clearly related to a shift in bacterial community structure, as shown by multivariate ordination statistics. The decrease in H₂ yield was associated with a decrease in the relative abundance of several H₂-producing clostridial species. Interestingly, Clostridium beijerinkii was found to be more resistant to the inhibitors, making this bacterium an ideal candidate for H₂ production from hydrolyzates of lignocellulosic biomass.     

Kinetic and thermodynamic studies of the Bunsen reaction in the sulfur–iodine thermochemical process

This work presents the kinetic and thermodynamic studies of the Bunsen reaction in the sulfur–iodine thermochemical cycle for hydrogen production by water splitting. A series of experimental runs have been carried out by feeding the gas mixture SO₂/N₂ in an I₂/H₂O medium in the temperature range of 336–358 K. The effects of the various operating parameters on the SO₂ conversion ratio have been evaluated. The results showed that the efficiency of SO₂ conversion into H₂SO₄ increased with the amount of I₂ or H₂O increase. The increasing reaction temperature impeded SO₂ conversion into H₂SO₄. A kinetic model has been developed to fit to the experimental data obtained in a semi-batch reactor. A good fitting can be observed for each experiment, which discloses the overall kinetic mechanism of the complex Bunsen reaction. The apparent activation energies were found to be 23.513 kJ mol⁻¹ and 9.212 kJ mol⁻¹ for the sequential reactions  and , respectively.     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.