Interrelationships between bioreactor volume,effluent recycle rate,temperature, pH, %H2, hydrogen productivity and hydrogen yield with undefined bacterial cultures

Simultaneous achievement of high volumetric productivities (HP = 231.3 mmol H₂/L/h) and high hydrogen yields (HY = 3.55 mol H₂/mol glucose) was obtained by increasing the temperature to 70 °C and by reducing the total bioreactor system volume (V) to 5.74 L and increasing the degassed effluent recycle rate (F_er) to 3.2 L/min, giving a V/F_er value of 1.8 min. The bioprocess involved the recycling of degassed effluent at a high flow rate through a quasi-stationary fluidized granular bed. In this process the rate of physical removal of H₂ trapped in the bulk liquid phase surrounding the fluidized granules reduced the thermodynamic constraints preventing the simultaneous achievement of high HPs and high HYs in the anaerobic fluidized granular bed bioreactor. Energy balance analysis showed that with heat recycling the bioreactor system could achieve a net positive volumetric energy output of 11.76 W/L at an energy efficiency of 49.3%.     

Production of biohydrogen from waste wheat in continuously operated UPBR: The effect of influent substrate concentration

Utilization of waste materials is one of the most economical approaches to biohydrogen production. Continuous generation of biohydrogen in a bioreactor makes the process more economical with respect to the conventional physical and chemical method. The two main parameters that affect the biohydrogen production in a continuously operated bioreactor are hydraulic retention time (HRT) and influent substrate concentrations. The effect of influent substrate concentration on biohydrogen generation in an up-flow packed bed reactor (UPBR) at HRT = 3 h was investigated in this study. The substrate was waste wheat which was acid hydrolyzed in H₂SO₄ by adjusting the pH value to pH = 2, under high temperature as T = 90 °C in an autoclave to obtain fermentable sugar solution. A natural and porous support particle namely, aquarium biological sponge (ABS) was the microbial immobilization surface in the reactor. Total and hydrogen gas volumes, hydrogen percentage, influent and effluent substrate concentrations, VFA concentrations were monitored. The influent substrate concentration (TS_o) was varied between TS_o = 10 g/L and TS_o = 35 g/L. The process performance was evaluated as biohydrogen volume, percentages, rate and yield under varying operating conditions. The production volume (4275 ml/day) and the rate (3.05 L H₂/L day) were maximum at influent sugar concentration of TS_o = 25 g/L, but the yield reached to its maximum value as Y = 1.22 mol H₂/mol glucose at TS_o = 19 g/L. Substrate limitation and inhibitions were observed at influent concentrations of TS_o = 10 g/L and TS_o = 35 g/L, respectively. The results indicated that ABS could be suggested as a microbial support particle for hydrogen generation in immobilized systems.     

Continuous biohydrogen production in immobilized biofilm system versus suspended cell culture

In this study, the performance of a new cell immobilization material, namely ceramic ball, was examined for continuous biohydrogen production in comparison to suspended cell culture system (CSTR). Production of biohydrogen in both systems was assessed under thermophilic conditions. Both systems were operated at varying hydraulic retention times (HRT) by shortening HRT values from 24 to 1.5 h at an influent sucrose concentration of 10 g/l. The immobilized bioreactor configuration outcompeted the CSTR bioreactor in terms of both volumetric hydrogen production (2.7 l H₂/l/day for immobilized system @ HRT = 3 h and 0.5 l H₂/l/day for CSTR @ HRT = 24 h) and resistance to cell-washout (CSTR reactor lost significant amount of biomass at short HRT values). It was concluded that immobilized bioreactor configuration is much more robust than CSTR against high organic loading rates and 5 fold more volumetric hydrogen production was achieved in 8 fold smaller immobilized bioreactor.     

Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions

Hydrogen (H₂) production from cheese processing wastewater via dark anaerobic fermentation was conducted using mixed microbial communities under thermophilic conditions. The effects of varying hydraulic retention time (HRT: 1, 2 and 3.5 days) and especially high organic load rates (OLR: 21, 35 and 47 g chemical oxygen demand (COD)/l/day) on biohydrogen production in a continuous stirred tank reactor were investigated. The biogas contained 5–82% (45% on average) hydrogen and the hydrogen production rate ranged from 0.3 to 7.9 l H₂/l/day (2.5 l/l/day on average). H₂ yields of 22, 15 and 5 mmol/g COD (at a constant influent COD of 40 g/l) were achieved at HRT values of 3.5, 2, and 1 days, respectively. On the other hand, H₂ yields were monitored to be 3, 9 and 6 mmol/g COD, for OLR values of 47, 35 and 21 g COD/l/day, when HRT was kept constant at 1 day. The total measurable volatile fatty acid concentration in the effluent (as a function of influent COD) ranged between 118 and 27,012 mg/l, which was mainly composed of acetic acid, iso-butyric acid, butyric acid, propionic acid, formate and lactate. Ethanol and acetone production was also monitored from time to time.To characterize the microbial community in the bioreactor at different HRTs, DNA in mixed liquor samples was extracted immediately for PCR amplification of 16S RNA gene using eubacterial primers corresponding to 8F and 518R. The PCR product was cloned and subjected to DNA sequencing. The sequencing results were analyzed by using MegaBlast available on NCBI website which showed 99% identity to uncultured Thermoanaerobacteriaceae bacterium.     

Design of a novel biohythane process with high H2 and CH4 production rates

A biohythane process based on wheat straw including: i) pretreatment, ii) H₂ production using Caldicellulosiruptor saccharolyticus, iii) CH₄ production using an undefined consortium, and iv) gas upgrading using an amine solution, was assessed through process modelling including cost and energy analysis. According to simulations, a biohythane gas with the composition 46–57% H₂, 43–54% CH₄ and 0.4% CO₂, could be produced at high production rates (2.8–6.1 L/L/d), with 93% chemical oxygen demand (COD) reduction, and a net energy yield of 7.4–7.7 kJ/g dry straw. The model was calibrated and verified using experimental data from dark fermentation (DF) of wheat straw hydrolysate, and anaerobic digestion of DF effluent. In addition, the effect of gas recirculation was investigated by both wet experiments and simulation. Sparging improved H₂ productivities and yields, but negatively affected the net energy gain and cost of the overall process.     

Evaluation of hidden H2-consuming pathways using metabolic flux-based analysis for a fermentative side-stream dynamic membrane bioreactor using untreated seed sludge

This study investigates the performance and hidden hydrogen consuming metabolic pathways of a fermentative side stream dynamic membrane (DM) bioreactor using flux balance analysis (FBA). The bioreactor was inoculated with untreated methanogenic seed sludge. It was found that fouling rate aggravated with increasing COD concentration (10–30 g/L) and was positively correlated to it rather than to the applied solid flux on the DM module. Due to increased fouling rate the hydraulic retention time (HRT) could not be reduced less than 0.82 ± 0.02 d. An increase in the organic loading rate (OLR) led to an increase in H₂ yield from 0.01 to 0.76 mol H₂/mol of sucrose. FBA revealed that homoacetogenesis was the main H₂-consuming pathway at lower OLRs (corresponding to 10 and 15 g COD/L), while for the OLR corresponding to 30 g COD/L, homoacetogens were suppressed. More importantly, caproic acid production pathway was identified for the first time as another H₂-consuming pathway at high OLR which was not significant at lower OLRs during fermentative dynamic membrane bioreactor operations.     

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H₂/g-substrate, hydrogen production rate of 39 mL/g h⁻¹ and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0-5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.     

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.     

Bioreactor for glycerol conversion into H2 by bacterium Enterobacter aerogenes

Glycerol was used as a substrate for H₂ production by bacterium Enterobacter aerogenes in the test tubes and bioreactor. A BioFlo/CelliGen 115 bioreactor (10 L working volume) was utilized to conduct the experiments for conversion of glycerol into H₂ by E. aerogenes cells. The highest H₂ production rate was observed under 2% glycerol in the culture medium. The glycerol uptake efficiency by bacteria in the bioreactor was found to be 65% during the 6 day period, matching glycerol uptake efficiency observed in the test tubes experiment (65%).Hydrogen production from glycerol (2% glycerol, v/v) by E. aerogenes in the bioreactor and test tubes was measured over the 6 days, showing the maximal H₂ rate at 650 mL g⁻¹ dry weight h⁻¹. The yield of H₂ production from glycerol at 0.89 mol/mol in the bioreactor was high, corresponding to the theoretical yield of 1 mol of H₂ per 1 mol of glycerol.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Biotechnological approach to generate green biohydrogen through the utilization of succinate-rich fermentation wastewater

Photofermentation seems to be an attractive mode of generating biohydrogen from fermentation effluent. Use of succinate fermentation effluent, however, has not been reported. Rhodobacter sphaeroides KKU-PS1 and Rhodopseudomonas palustris were acclimatised in succinate. It was determined that the KKU-PS1 was superior with respect to hydrogen productivity and was selected for further experiments. Photofermentation in succinate by the KKU-PS1 validated, generating 1217 mL H₂/L of cumulative hydrogen at a maximum rate of 6.7 mL H₂/L/h. Photofermentation from each single carbon sources that are components of effluent was performed and it was determined that acetate and succinate promoted the fastest growth of KKU-PS1 and hydrogen evolution, respectively. Photofermentation by the strain using mixed substrates mimicking diluted bio-succinate effluent produced yielded 1005 mL H₂/L cumulative hydrogen at a maximum rate of 4.1 mL H₂/L/h. The study highlighted potential of utilizing bio-succinate fermentation effluent for biohydrogen production, with further optimization required.     

Sequential dark and photo-fermentative hydrogen gas production from agar embedded molasses

In this study, molasses and dark fermentation effluent were solidified using agar and used for H₂ production by dark and photo-fermentation. During dark fermentation, the solid jelly form of molasses enabled a slow release of the substrate to the liquid broth hindering fast pH decreases. The initial total sugar concentration, H₂ yield, H₂ rate and lag phase in dark fermentation were 36.2 g/L, 226.24 mL H₂/g TS, 29.85 mL H₂/h and 4.37 h, respectively. Photo-fermentation of 5.77 g TVFA/L embedded dark fermentation effluent did not lead to efficient H₂ production. The best performance in photo-fermentation was obtained with 1.55 g TVFA/L containing diluted dark fermentation effluent. The H₂ yield, H₂ rate and lag phase in photo-fermentation were 870.26 mL H₂/g TVFA, 0.913 mL H₂/h and 54.07 h, respectively. Embedding concentrated substrate using agar can enhance H₂ production performance but only if the release of the substrate does not exceed inhibitory levels and if the rate of diffusion is tolerable for microbial activity.     

Improvement of fermentative biohydrogen production by Clostridium butyricum CWBI1009 in sequenced-batch,horizontal fixed bed and biodisc-like anaerobic reactors with biomass retention

A horizontal tubular fixed bed bioreactor (HFBR) and an anaerobic biodisc-like reactor (AnBDR) were designed to both fix Clostridium biomass and enable rapid transfer of the hydrogen produced to gas phase in order to decrease the strong effect of H₂ partial pressure and H₂ supersaturation on the performances of Clostridium strains. The highest H₂ production rate (703 mL H₂/L h) and yield (302 mL/g glucose consumed i.e. 2.4 mol/mol) with the pure culture were recorded in the AnBDR with 300 mL culture medium (total volume 2.3 L) at pH 5.2 and a glucose loading rate of 2.87 g/L h. These results are about 2.3 and 1.3-fold higher than those achieved in the same bioreactor with 500 mL liquid medium and with the same glucose consumption rate. Therefore, our experimentations and a short review of the literature reported in this paper emphasize the relevance of performing bioreactors with high L/G transfer.     

Two-stage UASB reactor converting coffee drink manufacturing wastewater to hydrogen and methane

In this study, the feasibility of a continuous two-stage up-flow anaerobic sludge blanket (UASB) reactor system, consisted of thermophilic (55 °C) dark fermentative H₂ production and mesophilic (35 °C) CH₄ production from coffee drink manufacturing wastewater (CDMW), was tested. A recently proposed operational strategy was used to overcome a major drawback of the long start-up period of the UASB reactor. Firstly, a completely stirred tank reactor (CSTR) was operated for 8 days to prepare seeding. The seed was then directly transferred to the UASB reactor. Microbial aggregation took place in the initial period, and the floc size was gradually increased over time. In UASB reactor, the maximum H₂ yield of 2.57 mol H₂/mol hexose_added and a stable H₂ production rate of 4.24 L H₂/L/h were observed at a hydraulic retention time (HRT) of 6 h and substrate concentration of 20 g Carbo. COD/L. In this novel method using CDMW, thermophilic H₂-producing granules with an average particle size of 1.3 mm was successfully developed after 100 days. The more bioenergy recovery was attempted in a post-treatment process using a mesophilic UASB reactor for CH₄ production from the H₂ fermented effluent. The maximum CH₄ yield of 325 mL of CH₄/g COD was achieved with removing 93% of the COD at an organic loading rate of 3.5 g COD/L/d. The developed two-stage UASB reactor system achieved biogas conversion by 88.2% (H₂ 15.2% and CH₄ 73%) and COD removal by 98%.     

Production of hydrogen from sewage biosolids in a continuously fed bioreactor: Effect of hydraulic retention time and sparging

Hydrogen was produced from primary sewage biosolids via mesophilic anaerobic fermentation in a continuously fed bioreactor. Prior to fermentation the sewage biosolids were heated to 70 °C for 1 h to inactivate methanogens and during fermentation a cellulose degrading enzyme was added to improve substrate availability. Hydraulic retention times (HRT) of 18, 24, 36 and 48 h were evaluated for the duration of hydrogen production. Without sparging a hydraulic retention time of 24 h resulted in the longest period of hydrogen production (3 days), during which a hydrogen yield of 21.9 L H₂ kg⁻¹ VS added to the bioreactor was achieved. Methods of preventing the decline of hydrogen production during continuous fermentation were evaluated. Of the techniques evaluated using nitrogen gas to sparge the bioreactor contents proved to be more effective than flushing just the headspace of the bioreactor. Sparging at 0.06 L L min⁻¹ successfully prevented a decline in hydrogen production and resulted in a yield of 27.0  L H₂ kg⁻¹ VS added, over a period of greater than 12 days or 12 HRT. The use of sparging also delayed the build up of acetic acid in the bioreactor, suggesting that it serves to inhibit homoacetogenesis and thus maintain hydrogen 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.     

Rapid formation of hydrogen-producing granules in an up-flow anaerobic sludge blanket reactor coupled with high-rate recirculation

Application of an up-flow anaerobic sludge blanket (UASB) reactor to dark fermentative H₂ production greatly improves H₂ productivity due to the maintenance of high biomass concentration. However, a long start-up HRT and start-up period are required to develop the H₂-producing granules (HPGs) and to avoid washing out the suspended sludge at the start of the process. In the present work, a novel strategy to rapidly form HPGs was developed in UASB reactor. To induce highly active mass transfer in the UASB reactor, a high recirculation rate (15 times the influent) was adopted over 10 days, then recirculation was stopped. As the operation progressed, self-flocculation took place and HPGs developed after 90 h of operation. A stable production of H₂ was observed after 20 days of operation. The thickness of the HPGs layer in the sole UASB reactor increased progressively, and consequently the average HPG diameter and concentration were 1.86 mm (0.1–3.9 mm) and 52 g/L, respectively, after 60 days of operation. These findings seem to suggest that high-rate recirculation plays a crucial role in accelerating the formation of HPGs in such UASB reactors through high up-flow velocity, providing active mass transfer.     

Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture

Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H₂ from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H₂ yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H₂ mol⁻¹ xylose_consumed and 3.2 ± 0.16 mol-H₂ mol⁻¹ glucose_consumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H₂ yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H₂ mol⁻¹ sucrose_consumed. The effects of stirring speed and different pH levels on growth and H₂ production were studied in the CSABR for highly efficient H₂ production. Growth and H₂ production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H₂ production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H₂ L⁻¹ h⁻¹ at 32, 52, 67, and 86 h, respectively.     

Biohydrogen production performance in a draft tube bioreactor with immobilized cell

The research was carried out using a draft tube fluidized bed bioreactor (DTFBR) system with immobilized cell. The total working volume of the DTFBR including the buffer tank and pipe was 2.4 L. The pH, volatile suspended solid (VSS) and total solid (TS) concentrations of the seed sludge were 6.8, 33.3 and 65.1 g/L, respectively. The thermally treated sludge was used for immobilization. Immobilization of cell was essentially achieved by silicone gel (SC) entrapment approaches. The entrapped-cell system operated stably at a low HRT without any cell washout. Hence, the H₂ production rate was enhanced via increasing organic loading rates. The system was fed with sucrose-based synthetic medium, and was examined for its H2 production performance under different influent sucrose concentrations, different solid volume volumetric content and hydraulic retention times.     

Hydrogen and methane production via a two-stage processes (H2-SBR + CH4-UASB) using tequila vinasses

The feasibility of producing hydrogen and methane via a two-stage fermentation of tequila vinasses was evaluated in sequencing batch (SBR) and up-flow anaerobic sludge blanket (UASB) reactors. Different vinasses concentrations ranging from 500 mg COD/L to 16 g COD/L were studied in SBR by using thermally pre-treated anaerobic sludge as inoculum for hydrogen production. Peak volumetric hydrogen production rate and specific hydrogen production were attained as 57.4 ± 4.0 mL H₂/L-h and 918 ± 63 mL H₂/gVSS-d, at the substrate concentration of 16 g COD/L and 6 h of hydraulic retention time (HRT). Increasing substrate concentration has no effect on the specific hydrogen production rate. The fermentation effluent was used for methane production in an UASB reactor. The higher methane composition in the biogas was achieved as 68% at an influent concentration of 1636 mg COD/L. Peak methane volumetric, specific production rates and yield were attained as 11.7 ± 0.7 mL CH₄/L-h, 7.2 ± 0.4 mL CH₄/g COD-h and 257.9 ± 13.8 mL CH₄/g COD at 24 h-HRT and a substrate concentration of 1636 mg COD/L. An overall organic matter removal (SBR + UASB) in this two-stage process of 73–75% was achieved.