Evaluation of process performance on biohydrogen production in continuous fixed bed reactor (C-FBR) using acid algae hydrolysate (AAH) as feedstock

In this study, a novel inoculation method to mitigate the inhibition of 5-hydroxymethylfurfural (5-HMF) is proposed. Acid algae hydrolysate containing 1.5 g 5-HMF/L and 15 g hexose/L hexose was fed to a continuous fixed bed reactor (C-FBR) partially packed with hybrid-immobilized beads. The inoculation method enabled a high rate of H₂ production, due to the reduction of 5-HMF inhibition and enhanced biofilm formation. Maximum hydrogen production was achieved at a hydraulic retention time of 6 h with a hydrogen production rate (HPR) of 20.0 ± 3.3 L H₂/L-d and a hydrogen yield (HY) of 2.3 ± 0.4 mol H₂/mol hexose _added. Butyrate and acetate were the major soluble metabolic products released during fermentation. Quantitative real-time polymerase chain reaction analysis revealed that Clostridium butyricum comprised 94.3% of the total bacteria, which was attributed to the high rate of biohydrogen production.     

Impact of furfural on biological hydrogen production kinetics from synthetic lignocellulosic hydrolysate using mesophilic and thermophilic mixed cultures

The impact of furfural on hydrogen production and microbial growth kinetics was assessed using mixed anaerobic cultures at mesophilic and thermophilic conditions. Mesophilic experiments showed a hydrogen yield of 1.6 mol H₂/mol initial sugars at 1 g/L furfural which is a 45% enhancement from the control (0 g/L furfural) at a substrate-to-biomass ratio (S°/X°) of 4 gCOD/gVSS. On the other hand, thermophilic experiments showed no enhancement at 1 g/L furfural but rather a 53% decrease in hydrogen yield from its control. Furfural inhibition threshold limit was observed to be greater than 1 g/L for mesophilic experiments and less than 1 g/L for thermophilic experiments. In both cases, 4 g/L was the most recalcitrant furfural concentration, with propionate and lactate the most predominant soluble metabolites in the mesophilic and thermophilic experiments respectively. It was also noted that in the presence of furfural, hydrogen-producers in both mesophilic and thermophilic mixed cultures were inactivated as no hydrogen was produced until furfural was completely degraded irrespective of sugars degradation. This study also presents the kinetics of microbial growth and substrate degradation obtained using the Monod model on MATLAB^®, ignoring an inhibition term. IC₅₀ of the mesophilic and thermophilic experiments were 1.03 g/L and 0.5 g/L respectively indicating that the thermophilic hydrogen producers were more strongly affected by furfural than the mesophilic cultures.     

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.     

Performance and population analysis of hydrogen production from sugarcane juice by non-sterile continuous stirred tank reactor augmented with Clostridium butyricum

Non-sterile operation of continuous stirred tank reactor (CSTR) augmented with Clostridium butyricum and fed with sugarcane juice was studied at various hydraulic retention time (HRT). The maximum hydrogen production rate and yield of 3.38 mmol H₂/L/h and 1.0 mol H₂/mol hexose consumed, respectively, were achieved at HRT 4 h. The relationship of the augmented microorganism and normal flora in the fermentation system under non-sterile condition were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Initially, at HRT 36 h, other species related to Lactobacillus harbinensis and Klebseilla pneumoniae were present as a major group in the reactor. When HRT was decreased to 12, 6 and 4 h, C. butyricum was present with a competition between L. harbinensis and K. pneumoniae. Results indicated that augmented C. butyricum could compete with contaminated microorganisms during non-sterile operation at low HRT (12-4 h) with the support of normal flora (K. pneumoniae).     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

Effect of temperature on continuous hydrogen production of cellulose

The effect of temperature on the hydrogen fermentation of cellulose was evaluated by a continuous experiment using a mixed culture without pretreatment. The experiments were conducted at three different temperatures, which were mesophilic [37 ± 2 °C], thermophilic [55 ± 2 °C] and hyper-thermophilic [80 ± 2 °C], with an influent concentration of cellulose of 5 g/l and a hydraulic retention time [HRT] of 10 days. A stable hydrogen production was observed at each condition. At 37 ± 2 °C, the maximum hydrogen yield was 0.6 mmol H₂/g cellulose. However, at 55 ± 2 °C and 80 ± 2 °C, the maximum hydrogen yields were 15.2 and 19.02 mmol H₂/g cellulose, respectively. While 26% of the biogas was methane under the mesophilic temperature, no methane gas was detected under both the thermophilic and hyper-thermophilic temperatures. The results show that operational temperature is a key to sustainable bio-hydrogen production and that the thermophilic and hyper-thermophilic conditions produced better results than mesophilic condition.     

Effects of 5-hydromethylfurfural,levulinic acid and formic acid,pretreatment byproducts of biomass,on fermentative H2 production from glucose and galactose

In this study, the effects of 5-hydromethylfurfural (5-HMF), levulinic acid, and formic acid on fermentative H₂ production have been investigated. The major byproducts of physicochemical pretreatment of lignocellulosic or algal biomass inhibited H₂ production from glucose and galactose. The half maximal inhibitory concentrations (IC₅₀) of 5-HMF on glucose, levulinic acid on glucose, formic acid on glucose, levulinic acid on galactose and formic acid on galactose were 0.59, 1.55, 12.50, 1.33 and 2.99 g/L, respectively. The pretreatment byproducts levels should be mitigated by controlling the severity of a physicochemical treatment or an additional selective removal process prior to H₂ fermentation.     

Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor

pH is considered as one of the most important factors governing the hydrogen fermentation process. In this project, five pH levels, ranging from 4.4 to 5.6 at 0.3 increments, were tested to evaluate the pH effect on hydrogen production from swine manure supplemented with glucose in an anaerobic sequencing batch reactor system with 16 h of hydraulic retention time (HRT). The optimal hydrogen yield (1.50 mol H₂/mol glucose) was achieved at pH 5.0 when the maximum production rate of 2.25 L/d/L was obtained. Continuous hydrogen production was achieved for over 3 weeks for pH 5.0, 4.7, and 4.4, with no significant methane produced. However, as pH increased to 5.3 and 5.6, methane production was observed in the biogas with concurrent reductions in hydrogen production, indicating that methanogens could become increasingly activated for pH 5.3 or higher. Acetate, propionate, butyrate, valerate, and ethanol were the main aqueous products whose distribution was significantly affected by pH as well.     

Hydrogen production from glucose by a mutant strain of Rhodovulum sulfidophilum P5 in single-stage photofermentation

Single-stage hydrogen production from glucose was investigated using the marine photosynthetic bacterium Rhodovulum sulfidophilum TH-79, a Tn7 transposon mutant of strain P5. The mutation in strain TH-79 did not affect its cell growth in glucose medium compared with the parent strain. TH-79 displayed improved photoheterotrophic hydrogen production performance when the medium contained glucose or galactose as the sole carbon source. The mutant produced about 7.07 mol H₂/mol glucose, which is similar to the yields of more complicated integration systems. A one-stage photofermentation system using a seawater culture medium appears to be a promising alternative to the integration of dark- and photofermentation systems.     

Influence of pH on fermentative hydrogen production from sweet sorghum extract

The present study focused on the influence of pH on the fermentative hydrogen production from the sugars of sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time of 12 h and a pH range of 3.5–6.5. The maximum hydrogen production rate and yield were obtained at pH 5.3 and were 1752 ± 54 mL H₂/d or 3.50 ± 0.07 L H₂/L reactor/d and 0.93 ± 0.03 mol H₂/mol glucose consumed or 10.51 L H₂/kg sweet sorghum, respectively. The main metabolic product at this pH value was butyric acid. The hydrogen productivity and yield were still at high levels for the pH range of 5.3–4.7, suggesting a pH value of 4.7 as optimum for hydrogen production from an economical point of view, since the energy demand for chemicals is lower at this pH. At this pH range, the dominant fermentation product was butyric acid but when the pH culture sharply decreased to 3.5, hydrogen evolution ceased and the dominant metabolic products were lactic acid and ethanol.     

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.     

Biohydrogen production from pure and natural lignocellulosic feedstock with chemical pretreatment and bacterial hydrolysis

Pretreatment and saccharification of lignocellulosic materials is the key technology affecting the efficiency of cellulosic biohydrogen production. In this work, two pure cellulosic materials (i.e., carboxymethyl-cellulose (CMC) and xylan) were directly hydrolyzed (without pretreatment) by a cellulolytic isolate Cellulomonas uda E3-01 able to release extracellular cellulolytic enzymes. Natural cellulosic feedstock (i.e., sugarcane bagasse) was chemically pretreated prior to the bacterial hydrolysis.A temperature-shift strategy (35 °C for cellulolytic enzymes production and 45 °C for hydrolysis reaction) was used to increase the production of reducing sugars during the bacterial hydrolysis. The hydrolysates of CMC, xylan, and bagasse were efficiently converted to H₂ via dark fermentation with Clostridium butyricum CGS5. The maximum hydrogen yield was 8.80 mmol H₂/g reducing sugar (i.e., 1.58 mol H₂/mol hexose) for CMC, 6.03 mmol H₂/g reducing sugar (i.e., 0.91 mol H₂/mol pentose) for xylan, and 6.01 mmol H₂/g reducing sugar for bagasse.     

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Significance of carbon to nitrogen ratio on the long-term stability of continuous photofermentative hydrogen production

The stable and optimized operation of photobioreactors (PBRs) is the most challenging task in photofermentative biological hydrogen production. The carbon to nitrogen ratio (C/N) in the feed is a critical parameter that significantly influences microbial growth and hydrogen production. In this study, the effects of changing the C/N ratio to achieve stable biomass and continuous hydrogen production using fed-batch cultures of Rhodobacter capsulatus YO3 (uptake hydrogenase deleted, hup-) were investigated. The experiments were carried out in 8 L panel PBRs operated in indoor conditions under continuous illumination and controlled temperature. Culture media containing different acetate (40-80 mM) and glutamate (2-4 mM) concentrations were used to study the effects of changing the C/N ratio on biomass growth and hydrogen production. Stable biomass concentration of 0.40 g dry cell weight per liter culture (gDCW/L_c) and maximum hydrogen productivity of 0.66 mmol hydrogen per liter culture per hour (mmol/L_c/h) were achieved during fed-batch operation with media containing 40 mM acetate and 4 mM glutamate, C/N = 25, for a period of over 20 days. A study on the effect of biomass recycling on biomass growth and hydrogen production showed that the feedback of cells into the photobioreactor improved biomass stability during the fed-batch operation but decreased hydrogen productivity.     

Effect of enzyme addition on fermentative hydrogen production from wheat straw

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H₂), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H₂ usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H₂ production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H₂ production from untreated wheat straw ranged from 5.18 to 10.52 mL-H₂ g-VS⁻¹. Whatever the experimental enzyme addition procedure, a two-fold increase in H₂ production yields ranging from 11.06 to 19.63 mL-H₂ g-VS⁻¹ was observed after enzymatic treatment of the wheat straw. The high variability in H₂ yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H₂ production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw⁻¹.     

Evaluation of biohydrogen production from glucose and protein at neutral initial pH

Organic wastes are considered as potential substances for economical biohydrogen production, because the carbohydrate and protein are main components. Previous investigations indicate that an optimum hydrogen production appear in acidic conditions to carbohydrates, or in alkali condition to protein. However, in practice, the treatment of these organic wastes by anaerobic fermentation usually carries out at neutral pH condition, in which biohydrogen production is only a middle process. So, the purpose of this paper is to evaluate the biohydrogen production at neutral pH condition from carbohydrates or protein. Batch tests were conducted to investigate the differences in biohydrogen production by anaerobic fermentation at neutral initial pH using carbohydrate and protein (glucose and peptone) as the sole carbon source. The experimental results showed that the maximal hydrogen yields of two substrates were about 0.14 ml H₂/mg glucose and 0.077 ml H₂/mg protein, respectively, at neutral initial pH. Although the hydrogen yields of glucose is far greater than that of protein at neutral pH, they were lower than previous results of hydrogen production in acidic condition to carbohydrate or in alkali condition to protein. This result shows that the neutral pH is not an optimal condition for biohydrogen production. In this experiment of biohydrogen production, a phenomenon has been observed that the hydrogen production and hydrogen consumption occurred simultaneously in the fermentation of protein, whereas the hydrogen production occurred only in the fermentation of glucose. Furthermore, the different evaluation of the main components of the organic liquid by-products produced by fermentation of each substrate implied that the biohydrogen production pathways of these two substrates were different. Molecular analysis indicated that the dominant microorganisms during the anaerobic fermentation of these two substrates are greatly different.     

Comparison of suspended and granular cell anaerobic bioreactors for hydrogen production from acid agave bagasse hydrolyzates

Acid agave bagasse hydrolyzates have been used as a substrate for hydrogen production, however, bioreactors are unstable and with poor performance. Granular biomass could be more successful in producing hydrogen from acid agave bagasse hydrolyzates in comparison with suspended biomass. Thus, this study aimed to evaluate the effect of increasing concentrations of acid agave hydrolyzates on hydrogen production, to compare the hydrogen productivity and stability of granular biomass in an expanded granular sludge bed (EGSB) reactor and suspended biomass in an anaerobic sequencing batch reactor (AnSBR) fed with acid hydrolyzates, and finally to determine the variation of microbial communities established in both bioreactor configurations. In batch tests, the heat-treated inoculum produced hydrogen from acid agave hydrolyzates without observing inhibition at 6.3 g/L of carbohydrates (_CHO). This hydrolyzate concentration was used to start up the AnBSR, which reached a productivity of 226 ± 53 mL H₂/L⋅d at organic loading rates (OLR) from 3.2 to 4.5 g_CHO/L⋅d. The hydrogen production stability index decreased from 0.8 to 0.6 at increasing OLR, and the AnSBR failed at the highest OLR of 5.7 g/L⋅d. The EGSB reactor reached the highest productivity of 361 ± 130 mL H₂/L⋅d at an OLR of 7.4 g_CHO/L⋅d, but with a low stability index of 0.6. Independently of the bioreactor configuration, microbial communities associated with the production of acetate/lactate were successfully established in both configurations with the prevalence of Lactobacillus spp. A low abundance of typical H₂ producers like Clostridium was always observed over the whole period of operation (<10% of the total abundance). In sum, the hydrogen productivity from acid agave hydrolyzates was higher for the EGSB reactor than for the AnSBR, but with much lower stability. The evidence provided by this study suggests the establishment of metabolic pathways for hydrogen production from organic acids.     

Kinetics of cotton cellulose hydrolysis using concentrated acid and fermentative hydrogen production from hydrolysate

The kinetics of cotton cellulose hydrolysis using concentrated sulfuric acid and the performance of fermentative hydrogen production from the hydrolysate in the batch system was carried out in this study. Effects of sulfuric acid concentrations, cotton cellulose concentrations and operating temperatures on the cotton cellulose hydrolysis were investigated. It was found that cotton cellulose can dissolve completely in sulfuric acid concentration above 55% (by volume) at room temperature. The reduced sugar yields were varied from 64.3 to 73.9% (g R-sugar/g cotton cellulose) with the initial cotton cellulose concentrations of 30-70 g/L at a temperature of 40 °C.The reduced sugar concentrations and the initial pH of biohydrogen production were investigated at 37 °C. It was found that the optimal values of the hydrogen yield and substrate utilization were 0.95 mol H₂/mol R-sugar and 98% with an initial pH of 8.2, when substrate concentration was fixed at 20 g R-sugar/L. The maximum hydrogen yield was 0.99 mol H₂/mol R-sugar at a substrate concentration of 15 g R-sugar/L. Using the Gompertz Equation Model simulation, the maximum hydrogen production rate was 253 mL H₂/h/L at a substrate of 30 g/L and initial pH of 8.4.     

Thermophilic hydrogen production from cellulose with rumen fluid enrichment cultures: Effects of different heat treatments

Elevated temperatures (52, 60 and 65 °C) were used to enrich hydrogen producers on cellulose from cow rumen fluid. Methanogens were inhibited with two different heat treatments. Hydrogen production was considerable at 60 °C with the highest H₂ yield of 0.44 mol-H₂ mol-hexose⁻¹ (1.93 mol-H₂ mol-hexose-degraded⁻¹) as obtained without heat treatment and with acetate and ethanol as the main fermentation products. H₂ production rates and yields were controlled by cellulose degradation that was at the highest 21%. The optimum temperature and pH for H₂ production of the rumen fluid enrichment culture were 62 °C and 7.3, respectively. The enrichments at 52 and 60 °C contained mainly bacteria from Clostridia family. At 52 °C, the bacterial diversity was larger and was not affected by heat treatments. Bacterial diversity at 60 °C remained similar between heat treatments, but decreased during enrichment. At 60 °C, the dominant microorganism was Clostridium stercorarium subsp. leptospartum.     

Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill

The fermentative hydrogen production capability of the newly isolated Clostridium sp. 6A-5 bacterium was studied in a batch cultivation experiment. Various culture conditions (temperature, initial pH, and glucose concentration) were evaluated for their effects on cell growth and hydrogen production (including the yield and rate) of Clostridium sp. 6A-5. Optimal cell growth was observed at 40 °C, initial pH 7.5–8, and glucose concentration 16–26 g/L. The optimal hydrogen yield was obtained at 43 °C, initial pH 8, and glucose concentration 10–16 g/L. Hydrogen began to evolve when cell growth entered the mid-exponential phase and reached the maximum production rate at the late exponential and stationary phases. The maximum hydrogen yield, and rate were 2727 mL/L, and 269.3 mL H₂/L h, respectively. These results indicate that Clostridium sp. 6A-5 is a good candidate for mesophilic fermentative hydrogen production.