Anaerobic fluidized bed reactor with expanded clay as support for hydrogen production through dark fermentation of glucose

This study evaluated hydrogen production in an anaerobic fluidized bed reactor (AFBR) fed with glucose-based synthetic wastewater. Particles of expanded clay (2.8–3.35 mm) were used as a support material for biomass immobilization. The reactor was operated with hydraulic retention times (HRT) ranging from 8 to 1 h. The hydrogen yield production increased from 1.41 to 2.49 mol H₂ mol⁻¹ glucose as HRT decreased from 8 to 2 h. However, when HRT was 1 h, there was a slight decrease to 2.41 mol H₂ mol⁻¹ glucose. The biogas produced was composed of H₂ and CO₂, and the H₂ content increased from 8% to 35% as HRT decreased. The major soluble metabolites during H₂ fermentation were acetic acid (HAc) and butyric acid (HBu), accounting for 36.1–53.3% and 37.7–44.9% of total soluble metabolites, respectively. Overall, the results demonstrate the potential of using expanded clay as support material for hydrogen production in AFBRs.     

Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

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.     

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.     

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.     

Metabolic engineering of Escherichia coli strains for co-production of hydrogen and ethanol from glucose

The co-production of H₂ and ethanol from glucose was studied to address the low H₂ production yield in dark fermentation. Several mutant strains devoid of ackA-pta, pfkA or pgi were developed using Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB as base strain. Disruption of ackA-pta eliminated acetate production during glucose fermentation but resulted in the secretion of a significant amount of pyruvate (0.73 mol mol⁻¹ glucose) without improving the co-production of H₂ and ethanol. When pfkA or pgi was further disrupted to enhance NAD(P)H supply by diverting the carbon flux from Embden-Meyerhof-Parnas (EMP) pathway to the pentose phosphate pathway (PPP), the cell growth of both strains was severely impaired under anaerobic conditions, and only the ΔpfkA mutant could recover its growth after adaptive evolution. The production yields of the ΔpfkA strain (H_2, 1.03 mol mol⁻¹ glucose and ethanol, 1.04 mol mol⁻¹ glucose) were higher than those of the pfkA⁺ strain (H₂, 0.69 mol mol⁻¹ glucose and ethanol, 0.95 mol mol⁻¹ glucose), but pyruvate excretion was not reduced. The excessive excretion of pyruvate in the ΔpfkA mutant was attributed to an insufficient NAD(P)H supply caused by the diversion of carbon flux from the EMP pathway to the Entner-Doudoroff pathway (EDP), rather than the PPP as intended. This study suggests that co-production of H₂ and ethanol from glucose is possible, but further metabolic pathway engineering is required to fully activate PPP under anaerobic conditions.     

Hydrogen bioproduction with anaerobic bacteria consortium from brewery wastewater

Biohydrogen production is a cheap and clean way to obtain hydrogen gas. In subtropical countries such as Brazil the average temperatures of 27 °C can favor the hydrogen producing bacteria growth. A mixed culture was obtained from a subtropical sludge treating brewery wastewater and anaerobic batch reactors were fed with glucose, sucrose, fructose and xylose in low concentrations (2.0, 5.0 and 10.0 g L⁻¹) at 37 °C, initial pH 5.5 and headspace with N₂ (99%) to maintain the anaerobic conditions. The inoculum was a subtropical granulated sludge from UASB (Upflow Anaerobic Sludge Blanket) reactor treating brewery wastewater. The higher H₂ yields were obtained in reactors operated with 2 and 5 g L⁻¹ of fructose and they were 1.5 mol H₂ mol⁻¹ of fructose and 1.3 mol H₂ mol⁻¹ of sucrose, respectively. The volatile fatty acids (VFA) generated at the end of operation were, predominantly, butyric and acetic acid, indicating the favoring of the metabolic route of hydrogen generation by the consortium of anaerobic bacteria from the brewery wastewater. Biomolecular analyses revealed the predominance of hydrogen producing bacteria from Firmicutes phylum distributed in the families Streptococcaceae, Veillonellaceae and uncultured bacteria. These results confirm future applications of subtropical sludges with agroindustrial wastewaters containing low concentrations of sugars on hydrogen generation.     

Effect of furans and linoleic acid on hydrogen production

The effects of furans (furfural and 5-hydroxymethylfurfural (HMF)) on hydrogen (H₂) production using mixed anaerobic cultures were evaluated by conducting batch experiments. Two mixed anaerobic cultures (culture A and B) fed furans plus glucose and treated with and without linoleic acid (LA) at pH 5.5 were maintained at 37 °C. In the LA inhibited cultures A and B fed 0.75 g L⁻¹ furfural and 0.25 g L⁻¹ HMF, the maximum H₂ yields observed were 1.89 ± 0.27 mol mol⁻¹ glucose and 1.75 ± 0.22 mol mol⁻¹ glucose, respectively. In cultures with maximum H₂ yields, Clostridium sp. and Flavobacterium sp. were dominant. Acetate, butyrate and ethanol were the major soluble metabolites detected in cultures A and B whereas propionate was also dominant in culture B. A canonical correspondence analysis based on the byproducts and the relative abundance of the terminal-restriction fragments revealed less variation between cultures treated with LA and low correlation value between the factors and the species composition.     

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.     

Effects of linoleic acid and its degradation by-products on mesophilic hydrogen production using flocculated and granular mixed anaerobic cultures

The effects of linoleic acid (LA (C18:2)) and its degradation by-products on hydrogen (H₂) production were examined at 37 °C and an initial pH value of 5.0 using granular and flocculated mixed anaerobic cultures from the same source. In the flocculated cultures, the H₂ consumers were inhibited to a greater extent when compared to the granular cultures. The maximum H₂ yields were 2.52 ± 0.2 and 1.9 ± 0.2 mol mol⁻¹ glucose in the flocculated and granular cultures, respectively. The major long chain fatty acids (LCFAs) detected at which H₂ attained a maximum value were LA (750 mg L⁻¹) and myristic acid (MA) (500 mg L⁻¹).     

Hydrogen production from rotten dates by sequential three stages fermentation

This study was devoted for H₂ production from rotten fruits of date palm (Phoenix dactylifera L.) by three fermentation stages. A facultative anaerobe, Escherichia coli EGY was used in first stage to consume O₂ and maintain strict anaerobic conditions for a second stage dark fermentative H₂ production by the strictly anaerobic Clostridium acetobutylicum ATCC 824. Subsequently, a third stage photofermentation using Rhodobacter capsulatus DSM 1710 has been conducted for the H₂ production. The maximum total H₂ yield of the three stages (7.8 mol H₂ mol⁻¹ sucrose) was obtained when 5 g L⁻¹ of sucrose was supplemented to fermentor as rotten date fruits. A maximum estimated cumulative H₂ yield of the three stages (162 LH₂ kg⁻¹ fresh rotten dates) was estimated at the (5 g L⁻¹) sucrose concentration. These results suggest that rotten dates can be efficiently used for commercial H₂ production. The described protocol did not require addition of a reducing agent or flashing with argon which both are expensive.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

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.     

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.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana

Anaerobic growth of Thermotoga neapolitana led maximum to hydrogen yield of 3.85 ± 0.07 mol H₂/mol glucose and production rate of 51 ml/l/h. This productivity is strongly affected by stirring, pH buffering, N₂ sparging and culture/headspace volume ratio. Embden–Meyerhoff pathway is the only glycolytic route in T. neapolitana but, under the conditions used in this study, about 12–15% of the biogas requires consumption of protein source. Reduction of the hydrogen yields below the theoretical 4 mol H₂/mol glucose is mainly due to production of lactate and alanine that affect the availability of pyruvate/NADH for the hydrogenase, as well as to loss of part of glucose by conversion to fructose that is eventually released in the medium. Hydrogen productivity is modulated during the bacterial growth and major biogas synthesis is recorded in the stationary phase in concomitance with reduction of lactate synthesis. Apparently, this event is not consistent with an equal increase in acetate production. In agreement with the hydrogenase model recently proposed for the sister species Thermotoga maritima, this suggests that cellular NADH⁺ ratio has a crucial role on biogas synthesis.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.     

Flux balance analysis of mixed anaerobic microbial communities: Effects of linoleic acid (LA) and pH on biohydrogen production

The internal fluxes of mixed anaerobic cultures fed 2000 mg l⁻¹ linoleic acid (LA) plus glucose at 6 initial pH conditions and maintained at 37 °C were estimated using a flux balanced analysis (FBA). In cultures fed LA at pH 7, less than 8% of the flux was diverted to CH₄. At an initial pH ≥ 5.5, the quantity of glucose removed was greater than 95%; however, at pH 4.5 and 5.0 the quantity consumed were 38% and 75%, respectively. The FBA output showed that the acetogenic H₂-consumers were responsible for more than 20% of the H₂ consumed. Adding LA and decreasing the pH was ineffective in reducing the activity of acetogenic H₂-consumers. As the initial pH decreased, the acetogenic H₂-consuming flux decreased in the presence of 2000 mg l⁻¹ LA. A maximum H₂ yield of 1.55 mol mol⁻¹ glucose consumed (peak hydrogenase flux (R12)) was attained when the acetogenic H₂-consuming flux reached 0.42 mol at a pH of 5.5.