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.     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

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.     

Thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19: Optimization of media composition

The composition of media for thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19 were optimized in order to maximize the hydrogen production potential (P_s). Results from Plackett-Burman design indicated that FeSO₄, CaCl₂, NaHCO₃, and MgCl₂ had a significantly effect (P ≤ 0.05) on P_s. The optimum media composition obtained from the response surface methodology (RSM) with central composite design (CCD), using the hydrolysate at a total sugar concentration of 8.98 g/L, were (all in mg/L): FeSO₄, 1454.65; MgCl_2, 511.36; CaCl_2, 278.62; and NaHCO_3, 2186.41 in which the P_s of 2397 mL H₂/L were obtained. Verification experiment using the optimum media composition in a continuous stirred tank reactor indicated a highly reproducible result in which the P_s of 2608 mL H₂/L was achieved at a hydraulic retention time of 32 h. The results demonstrated that the media composition obtained from the batch experiment using RSM with CCD can be practically applied to continuously produce hydrogen from the hydrolysate with the least error.     

Biohydrogen production from xylose by Thermoanaerobacterium thermosaccharolyticum KKU19 isolated from hot spring sediment

A thermophilic hydrogen producer was isolated from hot spring sediment and identified as Thermoanaerobacterium thermosaccharolyticum KKU19 by biochemical tests and 16S rRNA gene sequence analysis. The strain KKU19 showed the ability to utilize various kinds of carbon sources. Xylose was the preferred carbon source while peptone was the preferred organic nitrogen source. The optimum conditions for hydrogen production and cell growth on xylose were an initial pH of 6.50, temperature of 60 °C, a carbon to nitrogen ratio of 20:1, and a xylose concentration of 10.00 g/L. This resulted in a maximum cumulative hydrogen production, hydrogen production rate and hydrogen yield of 3020 ± 210 mL H₂/L, 3.95 ± 0.20 mmol H₂/L h and 2.09 ± 0.02 mol H₂/mol xylose consumed, respectively. Acetic and butyric acids were the main soluble metabolite products suggesting acetate and butyrate type fermentation.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

Enhanced bio-hydrogen production from cornstalk hydrolysate pretreated by alkaline-enzymolysis with orthogonal design method

Bio-hydrogen (H₂) production from renewable biomass has been accepted as a promising method to produce an alternative fuel for the future. In this study, fermentative hydrogen production from cornstalk (CS) hydrolysate pretreated by alkaline-enzymolysis method was investigated. Meanwhile, a five-factor and five-level orthogonal experimental array was designed to study the influences of Ca(OH)₂ concentration, alkaline hydrolysis time, alkaline hydrolysis temperature, cellulase and xylanase dosages on cornstalk pretreatment and hydrogen production. A maximum reducing sugar yield of 0.59 g/g-CS was obtained at Ca(OH)₂ 0.5%, hydrolysis temperature 115 °C, hydrolysis time 1.5 h, cellulase dosage 4000 U/g-CS and xylanase 4000 U/g-CS. Under this same condition, the maximum hydrogen yields of 168.9 mL/g-CS, 357.6 mL/g-CS, and 424.3 mL/g-CS were obtained at dark-fermentation, photo-fermentation, and two-stage fermentation respectively. It's also found that the significance of these five parameters on H₂ production followed from high to low order as: Ca(OH)₂ concentration, cellulase dosage, xylanase dosage, hydrolysis time, and hydrolysis temperature. By comparing the energy produced with the energy spent, the maximum Energy Sustainability Index (ESI) value of 1.11 was obtained at the two-stage fermentation. The results suggested that two-stage fermentation is a promising and efficient way for hydrogen production from lignocellulosic biomass.     

Co-digestion of oil palm trunk hydrolysate with slaughterhouse wastewater for thermophilic bio-hydrogen production by Thermoanaerobacterium thermosaccharolyticm KKU19

The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H₂SO₄ method using 1.56% (w/v) H₂SO₄ and 7.50 min reaction time at 450 W. The Plackett–Burman method was used to screen the key factors that influenced the hydrogen production potential (P_s). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO₄ concentration influenced the P_s. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum P_s of 2604 ± 86 mL H₂/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO₄ concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The P_s obtained when the hydrolysate was supplemented with SHW (2604mL ± 86 mL H₂/L substrate) was comparable to the P_s obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 ± 87 mL H₂/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source.     

A pilot-scale study of biohydrogen production from distillery effluent using defined bacterial co-culture

We evaluated the feasibility of improving the scale of hydrogen (H₂) production from sugar cane distillery effluent using co-cultures of Citrobacter freundii 01, Enterobacter aerogenes E10 and Rhodopseudomonas palustris P2 at 100 m³ scale. The culture conditions at 100 ml and 2 L scales were optimized in minimal medium and we observed that the co-culture of the above three strains enhanced H₂ productivity significantly. Results at the 100 m³ scale revealed a maximum of 21.38 kg of H₂, corresponding to 10692.6 mol, which was obtained through batch method at 40 h from reducing sugar (3862.3 mol) as glucose. The average yield of H₂ was 2.76 mol mol⁻¹ glucose, and the rate of H₂ production was estimated as 0.53 kg/100 m³/h. Our results demonstrate the utility of distillery effluent as a source of clean alternative energy and provide insights into treatment for industrial exploitation.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

Photofermentive production of biohydrogen from oil palm waste hydrolysate

Oil palm empty fruit bunch (OPEFB) was hydrolyzed with dilute sulfuric acid (6% v/v; 8 mL acid per g dry OPEFB) at 120 °C for 15-min to release the fermentable sugars. The hydrolysate contained xylose (23.51 g/L), acetic acid (2.44 g/L) and glucose (1.80 g/L) as the major carbon components. This hydrolysate was used as the sole carbon source for photofermentive production of hydrogen using a newly identified photosynthetic bacterium Rhodobacter sphaeroides S10. A Plackett–Burman experimental design was used to examine the influence of the following on hydrogen production: yeast extract concentration, molybdenum concentration, magnesium concentration, EDTA concentration and iron concentration. These factors influenced hydrogen production in the following decreasing order: yeast extract concentration > molybdenum concentration > magnesium concentration > EDTA concentration > iron concentration. Under the conditions used (35 °C, 14.6 W/m² illumination, initial pH of 7.0), the optimal composition of the culture medium was (per L): mixed carbon in OPEFB hydrolysate 3.87 g, K₂HPO₄ 0.9 g, KH₂PO₄ 0.6 g, CaCl₂⋅2H₂O 75 mg, l-glutamic acid 795.6 mg, FeSO₄⋅7H₂O 11 mg, Na₂MoO₂⋅2H₂O 1.45 mg, MgSO₄⋅7H₂O 2.46 g, EDTA 0.02 g, yeast extract 0.3 g). With this medium, the lag period of hydrogen production was 7.65 h, the volumetric production rate was 22.4 mL H₂/L medium per hour and the specific hydrogen production rate was 7.0 mL H₂/g (xylose + glucose + acetic acid) per hour during a 90 h batch culture of the bacterium. Under optimal conditions the conversion efficiency of the mixed carbon substrate to hydrogen was nearly 29%.     

Effect of lignocellulose-derived inhibitors on growth and hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16

In the process of producing H₂ from lignocellulosic materials, inhibitory compounds could be potentially formed during pre-treatment. This work experimentally investigated the effect of lignocellulose-derived inhibitors on growth and hydrogen production by Thermoanaerobacterium thermosaccharolyticum W16. Representative compounds presented in corn stover acid hydrolysate were added in various concentrations, individually or in various combinations and subsequently inhibitions on growth and H₂ production were quantified. Acetate sodium was not inhibitory to T. thermosaccharolyticum W16, rather than it was stimulatory to the growth and H₂ production. Alternatively, furfural, hydroxymethylfurfural (HMF), vanillin and syringaldehyde were potent inhibitors of growth and hydrogen production even though these compounds showed inhibitory effect depending on their concentrations. Synergistic inhibitory effects were exhibited in the introduction of combinations of inhibitors to the medium and in hydrolysate with concentrated inhibitors. Fermentation results from hydrolysates revealed that to increase the efficiency of this bioprocess from corn stover hydrolysate, the inhibitory compounds concentration must be reduced to the levels present in the raw hydrolysate.     

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h^?1 and the average pseudo-steady state hydrogen production rate was 36 mL H₂ L^?1 medium h^?1. The cumulative hydrogen production was 430 mL H₂ L^?1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H₂ L^?1 h^?1 and the cumulative hydrogen production was 517 mL H₂ L^?1 medium. Reducing the feed rate to less than 48 mL h^?1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.     

Influence of Cu concentration on the biohydrogen production of continuous stirred tank reactor

In this paper, the effect of hydraulic retention time (HRT, 16 h–4 h) on fermentative hydrogen production by mixed cultures was firstly investigated in a sucrose-fed anaerobic continuous stirred tank reactor (CSTR) at 35 °C and initial pH 8.79. After stable operations at HRT of 16–6 h, the bioreactor became unstable when the HRT was lowered to 4 h. The maximum hydrogen yield reached 3.28 mol H₂/mol-Sucrose at HRT 4 h. Supplementation of Cu²⁺ at HRT 4 h improved the operation stability through enhancement of substrate degradation efficiency. The effect of Cu²⁺ concentration ranging from 1.28 to 102.4 mg/L on fermentative hydrogen production was studied. The results showed that Cu²⁺ was able to enhance the hydrogen production yield with increasing Cu²⁺ concentration from 1.28 to 6.4 mg/L. The maximum hydrogen yield of 3.31 mol H₂/mol-Sucrose and the maximum hydrogen production rate of 14.44 L H₂/Day/L-Reactor were obtained at 6.4 mg/L Cu²⁺ and HRT 4 h Cu²⁺ at much higher concentration could inhibit the hydrogen production, but it could increase substrate degradation efficiency (12.8 and 25.6 mg/L Cu²⁺). The concentration of Cu²⁺ had effect on the distribution of soluble metabolite.     

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

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

Extreme-thermophilic biohydrogen production by an anaerobic heat treated digested sewage sludge culture

Biohydrogen production process from glucose using extreme-thermophilic H₂-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 °C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H₂/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H₂/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Deoiled algal cake as feedstock for dark fermentative biohydrogen production: An integrated biorefinery approach

An integrated biorefinery approach utilizing deoiled algal cake (after lipid extraction) as potential feed-stock for biohydrogen (H₂) production using selectively enriched acidogenic consortia as biocatalyst was evaluated. Algae pretreated extract (AP-E) documented maximum H₂ production rate (HPR), cumulative H₂ production (CHP) and specific H₂ yield (SHY) with higher substrate degradation (65%) in terms of COD removal efficiency than other conditions, which is a good sign for waste remediation. Along with the biohydrogen production and substrate removal the consortia also produced good amount of volatile fatty acids (VFA). VFA production in fermentation media resulted in reactor pH drop. The study depicted the feasible use of deoiled algal biomass as feed-stock for H₂ production in the framework of biorefinery.