Isolation and evaluation of a high H2-producing lab isolate from cow dung

Hydrogen producing bacterial strain was isolated from Indian cow dung and identified of the bacterial family Enterobacteriaceae. This lab isolate was differentiated from Citrobacter Y-19 at molecular level by using RAPD, PCR based technique, and OPO-03₄₆₀ and OPO-17₈₀₀ RAPD marker for this specific strain (lab isolate) was identified. Fermentative studies were investigated for important parameters, starting with pH of the culture, temperature, inoculum age and inoculum volume, initial substrate concentration and different substrates. Among different substrates, dextrose and sucrose were the preferred substrates for hydrogen production. The optimal starting pH of the culture was found to be 5.0. The H₂ production increased with increase in temperature up to 30 °C. The maximum value of H₂ production was recorded when inoculum volume was 12.5% of the culture broth and inoculum age was 14 h. Under batch fermentation conditions, the maximum hydrogen production rate and yield were 355.2 ml l⁻¹ h⁻¹ and 2.1 mol/mol glucose (conversion 35%), respectively. These results indicate that this lab isolate is an ideal hydrogen producer.     

Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture

We have analyzed the filamentous cyanobacterium Anabaena PCC 7120 (wildtype) containing one nitrogenase, one uptake hydrogenase and one bidirectional hydrogenase and its hydrogen uptake deficient mutant AMC 414 for their H₂ production capacities. Anabaena PCC 7120 and AMC 414 had similar growth rates in turbidostat mode with increased growth rates at higher light intensity. Rates of C₂H₂ reduction were similar for both strains. In contrast to the wildtype, AMC 414 produced H₂ in a PhotoBioReactor (PhBR) using air as the lifting gas. The rate of H₂ production increased with light intensity and was not even saturated at 456 μEm⁻² s⁻¹. H₂ production increased significantly when replacing the air with argon. The maximal H₂ production during outdoor conditions was recorded using AMC 414 with a peak at 14.9 ml H₂ h⁻¹ l⁻¹. Despite the relatively high production, maximal efficiency of solar energy to H₂ conversion was only 0.042%. A molecular method was developed to analyze the relative abundancies of weight and mutant in competition experiments in the PhBR.     

Strategy for enhancing photo-hydrogen production yield by repeated fed-batch cultures

In order to obtain the high H₂ yield, photo-hydrogen production by Rhodopseudomonas faecalis strain RLD-53 in fed-batch culture using acetate as the sole carbon was studied. In repeated fed-batch culture, biomass increased rapidly from 0.05 to 0.65 g/l within 120 h, with the specific maximal growth rate estimated 0.68 × 10⁻³ g/h. After 120 h, biomass increased slowly and after each time feeding biomass increased slightly. The specific cumulative H₂ volumes in each phase were 2791.3, 1161.7, 1445.5 and 840.6 ml H₂/l-culture, respectively. The average H₂ yield was 3.17 mol H₂/mol acetate based on the whole process while the average substrate conversion efficiency reached 79.3%. Specific maximum H₂ production rate and H₂ content was 37.2 ml H₂/l/h and 95.5%, respectively. The results demonstrated the repeated fed-batch mode obtained higher efficiency for hydrogen production, feeding acetate concentration and control of pH were important to fed-batch culture hydrogen production.     

Fed-batch operation for bio-H2 production by Rhodopseudomonas palustris (strain 42OL)

Interest in renewable and clean energies such as hydrogen has increased because of the high level of polluting emissions, increasing costs associated with petroleum and the escalating problems of global climate change. In the presence of a light source, a microbial photosynthetic process provides a system for the conversion of some organic compounds into biomass and hydrogen. Using Rhodopseudomonas palustris as a cell-factory, hydrogen photo-evolution was investigated in a photobioreactor (PBR) irradiated either from one or two opposite sides. Irradiating the photobioreactor from only one side, in the presence of malic acid, a reactor hydrogen production of 2.786 l(H₂) PBR⁻¹ was achieved. When the PBR was irradiated from two opposite sides, hydrogen photo-evolution increased to 3.162 l(H₂) PBR⁻¹. Experiments were carried out using inoculum from either the retardation or the exponential growth phases. Using the latter, the highest hydrogen photo-evolution rate based on the bacteriochlorophyll (Bchl) concentration was achieved (3295 μl(H₂) mg (Bchl⁻¹ h⁻¹). The hydrogen to biomass ratio (r_g) was 1.91 l g⁻¹ in the medium containing malic acid and 1.07 l g⁻¹ in that containing acetic acid. It was found that the hydrogen production rate was higher with malic than with acetic acid. Although photobiological hydrogen production cannot furnish alone the greater and greater world requirements of clean renewable energy, it is desirable that photobiological hydrogen technology will grow, in the near future, because photobioreactors for bio-hydrogen production can be positioned in fringe areas without competition with agricultural lands.     

Isolation of anoxygenic photosynthetic bacteria from Songkhla Lake for use in a two-staged biohydrogen production process from palm oil mill effluent

We are developing a process to produce biohydrogen from palm oil mill effluent. Part of this process will involve photohydrogen production from volatile fatty acids under low light conditions. We sought to isolate suitable bacteria for this purpose from Songkhla Lake in Southern Thailand. Enrichment for phototrophic bacteria from 34 samples was conducted providing acetate as a major carbon source and applying culturing conditions of anaerobic-low light (3000 lux) at 30 °C. Among the independent isolates from these enrichments 19 evolved hydrogen with productivities between 4 and 326 ml l⁻¹ d⁻¹. Isolate TN1 was the most efficient producer at a rate of 1.85 mol H₂ mol acetate⁻¹ with a light conversion efficiency of 1.07%. The maximum hydrogen production rate for TN1 was determined to be 43 ml l⁻¹ h⁻¹. Environmentally desirable features of photohydrogen production by TN1 included the absence of pH change in the cultures and no detectable residual CO₂.     

Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation

A two-step process of sequential anaerobic (dark) and photo-heterotrophic fermentation was employed to produce hydrogen from cassava and food waste. In dark fermentation, the average yield of hydrogen was approximately 199 ml H₂ g⁻¹ cassava and 220 ml H₂ g⁻¹ food waste. In subsequent photo-fermentation, the average yield of hydrogen from the effluent of dark fermentation was approximately 611 ml H₂ g⁻¹ cassava and 451 ml H₂ g⁻¹ food waste. The total hydrogen yield in the two-step process was estimated as 810 ml H₂ g⁻¹ cassava and 671 ml H₂ g⁻¹ food waste. Meanwhile, the COD decreased greatly with a removal efficiency of 84.3% in cassava batch and 80.2% in food waste batch. These results demonstrate that cassava and food waste could be ideal substrates for bio-hydrogen production. And a two-step process combining dark fermentation and photo-fermentation was highly improving both bio-hydrogen production and removal of substrates and fatty acids.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l⁻¹ d⁻¹ gave a maximum hydrogen yield of 0.27 l H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ l⁻¹ d⁻¹ (16.9 mmol l⁻¹ h⁻¹). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l⁻¹ d⁻¹) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).     

Pilot-scale hydrogen fermentation system start-up performance

A high-rate hydrogen production process able to produce H₂ at a maximum rate of 15 L/L/h was successfully developed by the Feng Chia University (FCU) biohydrogen research team. This highly efficient hydrogen fermentation system includes a 400 L pilot-scale system constructed for determining scale-up operation parameters for commercializing the bioH₂ production technology. The pilot-scale system is composed of a feedstock tank, mixing system, fermentor, gas/liquid separator and automatic control system. The fermentor is fed with sucrose (20 g COD/L) and operated at 35 °C. A batch strategy is used for system start-up. The fermentor was first operated in a batch mode for two days and then switched to a continuous-feeding mode (HRT 12 h) for one month. During the continuous operation, pH notably affected H₂ production efficiency and bacterial community. For the first 14-day operation, the H₂ production rate increased from 0.017 to 0.256 L/L/h with a pH variation from 5.0 to 7.0. The DGGE results indicate the presence of two Clostridium species (namely, Clostridium butyricum and Clostridium pasteurianum) in the fermenter. Stable hydrogen production rate was obtained at pH 5.5–6.0 when C. pasteurianum became dominant in the mixed culture.     

Enhanced hydrogen production by means of sulfur-deprived Chlamydomonas reinhardtii cultures grown in pretreated olive mill wastewater

Under sulfur-deprived conditions, the metabolism of Chlamydomonas reinhardtii switches to the photoproduction of hydrogen. This process is sustained by both photosystem II-driven water splitting and by the fermentation of stored carbohydrates. We investigated the possibility of using diluted pretreated olive mill wastewaters (OMW), which contain organic acids and sugars, as a substrate on which to grow Chlamydomonas, in order to obtain suitable biomass to produce hydrogen. The cells grown on a mixture of pretreated OMW and TAP (tris-acetate-phosphate) (50% dilution) were found to be richer in carbohydrates and exhibited a greater production of hydrogen (150 ml H₂ l⁻¹ culture), compared to the control cells (100 ml H₂ l⁻¹ culture). In these cultures, the hydrogen production process was characterized by a shorter aerobic phase and a longer hydrogen-production period. The results offer a useful perspective for the utilization of olive mill wastewaters, which constitute an environmental problem, particularly in Mediterranean areas, and for increasing the output for hydrogen production with Chlamydomonas.     

Utilization of spent copper-pickle liquor for recovery of metal values

A zero emission process was established by treatment of a spent liquor resulting from frequent pickling of copper alloy rods in sulfuric acid. The spent liquor contains 35 g l⁻¹ copper, 25 g l⁻¹ zinc, 1.1 g l⁻¹ chromium, 45 g l⁻¹ sulfuric acid, and 135 g l⁻¹ total sulfate. These pollutant materials, if discharged directly into water bodies, pose acute environmental and ecological problems. Recovery and separation of these metals have been studied. The solvent extraction technique was applied to separate copper selectively using 10% Acorga 5640 in kerosene, at pH 2.0 for 10 min. Three extraction stages with an organic/aqueous (O/A) phase ratio of 4/1 were applied for efficient removal of copper from the aqueous phase. A copper-loaded organic solution was stripped using 3 M sulfuric acid. Pure copper was obtained from the strip solution by electrowinning. From the raffinate, chromium was recovered as chromic III hydroxide at pH 5–6 by adding ammonium buffer solution (NH₄OH/NH₄Cl). Zinc was precipitated from the resulting solution either as carbonate or as sulfide by using ammonium carbonate at pH 7.6 or ammonium sulfide at pH 9, respectively. Ammonium sulfate by-product was crystallized from the final solution. At the optimum conditions, almost all contents of metals were recovered.     

Visible light-induced hydrogen over CuFeO2 via S2O3 oxidation

The properties of CuFeO₂ have been studied according to the catalytic hydrogen production upon visible light. CuFeO₂ with a low band gap E_g, a good chemical stability and a suitable flat band potential appears as a suitable candidate. The potential of photoelectrons allows favorably a thermodynamically H₂-evolution from alkaline thiosulfate S₂O₃²⁻ solution. There is a major difference between pure and loaded oxide with some metal catalysts. Our best results have been obtained with unloaded CuFeO₂ at 50 °C and pH 13.60. Thiosulfate S₂O₃²⁻ ions can be oxidized to sulfite SO₃²⁻ and subsequently to sulfate SO₄²⁻ and the electronic exchange occurs via mediation of surface states. The quite high H₂-formation at the beginning shows a tendency towards saturation, it competes with SO₃²⁻ produced by parallel oxidation of S₂O₃²⁻.     

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H₂ production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H₂ yield and specific H₂ production rates with xylose and glucose are 1.63 mol-H₂/mol xylose and 11.14-H₂ mmol/h g-cdw, and 2.02 mol-H₂/mol-glucose and 9.37 mmol-H₂/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H₂ yield and specific H₂ production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H₂/mol-mixed sugar and 12.56 mmol-H₂/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.     

Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O₂ consumption, lower O₂ content and higher H₂ output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O₂ contents and maximal H₂ evolution occurred. The highest H₂ production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H₂ production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H₂ yield by decreasing O₂ content in the medium. This protein had potential to be used in improvement of H₂ production in green algae.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

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

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

Thermophilic bio-energy process study on hydrogen fermentation with vegetable kitchen waste

An intermittent-continuous stirred tank reactor (I-CSTR) was evaluated for thermophilic anaerobic hydrogen fermentation with vegetable kitchen waste (VKW). The seeding sludge was enriched from kitchen waste compost. Because of different seasonal dietary habits, the quality of vegetable kitchen waste was unstable, and all variations of composition were in the range from 20 to 40%. The I-CSTR process was conducted under different volumetric loading rates (VLR) with different VKW-diluted concentrations. The hydrogen production rate and yield in Run 2 (VLR as 28 g-COD L⁻¹ day⁻¹) were 1.0 L-H₂ L⁻¹ day⁻¹ and 1.7 mmol-H₂ g-COD⁻¹, which were higher than those in Run1 (VLR as 19 g-COD/L-day). The hydrolysis efficiency of organic solids (VSS) was about 45% in Run 1 better than the 32% in Run 2. The carbohydrate component of VKW was clearly degraded with the accumulation of butyrate, while the organic nitrogen component was converted to ammonia. The vegetable cellulose was degraded from 3.2 g L⁻¹ and 3.6–1.8 and 3.2 g L⁻¹ in Runs 1 and 2, respectively. In addition, the high concentration of lactate from the acidified VKW could be degraded completely both in Runs 1 and 2. According to the results of the time series profile in day 59, oil and grease were not degraded significantly. The removal of oil and grease was superficially caused by stacking on the wall, pipe, and propeller of the reactor, or by floating on the liquid surface. The 16S rDNA cloning library and sequence were applied for analyzing microbial communities. The dominant OTU was closely affiliated to Thermoanaerobacterium thermosaccharolyticum, which is considered as the predominant hydrogen-producing bacteria. The OTUs closely related to Moorella thermoacetica and Clostridiaceae bacterium FH052 were considered as acetogenic bacterium and hydrogen-producing bacteria in the I-CSTR system.     

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

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

Hydrogen–methane production from swine manure: Effect of pretreatment and VFAs accumulation on gas yield

A two-phase anaerobic process to produce hydrogen and methane from swine manure was investigated, using pretreated sludge with heat, acid and alkali treatment as inoculum. The relative order of pretreatment methods of H₂ productivity effectiveness and CH₄ productivity effectiveness produced by the residua of the first phase was heat treatment > alkali treatment > acid treatment. When the inoculum sludge was heat-treated at 80°C for 30 min, the H₂ and CH₄ production rate was the highest of 36.6, 201.7 ml (g TS)_added⁻¹. There were significant correlations between biogas production and accumulation of acetic acid and butyric acids. When propionic acid and total VFA concentrations reached about 2850 mg L⁻¹ and 10.0 g L⁻¹, respectively, the average H₂ production rate and H₂ content decreased from 7.6 ml d⁻¹(g VS)_added⁻¹ and 55.3% to 1.4 ml d⁻¹(g VS)_added⁻¹ and 43.2%, respectively. The activity of methanogenic bacteria was inhibited to a significant extent when the total VFA concentration was above 10.0 g L⁻¹, but this inhibitory effect weakened when the VFA concentration fell to 6200–8500 mg L⁻¹. Correspondingly, average CH₄ production rate increased from 4.0 ml d⁻¹(g TS)_added⁻¹ to 12.5 ml d⁻¹(g TS)_added⁻¹. Propionic acid was degraded rapidly only when acetic and butyric acid concentrations dropped to 2500 mg L⁻¹ and 1000 mg L⁻¹, respectively.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.     

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.