Mixtures of 5-hydroxymethylfurfural,levulinic acid,and formic acid have different impact on H2-producing Clostridium strains

Fermentative hydrogen production allows the use of renewable biomasses as feedstocks. However, biomass saccharification results not only in carbohydrates, but also in products that can inhibit fermentation. Although biomass hydrolysates contain mixtures of inhibitors, most studies are performed with a single inhibitor. This study evaluates how 5-hydroxymethylfurfural (HMF, 0.60 g/L), levulinic acid (LA, 2.10 g/L), and/or formic acid (FA, 0.80 g/L) mixtures affect two H₂-producing clostridia, Clostridium beijerinckii Br21 and Clostridium acetobutylicum ATCC 824. Fermentation assays with and without (control) the inhibitors helped to calculate the specific H₂ production, substrate consumption, and bacterial cell growth rates for Clostridium beijerinckii Br21 or Clostridium acetobutylicum ATCC 824. HMF + AL, HMF + AF, AL + AF, and HMF + AL + AF mixtures inhibited H₂ production by C. beijerinckii Br21 by 58.7, 60.0, 46.9, and 83.0%, respectively, and by C. acetobutylicum ATCC 824 by 68.1, 71.4, 58.2, and 89.0%, respectively. Clostridium acetobutylicum ATCC 824 metabolized HMF more efficiently. However, organic acids and their combination with HMF inhibited H₂ production by C. beijerinckii Br21 to a lesser extent, which highlighted that this microorganism is robust for H₂ production from biomass hydrolysates.     

Enhancing saccharification of Agave tequilana bagasse by oxidative delignification and enzymatic synergism for the production of hydrogen and methane

Agave tequilana bagasse is a suitable lignocellulosic residue for energy production. However, the presence of lignin and the heterogeneous structure of hemicellulose may hinder the availability of polysaccharides. In this work, the pretreatment of A. tequilana bagasse with alkaline hydrogen peroxide (AHP) followed by enzymatic saccharification with hemicellulases and cellulases was assessed for the removal of lignin and extraction of fermentable sugars, respectively. Results of the AHP pretreatment indicated that it is possible to attain up to 97% delignification and recover 88% of cellulose and hemicellulose after only 1.5 h of treatment. Regarding the saccharification process, the total sugar yield and productivity were both increased by 2-fold using an enzymatic mixture (cellulases + hemicellulases) compared to single enzyme hydrolysis (cellulases), evidencing synergism. Further evaluation of the hydrolyzates as substrate for hydrogen and methane production, resulted in yields 1.5 and 3.6-times (215.14 ± 13 L H₂ and 393.4 ± 13 L CH₄ per kg bagasse, respectively) superior to those obtained with hydrolyzates of non-pretreated bagasse processed with a single enzyme. Overall, using AHP pretreatment and subsequent hydrolysis with enzymatic mixtures improves the saccharification of A. tequilana bagasse enhancing the production of hydrogen and methane.     

Pretreatment enhanced structural disruption,enzymatic hydrolysis,fermentative hydrogen production from rice straw

Rice straw (RS) is one of the major lignocellulosic wastes in the world and an abundant feedstock for producing biofuels and chemicals. However, RS is difficult to decompose. In this study, NaOH/urea and electrohydrolysis pretreated RS were used to enhance the structural disruption, enzymatic hydrolysis, and fermentative hydrogen production. Scanning electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy analyses demonstrated that both NaOH/urea and electrohydrolysis pretreatments could effectively disrupt the lignin structure and increase the cellulose crystallinity of RS. Following pretreatment, RS was hydrolyzed by cellulase. After 96 h of enzymatic hydrolysis, NaOH/urea- and electrohydrolysis-pretreated RS produced 3.2- and 1.7-fold higher total reducing sugars than the unpretreated RS (232.95 ± 3.60 mg/g), respectively. Finally, the obtained RS hydrolysates were used for fermentative hydrogen production. NaOH/urea- and electrohydrolysis-pretreatment hydrolysates produced 125.0 and 163.0 mL H₂/g RS, respectively, which is much higher than the hydrogen yield of unpretreated hydrolysates.     

Advances towards the understanding of microbial communities in dark fermentation of enzymatic hydrolysates: Diversity,structure and hydrogen production performance

Microbial communities involved in hydrogen (H₂) production from enzymatic hydrolysates of agave bagasse were analyzed through 16S rRNA sequencing. Two types of reactor configurations and four different enzymatic hydrolysates were evaluated. Trickling bed reactors led to highly-diverse microbial communities, but low volumetric H₂ production rates (VHPR, maximum: 5.8 L H₂/L-d). On the contrary, well-controlled environments of continuous stirred-tank reactors favored the establishment of low diverse microbial communities composed by Clostridium-Sporolactobacillus leading to high-performance H₂-production (VHPR maximum: 13 L H₂/L-d). Cellulase-Viscozyme and Celluclast-Viscozyme hydrolysates led to the co-dominance of Clostridium and Sporolactobacillus, possibly due to the presence of xylose and hemicellulose-derived carbohydrates. Cellulase hydrolysates were linked to communities dominated by Clostridium, while maintaining low abundance of Sporolactobacillus. Stonezyme hydrolysates favored microbial communities co-dominated by Clostridium-Lachnoclostridium-Leuconostoc. Moreover, contrary to the prevailing theory, it was demonstrated that H₂ production performance was inversely related to microbial diversity.     

Enzymatic saccharification of pretreated rice straw by cellulase produced from Bacillus carboniphilus CAS 3 utilizing lignocellulosic wastes through statistical optimization

A marine bacterium, Bacillus carboniphilus CAS 3 was subjected to optimization for cellulase production utilizing cellulosic waste through response surface methodology. Plackett – Burman and Central composite design was employed and the optimal medium constituents for maximum cellulase production (4040.45 U/mL) were determined as rice bran, yeast extract, MgSO₄·7H₂O and KH₂PO₄ at 6.27, 2.52, 0.57 and 0.39 g/L, respectively. The cellulase produced was purified to the specific activity of 434.94 U/mg and 11.46% of recovery with the molecular weight of 56 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 50 °C, 9 and 30% and more than 70% of its original activity was retained even at 80 °C, 12 and 35% respectively. Further, enzymatic saccharification of pretreated rice straw yielded about 15.56 g/L of reducing sugar at 96 h, suggesting that the purified cellulase could be useful for production of reducing sugars from cellulosic biomass into ethanol.     

Evaluation of semi-continuous hydrogen production from enzymatic hydrolysates of Agave tequilana bagasse: Insight into the enzymatic cocktail effect over the co-production of methane

This work addresses the hydrogen production from enzymatic hydrolysates of Agave tequilana bagasse and the valorization of the acidogenic effluent for methane production in anaerobic sequencing batch reactors (ASBRs). Regarding hydrogen production, the ASBR was operated at four organic loading rates (OLRs), which were modified by decreasing the cycle time (from 24 to 12 h) and increasing the COD concentration (from 8 to 12 and 16 g L^?1). Results showed that the highest OLR promoted the highest hydrogen production rate of 25.2 ± 2.1 NmL L^?1 h^?1. Conversely, the hydrogen molar yield remained constant, obtaining similar values to the highest reported for lignocellulosic hydrolysates in continuous reactors (1.6H₂-mol mol_{consumed sugar}^?1). Regarding methane production from the acidogenic effluent, an unexpected methane suppression was observed during the first 5 cycles of the ASBR operation. Such event was attributed to the disaggregation of the granular sludge due to the remaining hydrolytic activity of the enzymatic cocktail used for the hydrolysates production. This was corroborated by feeding acetate to an ASBR (positive control) and supplying the enzymatic cocktail. Overall, even though the ASBR configuration demonstrated its suitability for hydrogen production, further studies are needed to coupling the methanogenic phase in different reactor configurations.     

Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H₂ production after pretreatment. However, the pretreatment methods have different influence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/cellusase. The hydrolysis rates increased from 45.51% by ddH₂O-treatment to 60.79% by diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H₂ production vary among different hydrolysates. Photo-fermentative H₂ production of strain R. capsulatus JL1 and mutant JL1601 (cheR2^-) with hydrolysates were investigated. The maximum H₂ yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H₂ yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2^-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2^-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH₂O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H₂ production for mutant JL1601 (cheR2^-) but not for strain JL1.     

Fermentative hydrogen production from hydrolyzed cellulosic feedstock prepared with a thermophilic anaerobic bacterial isolate

Hydrogen gas was produced via dark fermentation from natural cellulosic materials and α-cellulose via a two-step process, in which the cellulosic substrates were first hydrolyzed by an isolated cellulolytic bacterium Clostridium strain TCW1, and the resulting hydrolysates were then used as substrate for fermentative H₂ production. The TCW1 strain was able to hydrolyze all the cellulosic materials examined to produce reducing sugars (RS), attaining the best reducing sugar production yield of 0.65 g reducing sugar/g substrate from hydrolysis of α-cellulose. The hydrolysates of those cellulosic materials were successfully converted to H₂ via dark fermentation using seven H₂-producing bacterial isolates. The bioH₂ production performance was highly dependent on the type of cellulosic feedstock used, the initial reducing sugar concentration (C_RS,o) (ranging from 0.7 to 4.5 mg/l), as well as the composition of sugar and soluble metabolites present in the cellulosic hydrolysates. It was found that Clostridium butyricum CGS5 displayed the highest H₂-producing efficiency with a cumulative H₂ production of 270 ml/l from α-cellulose hydrolysate (C_RS,o = 4.52 mg/l) and a H₂ yield of 7.40 mmol/g RS (or 6.66 mmol/g substrate) from napier grass hydrolysate (C_RS,o = 1.22 g/l).     

Biohydrogen production from thermochemically pretreated corncob using a mixed culture bioaugmented with Clostridium acetobutylicum

Bovine ruminal fluid (BRF) bioaugmented with Clostridium acetobutylicum (Clac) was assessed for hydrolyzing cellulose and produce biohydrogen (BioH₂) simultaneously from pretreated corncob in a single step, without the use of external hydrolytic biocatalysts. The corncob was pretreated using three thermochemical methods: H₂SO₄ 2%, 160 °C; NaOH 2%, 140 °C; NaOCl 2%, 140 °C; autohydrolysis: H₂O, 190 °C. Subsequently, BioH₂ production was carried out using the pretreated material with the highest digestibility applying a Taguchi experimental array to identify the optimal operating conditions. The results showed a higher glucose released from pretreated corncob with H₂SO₄ (134.7 g/L) compared to pretreated materials by autohydrolysis, NaOH and NaOCl (123 g/L, 89.8 g/L and 52.9 g/L, respectively). The mixed culture was able to hydrolyze the pretreated corncob and produce 575 mL of H₂ (at 35 °C, pH 5.5, 1:2 ratio of BRF:Clac and 5% of solids loading) equivalent to 132 L H₂/Kg of biomass.     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Hydrogen and methane production potential of agave bagasse enzymatic hydrolysates and comparative technoeconomic feasibility implications

In the present work, agave bagasse enzymatic hydrolysates obtained with newly locally-available commercial enzymatic preparations were explored for their corresponding hydrogen and methane production potential in batch mode. The major levels in chemical oxygen demand and total carbohydrates were provided by enzymatic hydrolysates made with Zymapect and Stonezyme, respectively. Batch experiments demonstrated that Celluclast 1.5L achieves the maximum hydrogen productivity (1.88 L H₂/L), from 1.6 to 2.0-fold higher than other alternatives, whereas Zymapect attains the highest methane productivity (1.32 L CH₄/L), with high specific yield reached by both Stonezyme and Zymapect (162 and 163 L CH₄/kg bagasse), from 1.7 to 2.0-fold higher than other options. Finally, a preliminary techno-economic analysis allowed to elucidate that the cheapest alternatives for hydrogen and methane production at batch scale are Celluclast 1.5L and Stonezyme, respectively. Overall, the present analysis could serve as groundwork for the selection of the best enzymatic alternatives for hydrogen and methane production.     

Hydrogen production from acid and enzymatic oat straw hydrolysates in an anaerobic sequencing batch reactor: Performance and microbial population analysis

Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H₂/mol sugar consumed) and hydrogen production rate (HPR, 278 mL H₂/L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H₂/mol sugar consumed and 29.6 mL H₂/L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.     

Dilution strategy to obtain H2 in the 2nd acidogenic phase from enzymatically pre-treated sugarcane bagasse

Sugarcane bagasse (SCB) pre-treated with laccase was used as substrate in two acidogenic phases (1 P and 2 P). In 1 P, the pretreatment conditions were statistically optimized (pH 4.0, 37 °C, 0.4 U of laccase/mL, 24.0 g SCB/L and 120 min of reaction), which allowed obtaining 84% more H₂ (166.8 mL/L) than SCB in natura (90.4 mL/L). In 2 P, the liquid fraction of the 1 P reactors was diluted (90%) and more 108.5 ml H₂/L was obtained. Furthermore, the quantification of enzymes genes responsible for the hydrolysis (Cel) and H₂ production (Hyd) was carried out. An increase in the expression of both genes was observed from the phase of highest H₂ production rates in P1. In 2 P, the 1 P low genic expression was indicative of the autochthonous bacteria activity. The 2nd acidogenic phase strategy proved to be a promising novelty for the use of pre-hydrolyzed substrates with concomitant production of more value-added products.     

Identification of factors that accelerate hydrogen production by Clostridium butyricum RAK25832 using casamino acids as a nitrogen source

The ability of Clostridium butyricum RAK25832 to use casamino acids as a nitrogen source was investigated. Strain RAK25832 showed the capacity to utilize different types of carbon sources. With glucose as a carbon source (10 g/L), the preferred final concentration of casamino acids was 26.67 g/L, with a cumulative hydrogen production, production rate, and yield of 2505 mL H₂/L, 160 mL/h, and 1.81 mol H₂/mol glucose, respectively. Eighteen metal elements were screened to identify the most important metals for biohydrogen production, and four elements were optimized. The optimal medium composition was MgCl₂·6H₂O (0.1 g/L), K₂HPO₄·3H₂O (6.67 g/L), NaHCO₃ (2.6 g/L), and FeCl₂·4H₂O (0.002 g/L). Vitamin supplementation of the medium showed no significant effect on hydrogen production. Under the optimized conditions, cumulative hydrogen production reached 3074 mL H₂/L. This is the first study to demonstrate the use of casamino acids as a nitrogen source by C. butyricum.     

Sequential fermentation of hydrogen and methane from steam-exploded sugarcane bagasse hydrolysate

The goal of this study was to sequential fermentation of hydrogen and methane from sugarcane bagasse (SCB). Steam explosion conditions for pretreating SCB were optimum at 195 °C and 1.5 min, which yielded 36.35 g/L of total sugar and 2.35 g/L of total inhibitors. Under these conditions (all in g/L): glucose, 11.33; xylose, 24.41; arabinose, 0.61; acetic acid, 2.33; and furfural, 0.02 were obtained. The resulting hydrolysate was used to produce hydrogen by anaerobic mixed cultures. A maximum hydrogen production rate of 396.50 mL H₂/L day was achieved at an initial pH of 6 and an initial total sugar concentration of 10 g/L. The effluent from the hydrogen fermentation process was further used to produce methane. Response surface methodology with central composite design was used to obtain the suitable conditions for maximizing methane production rate (MPR). An MPR of 185.73 mL/L day was achieved at initial pH, Ni and Fe concentrations of 7.59, 3.61 mg/L and 8.44 mg/L, respectively. Total energy of 304.11 kJ/L-substrate was obtained from a sequential fermentation of hydrogen and methane. This approach will not only add value to SCB, in the form of safe and clean energy, but also provide a solution for making use of this abundant waste.     

Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration

Pre-heated elephant dung was used as inoculum to produce hydrogen from sugarcane bagasse (SCB) hydrolysate. SCB was hydrolyzed by H₂SO₄ or NaOH at various concentrations (0.25-5% volume) and reaction time of 60 min at 121 °C, 1.5 kg/cm² in the autoclave. The optimal condition for the pretreatment was obtained when SCB was hydrolyzed by H₂SO₄ at 1% volume which yielded 11.28 g/L of total sugar (1.46 g glucose/L; 9.10 g xylose/L; 0.72 g arabinose/L). The maximum hydrogen yield of 0.84 mol H₂/mol total sugar and the hydrogen production rate of 109.55 mL H₂/L day were obtained at the initial pH 6.5 and initial total sugar concentration 10 g/L. Hydrogen-producing bacterium (Clostridium pasteurianum) and non hydrogen-producing bacterium (Flavobacterium sp.) were dominating species in the elephant dung and in hydrogen fermentation broth. Sporolactobacillus sp. was found to be responsible for a low hydrogen yield obtained.     

Research on optimization and performance of Na2HPO4·12H2O mixture system combining fuzzy optimization model based on analytic hierarchy process

In order to determine optimal salt hydrates matching and adding fraction of Na₂HPO₄ · 12H₂O, crystal characteristics, latent heat of phase change, supercooling degree, and phase change temperature were studied. According to experimental data, optimal salt hydrates matching and adding fraction of Na₂HPO₄ · 12H₂O are determined by adopting multi-objective fuzzy optimization model based on analytic hierarchy process. Aimed at 3% Na₂SiO₃ · 9H₂O-Na₂HPO₄ · 12H₂O optimized by the method, thermal cycling performance test is implemented. Test results show the following: the Na₂SiO₃ · 9H₂O-Na₂HPO₄ · 12H₂O system has lower supercooling degree than Na₂HPO₄ · 12H₂O. It forms stable crystal after multiple thermal cycles, the phase separation is eliminated completely, and supercooling degree is 3.6°C. Latent heat of phase change maintains at 164 J/g after 100 thermal cycles. It is a modified intermediate product of salt hydrates with development potential.     

Consolidated bioprocessing of hydrogen production from agave biomass by Clostridium acetobutylicum and bovine ruminal fluid

The biological production of H₂ represents a renewable and eco-friendly energy alternative compared to fossil fuels. However, its production from lignocellulose involves the use of expensive enzymatic complexes. In the present work, the production of H₂ from pretreated agave biomass was evaluated by means of a Consolidated Bioprocess (CBP). This strategy was carried through the interaction of cellulose-degrading microorganisms obtained from bovine ruminal fluid (BRF) capable of enhancing H₂ production by Clostridium acetobutylicum. The results obtained show the capacity of BRF to hydrolyze the acid pretreated agave, improving the production of H₂ in the experiments where the inoculum of Clostridium was greater. According to the results, production of H₂ is significantly affected by the increase of the solids loading, obtaining a maximum H₂ production at a 10% of solids loading, pH 5.5 and 35 °C, representing a yield of 150 L of H₂ per Kg of biomass in 264 h.     

Sustainable fermentation approach for biogenic hydrogen productivity from delignified sugarcane bagasse

Improper lignocellulosic wastes management causes severe environmental pollution and health damage. Conversion of such wastes particularly sugarcane bagasse (SCB) onto bioenergy is a sustainable approach due to a continuous depletion of conventional biofuels. The delignification of SCB is necessary to proceed for bio-genic H₂ productivity by anaerobic bacteria. The effect of autoclaving, pre-acidification/autoclaving and pre-alkalization/autoclaving of SCB on glucose recovery and subsequently H₂ productivity by dark fermentation was comprehensively investigated. Pre-acidified SCB with 1% H₂SO₄ (v/v) provided H₂ productivity of 8.5 ± 0.14 L/kg SCB and maximum H₂ production rate (R_m) of 105.9 ± 8.3 mL/h. Those values were dropped to 2.7 ± 0.13 L/kg SCB and 58.3 ± 12.9 mL/h for fermentation of delignified SCB with 2% H₂SO₄. This was linked to high levels of total phenolic compounds (1775.3 ± 212 mg/L) in the feedstock. Better H₂ productivity of 13.9 ± 0.58 L/kg SCB and R_m of 133.9 ± 3.6 mL/h was achieved from fermentation of pre-alkalized SCB with 1%KOH (v/v). 256.8 ± 9.8 U/100 mL of α-amylase, 165.7 ± 7.6 U/100 mL of xylanase, 232.8 ± 6.1 U/100 mL of CM-Cellulase, 176.5 ± 5.0 U/100 mL of polyglacturanase and 0.702 ± 0.013 mg M B. reduced/min. of hydrogenase enzyme was accounted for the batches supplied with delignified SCB by KOH. The Clostridium and Bacillus spp. was dominance and prevalence resulting a higher H₂ productivity and yield. A novel strain of Archea and alpha proteobacterium were also identified and detected.     

Enhanced hydrogen production through co-cultivation of Chlamydomonas reinhardtii CC-503 and a facultative autotrophic sulfide-oxidizing bacterium under sulfurated conditions

Photoproduction of H₂ using microalgae has been considered as a promising approach for developing sustainable hydrogen energy. The algae C. reinhardtii CC-503 was co-cultured with a facultative autotroph sulfur-oxidizing bacterium Thuomonas intermedia BCRC 17547 to improve H₂ production. The maximum H₂ production of co-culture at sulfur deficiency conditions was 122 μmol/mg Chl with algae/bacteria ratio as 60:1, which was 2.8-fold higher than that of the pure algal culture. Na₂S₂O₃ treatment can result in a maximum H₂ photoproduction rate of 255 μmol/mg Chl, which was 5.9 and 2.1 times higher than those of pure algae culture and co-culture without Na₂S₂O₃. Co-cultivation under sulphate condition can also significantly increase the biomass, respiratory rate, starch content and hydrogenase activity of C. reinhardtii. By supplement of Na₂S₂O₃, persistent (52 days) H₂ production of bacteria/algae co-culture can be achieved. Our results demonstrated that co-culture of C. reinhardtii CC-503 and bacteria BCRC17547 is a cost-effective strategy for improving photobiological H₂ production.