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.     

Fermentative hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture

In this study, production of hydrogen (H₂) from glucose, xylose, galactose, mannose, arabinose and rhamnose by a strain isolated from activated sludge was investigated. The strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter amalonaticus strain SA01 (99%). Optimal cultivation parameters for H₂ production and growth such as pH and temperature were investigated. H₂ yields from glucose at optimal conditions (pH 6.0 and 34 °C) were 1.82 ± 0.02 mol-H₂/mol-glucose. Strain CMC-1 fermented galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation period lowered residual formate level in the media and improved H₂ production for galactose, mannose and xylose (1.68 ± 0.24, 1.93 ± 0.14 and 1.63 ± 0.07 mol-H₂/mol-substrate respectively).     

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).     

Fermentative hydrogen production by microbial consortium

Heat pre-treatment of the inoculum associated to the pH control was applied to select hydrogen-producing bacteria and endospores-forming bacteria. The source of inoculum to the heat pre-treatment was from a UASB reactor used in the slaughterhouse waste treatment. The molecular biology analyses indicated that the microbial consortium presented microorganisms affiliated with Enterobacter cloacae (97% and 98%), Clostridium sp. (98%) and Clostridium acetobutyricum (96%), recognized as H₂ and volatile acids' producers. The following assays were carried out in batch reactors in order to verify the efficiencies of sucrose conversion to H₂ by the microbial consortium: (1) 630.0 mg sucrose/L, (2) 1184.0 mg sucrose/L, (3) 1816.0 mg sucrose/L and (4) 4128.0 mg sucrose/L. The subsequent yields were obtained as follows: 15% (1.2 mol H₂/mol sucrose), 20% (1.6 mol H₂/mol sucrose), 15% (1.2 mol H₂/mol sucrose) and 4% (0.3 mol H₂/mol sucrose), respectively. The intermediary products were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors.     

Clostridium strain co-cultures for biohydrogen production enhancement from condensed molasses fermentation solubles

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.     

Co-culture of Clostridium beijerinckii L9, Clostridium butyricum M1 and Bacillus thermoamylovorans B5 for converting yeast waste into hydrogen

In order to enhance anaerobic hydrogen production from yeast waste, a series of 120-mL batch co-cultures of Clostridium beijerinckii L9, Clostridium butyricum M1, and Bacillus thermoamylovorans B5 under mesophilic conditions were established according to full factorial design (FFD) and mixture design (MD). The experimental results were subjected to multivariate and response surface analyses to determine the relationships between bacteria converting yeast waste into hydrogen. The results indicated clearly that C. beijerinckii L9 and C. butyricum M1 had significant potential to convert yeast waste into hydrogen. There was no significant hydrogen generation when B. thermoamylovorancs B5 alone was cultured with yeast waste. However, B. thermoamylovorancs B5 could significantly shorten the co-culture’s hydrogen-producing lag phase. Response surface analyses demonstrate that B. thermoamylovorancs B5 can stimulate the specific hydrogen production rate of C. beijerinckii L9 and C. butyricum M1, greater in the case of the former than of the latter. An ultimate hydrogen yield of 46 mL H₂/g COD added yeast waste was obtained with an optimal volumetric ratio C. beijerinckii L9: C. butyricum M1: B. thermoamylovoranc B5 of 8.9:4.8:10.3. Highly reproducible co-culture results confirm that FFD and MD, via response surface analysis, are applicable to assess the roles of the individual microorganisms in the defined co-culture.     

Microbial diversity of hydrogen-producing bacteria in batch reactors fed with cellulose using leachate as inoculum

Hydrogen production using cellulosic residues offers the possibility of waste minimization with renewable energy recovery. In the present study, heat-treated biomass purified from leachate was used as inoculum in batch reactors for hydrogen production fed with different concentrations of cellulose (2.5, 5.0 and 10 g/L), in the presence and absence of exogenous cellulase. The heat-treated biomass did not degrade cellulose and hydrogen production was not detected in the absence of cellulase. In reactors with cellulase, the hydrogen yields were 1.2, 0.6 and 2.3 mol H₂/mol of hydrolyzed cellulose with substrate degradation of 41.4, 28.4 and 44.7% for 2.5, 5.0 and 10 g/L cellulose, respectively. Hydrogen production potentials (P) varied from 19.9 to 125.9 mmol H₂ and maximum hydrogen production rates (R_m) were among 0.8–2.3 mmol H₂/h. The reactor containing 10 g/L of cellulose presented the highest P and R_m among the conditions tested. The main acid produced in reactors were butyric acid, followed by acetic, isobutyric and propionic acids. Bacteria similar to Clostridium sp. (98–99%) were identified in the reactors with cellulase. The heat-treated leachate can be used as an inoculum source for hydrogen production from hydrolyzed cellulose.     

Continuous thermophilic hydrogen production and microbial community analysis from anaerobic digestion of diluted sugar cane stillage

The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 °C using a mixture of sugar cane stillage and glucose at approximately 5000–5300 mg COD L⁻¹. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H₂ yields of 5.73 mmol g COD_added⁻¹ were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m⁻³ d⁻¹). The maximum volumetric H₂ production of 0.78 L H₂ h⁻¹ L⁻¹ was achieved using stillage as carbon source. In all operational phases, the H₂ average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacterium sp. and Clostridium sp. were identified.     

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).     

Statistical optimization of fermentative hydrogen production from xylose by newly isolated Enterobacter sp. CN1

Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett–Burman design, xylose, FeSO₄ and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box–Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO₄ 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 ± 65 ml H₂/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H₂/L xylose medium, 1102 ml H₂/L glucose medium and 977 ml H₂/L sucrose medium; the maximum hydrogen yield were 2.0 ± 0.05 mol H₂/mol xylose, 0.64 mol H₂/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose.     

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.     

Enhanced hydrogen production from xylose and bamboo stalk hydrolysate by overexpression of xylulokinase and xylose isomerase in Klebsiella oxytoca HP1

Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H₂/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H₂/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.     

Performance and composition of bacterial communities in anaerobic fluidized bed reactors for hydrogen production: Effects of organic loading rate and alkalinity

This study evaluated the effects of the organic loading rate (OLR) and pH buffer addition on hydrogen production in two anaerobic fluidized bed reactors (AFBRs) operated simultaneously. The AFBRs were fed with glucose, and expanded clay was used as support material. The reactors were operated at a temperature of 30 °C, without the addition of a buffer (AFBR1) and with the addition of a pH buffer (AFBR2, sodium bicarbonate) for OLRs ranging from 19.0 to 140.6 kg COD m⁻³ d⁻¹ (COD: chemical oxygen demand). The maximum hydrogen yields for AFBR1 and AFBR2 were 2.45 and 1.90 mol H₂ mol⁻¹ glucose (OLR of 84.3 kg COD m⁻³ d⁻¹), respectively. The highest hydrogen production rates were 0.95 and 0.76 L h⁻¹ L⁻¹ for AFBR1 and AFBR2 (OLR of 140.6 kg COD m⁻³ d⁻¹), respectively. The operating conditions in AFBR1 favored the presence of such bacteria as Clostridium, while the bacteria in AFBR2 included Clostridium, Enterobacter, Klebsiella, Veillonellaceae, Chryseobacterium, Sporolactobacillus, and Burkholderiaceae.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Improvement of fermentative biohydrogen production by Clostridium butyricum CWBI1009 in sequenced-batch,horizontal fixed bed and biodisc-like anaerobic reactors with biomass retention

A horizontal tubular fixed bed bioreactor (HFBR) and an anaerobic biodisc-like reactor (AnBDR) were designed to both fix Clostridium biomass and enable rapid transfer of the hydrogen produced to gas phase in order to decrease the strong effect of H₂ partial pressure and H₂ supersaturation on the performances of Clostridium strains. The highest H₂ production rate (703 mL H₂/L h) and yield (302 mL/g glucose consumed i.e. 2.4 mol/mol) with the pure culture were recorded in the AnBDR with 300 mL culture medium (total volume 2.3 L) at pH 5.2 and a glucose loading rate of 2.87 g/L h. These results are about 2.3 and 1.3-fold higher than those achieved in the same bioreactor with 500 mL liquid medium and with the same glucose consumption rate. Therefore, our experimentations and a short review of the literature reported in this paper emphasize the relevance of performing bioreactors with high L/G transfer.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

Development and application of a functional CE-SSCP fingerprinting method based on [Fe–Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures

A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe–Fe]-hydrogenase genes was developed for monitoring the hydrogen (H₂)-producing clostridial population in mixed-culture bioprocesses. New non-degenerated primers were designed and then validated on their specific PCR detection of a broad range of clostridial hydA genes. The hydA-based CE-SSCP method gave a specific and discriminating profile for each of the Clostridium strains tested. This method was validated using H₂-producing mixed cultures incubated at temperatures ranging from 25 °C to 45 °C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H₂ production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H₂ production yields. The highest performances were related to the dominance of Clostridium sporogenes-like hydA sequences. This CE-SSCP tool offers highly reliable and throughput analysis of the functional diversity and structure of the hydA genes for better understanding of the H₂-producing clostridial population dynamics in H₂ dark fermentation bioreactors.     

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.     

Statistical optimization of medium components affecting simultaneous fermentative hydrogen and ethanol production from crude glycerol by thermotolerant Klebsiella sp. TR17

A thermotolerant Klebsiella sp. TR17 for production of hydrogen from crude glycerol was investigated. Results from Plackett–Burman design indicated that the significant variables, which influenced hydrogen production, were KH₂PO₄ and NH₄Cl (for buffer capacity and nitrogen source). Subsequently, the two selected variables and crude glycerol were optimized by the Central Composite design for achieving maximum hydrogen and ethanol yield. The concentration of crude glycerol, KH₂PO₄, and NH₄Cl had an individual effect on both hydrogen and ethanol yield (p < 0.05), while KH₂PO₄ and NH₄Cl had an interactive effect on ethanol yield (p < 0.05). The optimum medium components were 11.14 g/L crude glycerol, 2.47 g/L KH₂PO₄, and 6.03 g/L NH₄Cl. The predicted maximum simultaneous hydrogen and ethanol yield were 0.27 mol H₂/mol glycerol and 0.63 mol EtOH/mol glycerol, respectively. Validation of the predicted optimal conditions exhibited similar hydrogen and ethanol yield of 0.26 mol H₂/mol glycerol and 0.58 mol EtOH/mol glycerol, respectively.     

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.