A newly isolated green alga, Tetraspora sp. CU2551, from Thailand with efficient hydrogen production

A novel unicellular hydrogen-producing green alga was isolated from fresh water pond in Pathumthani province, Thailand. Under light microscope, this alga was identified as belonging to the genus Tetraspora. Phylogenetic analysis of 18S rRNA sequence revealed that the green alga, identified as Tetraspora sp. CU2551, is closely related to other unicellular green algal species. Tetraspora sp. CU2551 had the shortest doubling time when grown in Tris-acetate-phosphate (TAP) medium under a light intensity of 48–92 μE/m²/s and a temperature of 36 °C. Hydrogen production increased with increasing pH from 5.75 to 9.30; however, almost no production was observed at a pH of 5.25. Addition of 0.5 mM β-mercaptoethanol to the TAP medium stimulated hydrogen production about two-fold. During the hydrogen production phase, the use of TAP medium lacking both nitrogen and sulfur resulted in about 50% increase in the hydrogen production. This was in contrast to only a small increase in the production when either nitrogen or sulfur was omitted in TAP medium. The stimulation of hydrogen production by 0.5 mM β-mercaptoethanol under nitrogen- and sulfur-deprived conditions occurred only when the cells were grown at a light intensity lower than 5 μE/m²/s with no effects at higher intensities. Maximal calculated hydrogen production, 17.3–61.7 μmol/mg Chl a/h, is a very high production rate compared to other green algae and makes Tetraspora sp. CU2551 an interesting model strain for photobiological hydrogen production.     

Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii

The direct relationship between hydrogenase gene conformation and its function in green alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the gene region is present.     

Enhanced H2 photoproduction by down-regulation of ferredoxin-NADP reductase (FNR) in the green alga Chlamydomonas reinhardtii

Renewable H₂ photoproduction by green algae such as Chlamydomonas reinhardtii is a promising system for solar fuels. However, large-scale application of the system has lagged virtually due to lack of high H₂-producing strains. We previously identified ferredoxin-NADP⁺ reductase (FNR) among the 105 proteins differentially expressed in Chlamydomonas during sulfur-deprived H₂ photoproduction. In this work, we used an RNA interference (RNAi) approach to generate Chlamydomonas mutant strains with reduced levels of FNR. We found that fnr-RNAi strains exhibited higher rates of H₂ photoproduction (2.5-fold) than wild type under sulfur-deprived condition. To elucidate the basis for this increase, we analyzed the physiological characteristics of the fnr-RNAi strains under such condition. Major changes, due to the down-regulation of FNR, included the lower rates of photosynthetic O₂ evolution (44%), greater reduction of Rubisco amounts (60%) and higher rates of starch degradation (140%). These may result in an earlier onset of anaerobiosis and increased electron supply to the hydrogenases in the mutant strains. The results provide new information of FNR in regulating H₂ metabolism as well as potential strains for further improvement of the organism toward application in solar-powered systems.     

Enhancement of hydrogen production by the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005

The efficiency of hydrogen production by different cyanobacterial species depends on several external factors. We report here the factors enhancing hydrogen production by filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Cells adapted to dark-anaerobic conditions produced hydrogen consistent with increased hydrogenase activity when supplemented with Fe²⁺. Stimulation of hydrogen production could be achieved by addition of reductants, either dithiothreitol or β-mercaptoethanol with higher production observed with the latter. Additionally, Fe²⁺ and β-mercaptoethanol added to nitrogen- and sulphur-deprived cells significantly stimulated H₂ production with maximal value of 5.91 ± 0.14 μmol H₂ mg Chla⁻¹ h⁻¹. Glucose and a small increase of osmolality imposed by either NaCl or sorbitol enhanced hydrogen production. High rates of hydrogen production were obtained in cells adapted in nitrogen-deprived medium with neutral and alkaline external pH, significant decrease of hydrogen production occurred under acidic external pH.     

Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii

Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.     

Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii cultures: The effect of the H2 partial pressure

Photoproduction of H₂ gas has been examined in sulfur/phosphorus-deprived Chalmydomonas reinhardtii cultures, placed in photobioreactors (PhBRs) with different gas phase to liquid phase ratios (V_g.p./V_l.p.). The results demonstrate that an increase in the ratio stimulates H₂ photoproduction activity in both algal suspension cultures and in algae entrapped in thin alginate films. In suspension cultures, a 4× increase (from ∼0.5 to ∼2) in V_g.p./V_l.p results in a 2× increase (from 10.8 to 23.1 mmol l⁻¹ or 264–565 ml l⁻¹) in the total yield of H₂ gas. Remarkably, 565 ml of H₂ gas per liter of the suspension culture is the highest yield ever reported for a wild-type strain in a time period of less than 190 h. In immobilized algae, where diffusion of H₂ from the medium to the PhBR gas phase is not affected by mixing, the maximum rate and yield of H₂ photoproduction occur in PhBRs with V_g.p./V_l.p above 7 or in a PhBR with smaller headspace, if the H₂ is effectively removed from the medium by continuous flushing of the headspace with argon. These experiments in combination with studies of the direct inhibitory effect of high H₂ concentrations in the PhBR headspace on H₂ photoproduction activity in algal cultures clearly show that H₂ photoproduction in algae depends significantly on the partial pressure of H₂ (not O₂ as previously thought) in the PhBR gas phase.     

Aquifex aeolicus membrane hydrogenase for hydrogen biooxidation: Role of lipids and physiological partners in enzyme stability and activity

Hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus is a good candidate for biotechnological devices thanks to its ability to oxidize hydrogen at high temperature, even in the presence of oxygen and CO. In order to enhance the enzyme stability and the catalytic efficiency, we investigated the hydrogen oxidation process with hydrogenase I embedded in a physiological-like environment. Hydrogenase I partners in the metabolic chain, namely membrane quinone and cytochrome b, were purified and fully characterized. The complex hydrogenase I–cytochrome b was inserted into liposomes. Surface Plasmon Resonance revealed that quinone took part in the stabilization of the complex. By use of molecular modelization and electrochemistry analysis, enzyme stability has been demonstrated to be stronger and enzymatic efficiency to be five times higher when hydrogenase is embedded into the liposomes. This result raises the possibility of using hydrogenases as biocatalysts in fuel cells.     

Fermentative hydrogen production by a new alkaliphilic Clostridium sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay,New Caledonia

The hydrogen-producing strain PROH2 pertaining to the genus Clostridium was successfully isolated from a shallow submarine hydrothermal chimney (Prony Bay, New Caledonia) driven by serpentinization processes. Cell biomass and hydrogen production performances during fermentation by strain PROH2 were studied in a series of batch experiments under various conditions of pH, temperature, NaCl and glucose concentrations. The highest hydrogen yield, 2.71 mol H₂/mol glucose, was observed at initial pH 9.5, 37 °C, and glucose concentration 2 g/L, and was comparable to that reported for neutrophilic clostridial species. Hydrogen production by strain PROH2 reached the maximum production rate (0.55 mM-H₂/h) at the late exponential phase. Yeast extract was required for growth of strain PROH2 and improved significantly its hydrogen production performances. The isolate could utilize various energy sources including cellobiose, galactose, glucose, maltose, sucrose and trehalose to produce hydrogen. The pattern of end-products of metabolism was also affected by the type of energy sources and culture conditions used. These results indicate that Clostridium sp. strain PROH2 is a good candidate for producing hydrogen under alkaline and mesothermic conditions.     

The enhancement mechanism of hydrogen photoproduction in Chlorella protothecoides under nitrogen limitation and sulfur deprivation

The aim of this study was to understand the enhancement mechanism of H₂ photoproduction in Chlorella protothecoides under simultaneous nitrogen limitation and sulfur deprivation (LNS). Nitrogen limitation (LN) rather than sulfur deprivation significantly inhibited relative variable fluorescence at K-step (W_K) and J-step (V_J), photochemical efficiency of PSII (photosystem II), F_v/F_m, during the process of incubation in the light. Under such conditions, photosynthetic O₂ evolution decreased and the anaerobiosis was established after 12 h of incubation. The algae generated large amounts of H₂ under nitrogen limitation but generated only trace amounts under sulfur deprivation. Obviously, nitrogen limitation rather than sulfur deprivation was the decisive factor that induced H₂ photoproduction in C. protothecoides under LNS. The LNS culture generated much more H₂ than the LN culture in the presence of DCMU during incubation, suggesting that a PSII-independent electron source contributed many more electrons for transfer to hydrogenase in the LNS culture. PSII electron transport includes linear electron flow (LEF) and cyclic electron flow (CEF) of PSII in C. protothecoides. In the PSII-dependent electron source for H₂ photoproduction, PSII supplies electrons to hydrogenase through the LEF. The LNS culture showed much higher LEF and lower CEF than the LN culture during the H₂ photoproduction phase, as indicated by the large lower quantum yield of PSII electron transport (Φ_PSII) in the LNS culture in the presence of DCMU. Therefore, compared with nitrogen limitation, simultaneous nitrogen limitation and sulfur deprivation enhanced H₂ photoproduction in C. protothecoides mainly due to enhanced PSII-dependent and -independent electron sources.     

Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii

Sulfur deprivation, which is considered as an efficient way to trigger long-term hydrogen photoproduction in unicellular green algae has two major effects: a decrease in PSII which allows anaerobiosis to be reached and carbohydrate (starch) storage. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in the green alga Chlamydomonas reinhardtii, little remains known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify those steps likely to limit the starch-dependent hydrogen production, we have designed a genetic screening procedure aimed at isolating mutants of the green alga C. reinhardtii affected in starch mobilization. Using two different screening protocols, the first one based on aerobic starch degradation in the dark and the second one on anaerobic starch degradation in the light, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std1-8) with the first screen and ten (sda1-10) with the second. Most of the mutant strains isolated in this study showed a reduction or a delay in the PSII-independent hydrogen production. Further characterization of these mutants should allow the identification of molecular determinants of starch-dependent hydrogen production and supply targets for future biotechnological improvements.     

Biohydrogen production from cellobiose in phenol and cresol-containing medium using Clostridium sp. R1

Cellobiose fermentation in batch test using an isolated strain, Clostridium sp. R1, was investigated. The Clostridium sp. R1 achieved a maximum hydrogen yield of 3.5 mol H₂ mol⁻¹ cellobiose at pH 6 and 30 °C, higher than most yields reported in literature. This strain can generate hydrogen from a number of carbohydrates, including galactose, glucose, mannose, maltose, sucrose, and starch. This strain can also convert cellobiose to hydrogen in the presence of toxic phenol or cresol. The inhibition effects of phenolic compounds on strain R1 activity followed phenol > p-cresol > o-cresol > m-cresol. Co-culturing with another strain, Clostridium butyricum, can co-degrade some of the phenol as substrates. The new isolated strain can yield hydrogen from phenol-containing wastewaters.     

Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor

The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of downcomer and riser (A_d/A_r) in range of 0.4–3.2 were considered. Maximum biomass concentration of 1.63 g L⁻¹ in 9 d under light intensity of 120 μE m⁻² s⁻¹ was observed at A_d/A_r of 1.6. The mixing time of the reactors was inversely proportional to A_d/A_r. Maximal H₂ production was found to be 1600 mL L⁻¹ upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L⁻¹), autoclave (600 mL L⁻¹) and HCl treatment (1230 mL L⁻¹). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L⁻¹ as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H₂ production.     

Occurrence and characterisation of the hydrogen-evolving enzyme in Frankia sp.

An increase in hydrogen evolution from the hydrogen-evolving enzyme in the actinomycete Frankia was recorded in the presence of nickel. Immunogold localisation analysis of the intracellular distribution of hydrogenase proteins indicated that they were evenly distributed in the membranes and cytosol of both hyphae and vesicles. In addition, molecular characterisation of the hydrogen-evolving enzyme at the proteomic level, using two-dimensional gel electrophoresis combined with mass spectrometry, confirmed that the Frankia hydrogen-evolving enzyme is similar to the cyanobacterial bidirectional hydrogenase of Anabena siamensis.     

Response of H2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

The effects of external factors on both H₂ production and bidirectional Hox-hydrogenase activity were examined in the non-N₂-fixing cyanobacterium Synechocystis PCC 6803. Exogenous glucose and increased osmolality both enhanced H₂ production with optimal production observed at 0.4% and 20 mosmol kg⁻¹, respectively. Anaerobic condition for 24 h induced significant higher H₂ase activity with cells in BG11₀ showing highest activities. Increasing the pH resulted in an increased Hox-hydrogenase activity with an optimum at pH 7.5. The Hox-hydrogenase activity gradually increased with increasing temperature from 30 ^○C to 60 ^○C with the highest activity observed at 70 ^○C. A low concentration at 100 μM of either DTT or β-mercaptoethanol resulted in a minor stimulation of H₂ production. β-Mercaptoethanol added to nitrogen- and sulfur-deprived cells stimulated H₂ production significantly. The highest Hox-hydrogenase activity was observed in cells in BG11₀-S-deprived condition and 750 μM β-mercaptoethanol measured at a temperature of 70 °C; 14.32 μmol H₂ mg chl a⁻¹ min⁻¹.     

Treatment with moderate concentrations of NaHSO3 enhances photobiological H2 production in the cyanobacterium Anabaena sp. strain PCC 7120

In cyanobacteria, treatment with low concentrations of NaHSO₃ can enhance photosynthetic efficiency, whereas NaHSO₃ in high amounts often inhibits cell growth and photosynthesis may even cause death. In the present study, our results showed that treatment with moderate concentrations of NaHSO₃ considerably improved the yield of photobiological H₂ production in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. Under steady state conditions, the accumulated H₂ levels in cells treated with 1 mM NaHSO₃ were approximately 10 times higher than that in untreated cells. Such improvement occurred in heterocysts and was most likely caused by increases in the expression and activity of nitrogenase. The effects of treatment with low, moderate, and high concentrations of NaHSO₃ in cyanobacteria were proposed on the basis of the results obtained in the present study and from previous knowledge.     

Solar disinfection of wild Salmonella sp. in natural water with a 18 L CPC photoreactor: Detrimental effect of non-sterile storage of treated water

For the first time solar disinfection of liters of water containing wild Salmonella sp. and total coliforms was carried out in a compound parabolic collector (CPC) photoreactor at temperatures of almost 50 °C. Using surface water with high turbidity, this treatment was efficient in completely inactivating Salmonella sp. without regrowth during the subsequent 72 h of dark sterile storage. However if the solar treated water is poured in a non- sterile container, bacteria regrowth occurs even if 10 mg L⁻¹ of H₂O₂ is added before the storage. On the other hand, 30 mg L⁻¹ of H₂O₂ added when the irradiation started was completely depleted within 2 h and did not prevent bacterial regrowth during post-irradiation storage in non-sterile containers, demonstrating that storage of large volumes of water treated by solar irradiation was not optimal. Finally, total coliforms (Escherichia coli included) showed a far higher sensitivity than Salmonella sp. and demonstrated to be an inappropriate indicator for monitoring bacterial contamination in water during solar disinfection processes.