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.     

Genome based metabolic flux analysis of Ethanoligenens harbinense for enhanced hydrogen production

Ethanoligenens harbinense is a promising hydrogen producing microorganism due to its high inherent hydrogen production rate. Even though the effect of media optimization and inhibitory metabolites has been studied in order to improve the hydrogen productivity of these cultures, the identification of the underlying causes of the observed changes in productivity has not been targeted to date. In this work we present a genome based metabolic flux analysis (MFA) framework, for the comprehensive study of E. harbinense in culture, and the effect of inhibitory metabolites and media composition on its metabolic state. A metabolic model was constructed for E. harbinense based on its annotated genome sequence and proteomic evidence. This model was employed to perform MFA and obtain the intracellular flux distribution under different culture conditions. These results allow us to identify key elements in the metabolism that can be associated to the observed production phenotypes, and that can be potential targets for metabolic engineering in order to enhanced hydrogen production in E. harbinense.     

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.     

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.     

Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented.Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design.Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen.Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB.All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.     

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.     

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Escherichia coli can produce H₂ from glucose via formate hydrogen lyase (FHL). In order to improve the H₂ production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H₂ production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H₂ production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H₂ yield; 1.80 mol/mol glucose under high H₂ pressure or 2.11 mol/mol glucose under reduced H₂ pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdABdirected most of the carbons from glucose to the glycolytic pathway leading to H₂ production by FHL, not to the pentose phosphate pathway.     

A high yield mutant of Chlamydomonas reinhardtii for photoproduction of hydrogen

To identify genes important for the photoproduction of H₂ of Chlamydomonas reinhardtii, random insertional mutants, by the ble gene encoding the enzyme with zeomycin resistant properties, were screened for clones with changed H₂ production. One of the mutants, denoted T1, with 7.8-fold of H₂ yield and about 23% of respiration rate increased compared with the parental strain cc849 was obtained among the zeomycin-resistant transformants. The photosynthetic rate and Fv/Fm, the efficiency of PSII primary photochemistry of T1 declined. Therefore, the photosynthesis/respiration capacity ratio (P/R ratio) of T1 was reduced compared with strain cc849. However, the growth of the mutant T1 was not inhibited, indicating that T1 was a good mutant for further studies of the genes improving H₂ yield by lowering the photosynthetic capacity and/or enhancing the respiration capacity in C. reinhardtii, and had potential to be used in further improvement of H₂ generation by further biotechnological approaches.     

How bacteria get energy from hydrogen: a genetic analysis of periplasmic hydrogen oxidation in Escherichia coli

Dihydrogen oxidation is an important feature of bacterial energy conservation. In Escherichia coli hydrogen oxidation (‘uptake’) is catalysed by membrane-bound [NiFe] hydrogenase-1 and [NiFe] hydrogenase-2. The bulk of these uptake isoenzymes is exposed to the periplasm and biosynthesis of the proteins involves membrane transport via the twin-arginine translocation (Tat) pathway. Hydrogenase-2 is encoded by the hybOABCDEFG operon and the core enzyme is a heterodimer of HybO and HybC. HybO is synthesised with a twin-arginine signal peptide. HybOC is associated with two other proteins (HybA and HybB) that complete the respiratory complex. The HybOC dimer is bound to the cytoplasmic membrane and appears to be anchored via a hydrophobic transmembrane α-helix located at the C-terminus of HybO. Thus, hydrogenase-2 is an example of an integral membrane protein assembled in a Tat-dependent (Sec-independent) manner. Studies of the biosynthesis, targeting, and assembly of hydrogenase-2 would set a paradigm for all respiratory complexes of this type.     

Hydrogen production from a carbon-monoxide oxidation pathway in Rubrivivax gelatinosus

Upon feeding CO to the gas phase of a photosynthetic bacterium Rubrivivax gelatinosus CBS, a CO oxidation: H₂ production pathway is quickly induced. Hydrogen is produced according to the equation CO+H₂O→CO₂+H₂. Two enzymes are known to be involved in this pathway: a CO dehydrogenase (CODH) with a pH optimum of 8.0 and above, and a hydrogenase with a pH optimum near 7.5. Carbon monoxide dehydrogenase also displays a temperature optimum near 50°C. When CO mass transfer is not limited during a CO uptake measurement, an extreme fast rate of CO uptake was determined, allowing for the removal of near 87% of the dissolved CO from a bacterial suspension within 10 s. This process has therefore two potential applications, one in the production of H₂ gas as a clean renewable fuel using the linked CO oxidation: H₂ production pathway, and another in using the CODH enzyme itself as a fuel–gas conditioning catalyst. These applications thereby will improve the overall H₂ economy when gasified waste biomass serves as the inexpensive feedstock.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.     

Improvement of hydrogen production by over-expression of a hydrogen-promoting protein gene in Enterobacter cloacae

The gene of a hydrogen-promoting protein (which we term HPP) from Enterobacter cloacae IIT-BT 08 was cloned and over-expressed in E. cloacae CICC10017 for the first time in this study, and the overall hydrogen yield was greatly improved using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into a hydrogen producing strain of E. cloacae CICC10017 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the GST-tagged protein. An in vitro assay of cell lysates indicates hydrogenase activity of the recombinant strain is 534.78 ± 18.51 ml/(g-DW·h), nearly 2-fold higher than the wild strain. The hydrogen yield of the recombinant strain is 2.55 ± 0.1 mol/mol-glucose, also 2-fold higher than the wild strain. The recombinant strain produces more acetate and butyrate during hydrogen fermentation, but less ethanol, due to the higher hydrogenase activity with the over-expression of the hydrogen-promoting protein. Together, the results demonstrate that successful expression of a single structural gene improves the overall yield of hydrogen by directing metabolic fluxes away from formation of products that compete for NADH.     

Fermentative hydrogen production with xylose by Clostridium and Klebsiella species in anaerobic batch reactors

Hydrogen production was obtained from low concentrations of xylose metabolized by heat treated inoculum obtained from the slaughterhouse wastewater treatment UASB reactor installed in Brazil. The molecular biological analysis Clostridium and Klebsiella species, recognized as H₂ and volatile acid producers, in addition to Burkholderia species and uncultivated bacteria. The assays were carried out in batch reactors: (1) 630.0 mg xylose/L, (2) 1341.0 mg xylose/L, (3) 1848.0 mg xylose/L and (4) 3588.0 mg xylose/L. The following yields were obtained: 3% (0.2 mol H₂/mol xylose), 8% (0.5 mol H₂/mol xylose), 10% (0.6 mol H₂/mol xylose) and 14% (0.8 mol H₂/mol xylose), respectively. The end products obtained were acetic acid, butyric acid, methanol and ethanol in all of the anaerobic reactors. The concentrations of xylose did not inhibit microbial growth and hydrogen production. This suggested that low concentrations of xylose should be added to wastewater to produce hydrogen.     

Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: Effect of pH and glucose concentrations

Fermentative hydrogen production by strict anaerobes has been widely reported. There is a lack of information related to metabolic flux distribution and its variation with respect to fermentation conditions in the metabolic production system. This study aimed to get a better understanding of the metabolic network and to conduct metabolic flux analysis (MFA) of fermentative hydrogen production by a recently isolated Clostridium butyricum strain W5. We chose the specific growth rate as the objective function and used specific H₂ production rate as the criterion to evaluate the experimental results with the in silico MFA. For the first time, we constructed an in silico metabolic flux model for the anaerobic glucose metabolism of C. butyricum W5 with assistance of a modeling program MetaFluxNet. The model was used to evaluate metabolic flux distribution in the fermentative hydrogen production network, and to study the fractional flux response to variations in initial glucose concentration and operational pH. The MFA results suggested that pH has a more significant effect on hydrogen production yield compared to the glucose concentration. The MFA is a useful tool to provide valuable information for optimization and design of the fermentative hydrogen production process.