Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C., reinhardtii

An anaerobic incubation period of varying duration is required to induce hydrogenase activity in $\text{C.}\text{,}\text{reinhardtii}$. Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.     

Hydrogenase activity of Chlorella vulgaris cells

Cell suspensions of Chlorella vulgaris were found to possess the hydrogenase activity as was confirmed by their ability to absorb H2 in the presence of benzyl viologen, azocarmine and other hydrogen acceptors as well as to produce H2 from reduced methyl viologen. Incubation of the cells in the dark under anaerobic conditions in the atmosphere of H2, N2 or Ar stimulated the activity of hydrogenase and induced its de novo synthesis. Treatment of the cells adapted to anaerobiosis with dry ice or liquid nitrogen considerably increased their hydrogenase activity. The enzyme of the adapted cells was more resistant to the inactivation by O2 and temperature.     

Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoenzyme composition in Salmonella typhimurium.

Hydrogen metabolism in Salmonella typhimurium is differentially regulated by mutations in the two anaerobic regulatory pathways, defined by the fnr (oxrA) and oxrC genes, and is controlled by catabolite repression. The synthesis of the individual hydrogenase isoenzymes is also specifically influenced by fnr and oxrC mutations and by catabolite repression in a manner entirely consistent with the proposed role for each isoenzyme in hydrogen metabolism. Synthesis of hydrogenase isoenzyme 2 was found to be fnr dependent and oxrC independent, consistent with a role in respiration-linked hydrogen uptake which was shown to be similarly regulated. Also in keeping with such a respiratory role was the finding that both hydrogen uptake and the expression of isoenzyme 2 are under catabolite repression. In contrast, formate hydrogenlyase-dependent hydrogen evolution, characteristic of fermentative growth, was reduced in oxrC strains but not in fnr strains. Hydrogenase 3 activity was similarly regulated, consistent with a role in hydrogen evolution. Unlike the expression of hydrogenases 2 and 3, hydrogenase 1 expression was both fnr and oxrC dependent. Hydrogen uptake during fermentative growth was also both fnr and oxrC dependent. This provided good evidence for a distinction between hydrogen uptake during fermentation- and respiration-dependent growth and for a hydrogen-recycling process. The pattern of anaerobic control of hydrogenase activities illustrated the functional diversity of the isoenzymes and, in addition, the physiological distinction between the two anaerobic regulatory pathways, anaerobic respiratory genes being fnr dependent and enzymes required during fermentative growth being oxrC dependent.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production.

Escherichia coli can perform at least two modes of anaerobic hydrogen metabolism and expresses at least two types of hydrogenase activity. Respiratory hydrogen oxidation is catalysed by two 'uptake' hydrogenase isoenzymes, hydrogenase -1 and -2 (Hyd-1 and -2), and fermentative hydrogen production is catalysed by Hyd-3. Harnessing and enhancing the metabolic capability of E. coli to perform anaerobic mixed-acid fermentation is therefore an attractive approach for bio-hydrogen production from sugars. In this work, the effects of genetic modification of the genes encoding the uptake hydrogenases, as well as the importance of preculture conditions, on hydrogen production and fermentation balance were examined. In suspensions of resting cells pregrown aerobically with formate, deletions in Hyd-3 abolished hydrogen production, whereas the deletion of both uptake hydrogenases improved hydrogen production by 37% over the parent strain. Under fermentative conditions, respiratory H2 uptake activity was absent in strains lacking Hyd-2. The effect of a deletion in hycA on H2 production was found to be dependent upon environmental conditions, but H2 uptake was not significantly affected by this mutation.     

Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria

Abstract The enzyme activities responsible for the evolution and consumption of hydrogen in three unicellular cyanobacteria were investigated. Gloeothece sp. 6909 and Cyanothece sp. 7822 performed an oxygen-tolerant nitrogen fixation, whereas the nitrogenase activity of Synechococcus sp. 7425 was much more sensitive to oxygen. While in Gloeothece the net hydrogen production during nitrogen fixation was relatively low due to recycling by an uptake hydrogenase, little hydrogen consumption was detected in Cyanothece and Synechococcu . On the other hand a reversible hydrogenase was demonstrated in the latter strains. However, only Cyanothece shows hydrogenase-catalysed hydrogen production in vivo under anaerobic conditions in the dark. It is suggested that hydrogen is a fermentation product, and that the physiological function of this reversible hydrogenase is the removal of excess reduction equivalents under such conditions.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D₂ uptake and in H₂ and HD evolution. In contrast to the nitrogenase-mediated H₂ production, this anaerobic H₂ and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H₂ and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D₂ is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.     

Hydrogen metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12.

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel.     

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

Correlation between respiration and hydrogenase adaptation in Scenedesmus obliquus

Full activity of hydrogenase is induced in autotrophic or heterotrophic Scenedesmus obliquus [strain D3 (Gaffron)] cells after 2 h under anaerobic adaptation. Semi-aerobic conditions allow only an induction of 20% activity. Hydrogenase is completely inactivated by oxygen in cell free preparations. Semi-aerobic adaptation of hydrogenase is enhanced by glucose and inhibited by sodium azide. It is concluded that respiration enhances hydrogenase activation by creating an oxygen free microclimate and by providing energy in the form of ATP.     

Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium.

We found that Salmonella typhimurium strain LT2 (Z) possessed two immunologically distinct, membrane-bound hydrogenase isoenzymes, which were similar in electrophoretic mobilities and apoprotein contents to hydrogenase isoenzymes 1 and 2 of Escherichia coli. The S. typhimurium enzymes cross-reacted with antibodies raised to the respective hydrogenase isoenzymes of E. coli. As for E. coli, an additional membrane-bound hydrogenase activity (termed hydrogenase 3), which did not cross-react with antibodies raised against either hydrogenase 1 or 2, was also present in detergent-dispersed membrane preparations. The physiological role of each of the three isoenzymes in E. coli has remained unclear owing to the lack of mutants specifically defective for individual isoenzymes. However, analysis of two additional wild-type isolates of S. typhimurium revealed specific defects in their hydrogenase isoenzyme contents. S. typhimurium LT2 (A) lacked isoenzyme 2 but possessed normal levels of hydrogenases 1 and 3. S. typhimurium LT7 lacked both isoenzymes 1 and 2 but retained normal hydrogenase 3 activity. Characterization of hydrogen metabolism by these hydrogenase-defective isolates allowed us to identify the physiological role of each of the three isoenzymes. Hydrogenase 3 activity correlated closely with formate hydrogenlyase-dependent hydrogen evolution, whereas isoenzyme 2 catalyzed hydrogen uptake (oxidation) during anaerobic, respiration-dependent growth. Isoenzyme 1 also functioned as an uptake hydrogenase but only during fermentative growth. We postulate that this enzyme functions in a hydrogen-recycling reaction which operates during fermentative growth.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Engineering the Rhizobium leguminosarum bv. viciae Hydrogenase System for Expression in Free-Living Microaerobic Cells and Increased Symbiotic Hydrogenase Activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

谷氨酸废液培养莱茵衣藻的产氢研究

通过在一般培养基中添加尿素、谷氨酸、葡萄糖等有机物,考察了菜茵衣藻混合营养培养的产氢效果。结果表明,含尿素混合营养培养菜茵衣藻产氢是含铵氮的培养基培养的6倍,无硫含尿素的混合营养培养具有最佳的产氢效果,是有硫含铵氮的培养基的10倍。谷氨酸及葡萄糖的添加对其生长和产氢也有促进作用,对菜茵衣藻生长和产氢促进作用的尿素的最佳添加量为0．25g/L,谷氨酸的最佳添加量为0．7g/L,葡萄糖的添加量为0．2g/L。     

Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme.

The cellular contents of the nickel-containing, membrane-bound hydrogenase isoenzymes 1 and 2 (hydrogenases 1 and 2) were analyzed by crossed immunoelectrophoresis. Their expression was differentially influenced by nutritional and genetic factors. Hydrogenase 2 content was enhanced after growth with either hydrogen and fumarate or glycerol and fumarate and correlated reasonably with cellular hydrogen uptake capacity. Hydrogenase 1 content was negligible under the above conditions but was enhanced by exogenous formate. Its expression was greatly reduced in a pfl mutant, which is unable to synthesise formate, but was restored to normal levels when the growth medium included formate. A mutation in the anaerobic regulatory gene, fnr, led to low overall hydrogenase activity and greatly reduced levels of both isoenzymes and abolished the formate enhancement of hydrogenase 1 content. Formate hydrogenlyase activity was similarly reduced in the fnr strain but, in contrast, was restored, as was overall hydrogenase activity, to normal levels by growth in the presence of formate. Low H2 uptake activity was found for the fnr strain under all growth conditions examined. Hydrogenase 1 content, therefore, does not correlate with formate hydrogenlyase activity and its role is unclear. A third hydrogenase isoenzyme, immunologically distinct from hydrogenases 1 and 2, whose expression is enhanced by formate, is present and forms part of the formate hydrogenlyase. We suggest that the effect of the fnr gene product on formate hydrogenlyase expression is mediated via internal formate.     

Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.     

Hydrogen production by <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revisited: Rubisco as a biotechnological target

Hydrogen production by C. reinhardtii seems a promising alternative as a source of non-polluting biofuel. Hydrogen is generated as a result of combining free protons and electrons (supplied by ferredoxin) through the activity of an oxygen-sensitive hydrogenase. Thus, substantial hydrogen production is only observed in the light under anaerobic conditions. These require a reduced rate of photosynthetic oxygen evolution which is usually achieved by impairing photosystem II through sulphur starvation. Several approaches have been conducted to enhance and extend hydrogen production by addressing problems such as the mechanism of hydrogenase inhibition by oxygen, the stressing impact on the cells of the culture conditions, the use of starch as an alternate source of electrons under reduced photosynthetic activity, and the need of maintaining a balance between oxygen evolution and consumption. The photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) appears as suitable objective for biotechnological optimization of hydrogen production because of its relevance controlling the hydrogenase main competitor electron sink (the Calvin-Benson cycle), as well as starch accumulation and photorespiratory oxygen consumption. Possible strategies for increasing hydrogen generation based on alteration of Rubisco properties and/or catabolism through site-directed mutagenesis are discussed.