Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H₂ photoproduction. However, a mutation in a structural hup gene did not result in maximum H₂ production rates, indicating that the capacity to recycle H₂ was not completely lost. Highest H₂ production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H₂ recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Optimizing photoheterotrophic H2 production by Rhodobacter capsulatus upon interposon mutagenesis in the hupL gene

In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H₂ by activity of the nitrogenase enzyme complex. When grown with glutamate as an N source and either d,l-malate or l-lactate as carbon sources, the efficiency of H₂ production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibited efficiencies of 54% (with d,l-malate) and 64% (with l-lactate), respectively. With NH _inf4 ^sup+ as the N source, efficiencies of H₂ production were 70% (mutant) and 52% (wild type). Correspondence to: J. Oelze     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H₂ evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H₂ or ³H₂ in the presence of acetylene. In all cases, the rate of H₂ evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H₂ evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup⁻ strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H₂ evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup⁻ strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H₂ evolution by Hup⁺ strains were similar in magnitude to their H₂-saturated rates of Hup activity. The curvilinear relationship between H₂ evolution and acetylene reduction for a representative Hup⁺ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.     

Beneficial Effects of Nickel on Pseudomonas saccharophila under Nitrogen-Limited Chemolithotrophic Conditions

Addition of nickel stimulated growth and nitrogenase activity of Pseudomonas saccharophila under nitrogen-limited chemolithotrophic conditions, apparently because of a significant increase in expression of uptake hydrogenase activity. Inhibition of hydrogenase expression by 50 μM EDTA was relieved by nickel over a wide concentration range (1 to 200 μM). Co²⁺, Zn²⁺, Mn²⁺, and Cu²⁺ stimulated expression of hydrogenase activity, but to a much lesser degree than nickel, and Fe²⁺, Mg²⁺, SeO₄²⁻, and SeO₃²⁻ did not increase expression. Nickel in individual combination with Mg²⁺, Fe²⁺, SeO₃²⁻, and SeO₄²⁻ resulted in activities that were essentially the same as that with nickel alone. Hydrogenase synthesis required the presence of nickel, and repression by O₂ was alleviated by increasing the concentration of added nickel. Cells placed under hydrogenase derepression conditions showed progressive incorporation of radioactive nickel to a much greater extent than did cells which were not derepressed.     

Effect of anaerobiosis on photosynthetic reactions and nitrogen metabolism of algae with and without hydrogenase

Summary The effect of anaerobic (N₂+CO₂) pre-incubation in the dark on photosynthetic reactions (O₂ evolution, measured manometrically and with the oxygraph; fluorescence; and photoproduction of H₂, measured with the mass spectrometer) was studied in algae with hydrogenase (strains of Chlorella fusca, C. kessleri, C. vulgaris f. tertia, and Ankistrodesmus braunii) and in algae without hydrogenase (strains of Chlorella vulgaris, C. saccharophila, and C. minutissima).The inhibition by anaerobic incubation of photosynthetic O₂ evolution is much stronger in algae without hydrogenase than it is in algae with hydrogenase. The effect of anaerobiosis is most pronounced at rather low light intensity (about 1000 lux), in acid medium (pH 4), and after prolonged anaerobic incubation in the dark (about 20 h). These results indicate that the presence of hydrogenase might be ecologically advantageous for algae under certain conditions.Chlorophyll fluorescence showed the fastest response to anaerobic incubation, and the most pronounced difference between algae with and without hydrogenase. After only 30 min under N₂+CO₂, fluorescence in algae with hydrogenase starts with a peak and decreases within 10 to 20 sec to a rather low steady-state level which is only slightly higher than that found under aerobic conditions. In algae without hydrogenase, fluorescence is rather low during the first 1 to 2 sec and then rises to a higher steady-state level which is much higher than that of the aerobic controls. This indicates an inhibition due to anaerobiosis of photosystem II in algae without hydrogenase.Algae with hydrogenase can react in different ways during the first minutes of illumination. In some cases there is an immediate photoproduction of H₂, which is followed after a few minutes by photosynthetic O₂ evolution; in other algae there is a simultaneous production of H₂ and O₂ from the very beginning; in a few experiments there was no photoproduction of H₂ at all, and in this case there was no photosynthetic O₂ evolution either. Thus, photoproduction of H₂ seems to be the process which normally enables algae with hydrogenase to oxidise and thereby activate their photosynthetic electron transport system after anaerobic incubation.A mass spectrometric search for nitrogen fixation (using N₂ and acetylene) in eucaryotic green algae gave negative results, even with species containing hydrogenase and under anaerobic conditions.     

High Photobiological Hydrogen Production Activity of a <Emphasis Type="Italic">Nostoc</Emphasis> sp. PCC 7422 Uptake Hydrogenase-Deficient Mutant with High Nitrogenase Activity

We describe a strategy to establish cyanobacterial strains with high levels of H₂ production that involves the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering. Nostoc sp. PCC 7422 was chosen from 12 other heterocystous strains, because it has the highest nitrogenase activity. We sequenced the uptake hydrogenase (Hup) gene cluster as well as the bidirectional hydrogenase gene cluster from the strain, and constructed a mutant (ΔhupL) by insertional disruption of the hupL gene. The ΔhupL mutant produced H₂ at 100 μmoles mg chlorophyll a ^-1 h^-1, a rate three times that of the wild-type. The ΔhupL cells could accumulate H₂ to about 29% (v/v) accompanied by O₂ evolution in 6 days, under a starting gas phase of Ar + 5% CO₂. The presence of 20% O₂ in the initial gas phase inhibited H₂ accumulation of the ΔhupL cells by less than 20% until day 7.     

Properties of the solubilized membrane-bound hydrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The membrane-bound hydrogenase of the photosynthetic bacterium Rhodospirillum rubrum has been purified 490-fold with a yield of 5.8%. The enzyme was homogeneous by disc gel electrophoresis. A method for the permanent, oxygen-insensitive, staining of hydrogenase on polyacrylamide gels is described. The enzyme is a monomer of molecular weight about 66,000 containing four iron and four acid-labile sulfur atoms per molecule. The electron paramagnetic resonance spectrum at 20 °K exhibits a strong signal in the oxidized state only with g > 2—this is characteristic of high potential iron-sulfur protein. The hydrogenase is thermostable and also resistant to both denaturation agents and oxygen inactivation. Carbon monoxide reversibly inhibits the enzyme but metal-complexing and thiol-blocking reagents have little effect on activity. The enzyme will catalyze both H₂ evolution and H₂ uptake in the presence of many artificial electron carriers but the two activities differ in their pH optima. There is a correlation between H₂ evolution activity and the redox potential of the mediator dye. Ferredoxins and pyridine nucleotides do not readily interact with the hydrogenase. We have shown that irradiation of a solution containing methyl viologen, EDTA, proflavin, and R. rubrum hydrogenase will evolve hydrogen continuously for over 9 h. However, the enzyme evolves hydrogen at only very low rates from in vitro chloroplast-ferredoxin and chloroplast-methyl viologen systems. R. rubrum hydrogenase has a number of properties in common with the hydrogenases purified from two other photosynthetic bacteria, Chromatium and Thiocapsa, but is distinct from the hydrogenases of nonphotosynthetic bacteria.     

Role of calcium in the inhibition of nitrogenase activity by Methylparathion and Benthiocarb in the cyanobacterium Nostoc muscorum

Methylparathion and Benthiocarb inhibition of N₂ fixation in the cyanobacterium Nostoc muscorum was reversed by Ca²⁺ at 1 mm but not at 0.1 mm. The concentration of intracellular Ca²⁺ was relatively high in the presence of these pesticides when 1 mm Ca²⁺ was also present, indicating that intracellular Ca²⁺ may participate in protecting nitrogenase activity against Methylparathion and Benthiocarb.The authors are with the Department of Biochemistry, University College of Science, 35 Ballygunge Circular Road, Calcutta 700 019, India.     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

Expression of hup Genes in Azorhizobium caulinodans and Their Coregulation With RuBP Carboxylase

In Azorhizobium caulinodans strain IRBG 46, H₂ evolved by nitrogenase Induced uptake hydrogenase ex-planta. The strain expressed an efficient H₂ recycling system under both symbiotic and ex-planta conditions. For the first time, a comparable relative efficiency of electron transfer to N₂ via nitrogenase under symbiotic as well as ex-planta conditions for both Hup? strain B11 as well as Hup⁺ strain IRBG 46, has been reported. The study also suggested a coordinate regulatory relationship between rubisco and hup genes.     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

Differential Expression of Uptake Hydrogenase Activity in Free Living Cells and Nodule Bacteroids of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 in which N₂ fixation ability was affected, was previously isolated by NTG mutagenesis. Now, the mutation has been shown to affect H₂- uptake hydrogenase (Hup) activity under symbiotic conditions. However, free living Hup activity remained unaffected. Thus the mutant is Hup^- under symbiotic conditions and Hup⁺ under free living condtions. A possible regulatory link between N₂ fixation and H₂ uptake system has been discussed.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Assessment of Hg2+ toxicity to a N2-fixing cyanobacterium in long- and short-term experiments

Toxicological responses of the filamentous N₂-fixing cyanobacteriumNostoc calcicola Bréb. towards Hg²⁺ were studied to enumerate the decisive lethal events. In low-dose, long-term experiments (0.05–0.25 m Hg²⁺, 10 days), photoautotrophic growth was severely inhibited with concurrent loss of photosynthetic pigments (phycocyanin>chlorophyll >carotenoids) and nucleic acids. The termination of growth after a day 4 exposure to 0.25 m Hg²⁺ has been attributed to the complete inhibition ofin vivo photosynthetic activity in the cyanobacterium (O₂ evolution>¹⁴CO₂ incorporation). The elevated Hg²⁺ concentrations irreversibly damaged the cell membrance as observed under light microscopy, and as indicated by the leakage of intracellular electrolytes and phycocyanin. In high-dose, short-term experiments (0.5–20.0 m Hg²⁺, up to 6 h), thein vivo activities of selected enzymes (glutamine synthetase > nitrate reductase > nitrogenase) were less inhibited by Hg²⁺ than the uptake of nutrient ions (NH ₄ ⁺ >NO ₃ ^– >PO ₄ ^3– ).     

Metabolically Engineered <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes

Three differently metabolically engineered strains, 2 single PHA^- and Hup^- mutants and one double PHA^-/Hup^- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H₂ photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H₂ recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup^- and the double PHA^-/Hup^- mutants, but not the single PHA^- mutant, exhibited increased rates of H₂ photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup^- mutant showed higher rates of H₂ production, but all 3 mutants sustained a longer-term H₂ photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H₂ evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H₂ photoproduction using actual waste-derived substrates.