Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate,pH and the F0F1-ATPase

Molecular hydrogen (H₂) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H₂-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH]_out) and the presence of formate. FHL is related with the F₀F₁-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H₂-oxidizing enzymes during glucose fermentation at neutral and low [pH]_out. They operate in a reverse, H₂-producing mode during glycerol fermentation at neutral [pH]_out. Hyd-1 and Hyd-2 activity depends on F₀F₁. Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H₂ production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H₂ production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H₂ production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90° C with an optimum around 85° C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H₂S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO₂, NH₃, and H₂S (in the presence of S°) were detected as end products. Growth was not inhibited by H₂. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322) Received: 7 July 1995 / Accepted: 25 August 1995     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

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.     

Metabolic engineering to enhance bacterial hydrogen production

Hydrogen fuel is renewable, efficient and clean, and fermentative bacteria hold great promise for its generation. Here we use the isogenic Escherichia coli K‐12 KEIO library to rapidly construct multiple, precise deletions in the E. coli genome to direct the metabolic flux towards hydrogen production. Escherichia coli has three active hydrogenases, and the genes involved in the regulation of the formate hydrogen lyase (FHL) system for synthesizing hydrogen from formate via hydrogenase 3 were also manipulated to enhance hydrogen production. Specifically, we altered regulation of FHL by controlling the regulators HycA and FhlA, removed hydrogen consumption by hydrogenases 1 and 2 via the hyaB and hybC mutations, and re‐directed formate metabolism using the fdnG, fdoG, narG, focA, fnr and focB mutations. The result was a 141‐fold increase in hydrogen production from formate to create a bacterium (BW25113 hyaB hybC hycA fdoG/pCA24N‐FhlA) that produces the largest amount of hydrogen to date and one that achieves the theoretical yield for hydrogen from formate. In addition, the hydrogen yield from glucose was increased by 50%, and there was threefold higher hydrogen production from glucose with this strain.     

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production

Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal. To explore the reductant flux and possible competition for H₂ production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H₂ production and consumption. The results obtained establish that H₂ is generated predominantly by the membrane‐bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H₂ consumption results in a substantial net increase in H₂ production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H₂ production, revealing a second mechanism to increase H₂ production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild‐type growth and H₂ production. With S° present, they grew slower but synthesized more H₂ per unit biomass than T. kodakarensis TS517.     

Enhanced hydrogen production from glucose by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H₂/mol glucose (maximum, 2 mol H₂/mol glucose).     

Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH

Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4. These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H₂ and CO₂ during fermentative growth. FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH. In this study, regulation of activity of these pathways by formate has been investigated. In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H₂ became mostly Hyd-3 dependent. These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4. Received: 27 December 2001 / Accepted: 25 January 2002     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Synthetic biology for improved hydrogen production in Chlamydomonas reinhardtii

Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H₂ is almost exclusively produced from coal and natural gas. As such, methods to produce H₂ that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H₂ may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H₂ s⁻¹ for CaHydA from Clostridium acetobutyliticum). Yet to date, limitations in C. reinhardtii H₂ production pathways have hampered commercial scale implementation, in part due to O₂ sensitivity of hydrogenases and competing metabolic pathways, resulting in low H₂ production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H₂ production pathways in C. reinhardtii. We also summarize recent advancements of algal H₂ production using synthetic biology and describe valuable tools such as high-throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H₂ production.     

Transient Production of Formate During Chemolithotrophic Growth of Anaerobic Microorganisms on Hydrogen

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H₂ and CO₂ as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H₂ and CO₂ using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H₂ was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H₂ partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H₂. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H₂. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H₂. Received: 24 November 1998 / Accepted: 30 December 1998     

Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans

The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H₂ and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids

From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid – shaped and possess up to five flagella. They grow between 56° and 93°C (optimum 85°C) and pH 5.0–9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H₂S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H₂. DNA–DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type strain is isolate AEDII10 (DSM 11906). Received: September 24, 1997 / Accepted: January 1, 1998     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F₀F₁ ATP synthase and formate oxidation by formate hydrogen lyase (FHL) has been found in inside-out membrane vesicles of the Escherichia coli mutant JW 136 (Δhya/Δhyb) with double deletions of hydrogenases 1 and 2, grown anaerobically on glucose in the absence of external electron acceptors at pH 6.5. ATP synthesis was suppressed by the H⁺-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, sodium azide, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of the vesicles. The maximal rate of ATP synthesis (0.83 μmol/min per mg protein) was determined at simultaneous application of sodium formate, ADP, and inorganic phosphate, and was stimulated by K⁺ ions. The results confirm the assumption of a dual role of hydrogenase 3, the formate hydrogen lyase subunit that can couple the reduction of protons to H₂ and their translocation through membrane with chemiosmotic synthesis of ATP.     

Role of the Hya Hydrogenase in Recycling of Anaerobically Produced H2 in Salmonella enterica Serovar Typhimurium

Double and triple uptake-type hydrogenase mutants were used to determine which hydrogenase recycles fermentatively produced hydrogen. The Δhyb Δhya and Δhyd Δhya double mutants evolved H₂ at rates similar to that of the triple mutant strain, so Hya alone oxidizes the bulk of H₂ produced during fermentation. When only Hya was present, no hydrogen production was observed in nutrient-limited medium. H₂ uptake assays showed that Hya can oxidize both exogenously added H₂ and formate hydrogen lyase-evolved H₂ anaerobically. Even after anaerobic growth, all three uptake-type hydrogenases could function in the presence of oxygen, including using O₂ as a terminal acceptor.Due to the anticipated scarcity of fossil fuels, there has been a surge of interest in H₂ production for alternative energy means. Numerous studies have attempted to engineer H₂-producing organisms, such as photosynthetic bacteria, cyanobacteria, and Escherichia coli, to produce maximal amounts of H₂ while minimizing the H₂-oxidizing capability of the organism (4, 5, 9, 10, 19). Hydrogenase expression and activity are controlled by multiple regulatory pathways and respond to fluctuations in pH, oxygen levels, and availability of metabolites and metal cofactors (17). In addition, the presence of hydrogen uptake hydrogenases decreases the net H₂ yield even under conditions that favor H₂ production. It is therefore important to understand the interactions between H₂-oxidizing enzymes (i.e., respiratory hydrogenases) and H₂-producing enzymes.Gene sequence analysis has revealed that many enteric bacteria contain the genes necessary for hydrogen production and oxidation. The E. coli hydrogenases have been studied extensively, while Salmonella enterica serovar Typhimurium hydrogenases have been studied to a lesser extent. Both E. coli and Salmonella serovar Typhimurium contain the hydrogen-oxidizing hydrogenases Hya and Hyb. Salmonella serovar Typhimurium also contains Hyd, which is another hydrogen-oxidizing hydrogenase (2, 13, 15). Hyc and Hyf are hydrogen-evolving hydrogenases that are present in both E. coli and Salmonella serovar Typhimurium, although it is unknown whether Hyf is functional (1).The Salmonella serovar Typhimurium hydrogenases are important for cellular metabolism. Hyc produces H₂ in order to remove excess reductant generated during mixed-acid fermentation. Hyc and formate dehydrogenase constitute the formate hydrogen lyase (FHL) complex (16), which oxidizes formate to produce CO₂ and H₂ (12). The hyb genes in E. coli and Salmonella serovar Typhimurium are expressed at high levels under anaerobic respiration conditions, and Hyb probably contributes to energy conservation (11, 15, 20). Hyb oxidizes H₂ and generates electrons, which are passed through the electron transport chain to terminal acceptors such as fumarate. The protons generated contribute to the proton-motive force. The role of Hya is not as well characterized. Hya may be used to recycle Hyc-produced H₂, since the hya operon is expressed at high levels during fermentative growth, or it may play a role in acid stress resistance (6, 14, 20, 21). The hyb genes are expressed at high levels under aerobic conditions in Salmonella serovar Typhimurium, and Hyb may couple H₂ oxidation to O₂ reduction (20).Redwood et al. recently examined the roles of uptake-type hydrogenases on net hydrogen production in E. coli (10). Cells were pregrown aerobically or anaerobically with formate and then allowed to ferment in anaerobic bottles. H₂ gas was collected, and other fermentation products were measured. They found that H₂ production increased by 37% in an hya hyb double mutant (compared to that in the wild type) that was grown overnight aerobically with formate. This increase in production was associated with the loss of hyb and not hya. Therefore, in E. coli, Hyb may be responsible for recycling fermentatively produced H₂.In this study, we measured the effect of uptake-type hydrogenase mutations on H₂ production in Salmonella serovar Typhimurium. We found that the majority of H₂-recycling activity in fermenting cells was dependent on the presence of hya, and having only Hya was sufficient to prevent any detectable H₂ evolution. These results demonstrate yet another difference between H₂ metabolism in E. coli and H₂ metabolism in Salmonella serovar Typhimurium.