Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Abstract

When carbohydrates are fermented by the hyperthermophilic anaerobeThermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded byldh) with a truncatedldhfused to a kanamycin resistance cassette expressed from a native PgroESLpromoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded bymalK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of themalK3mutation into a clean genetic background also conferred increased H2production, confirming that the mutant allele was sufficient for increased H2synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2production, changing fermentation efficiency and shifting energy management.IMPORTANCEBiorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophileThermotoga maritimathat exceed the physiologic limits for H2formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

Metabolic Engineering and Fermentation Process Strategies for L-Tryptophan Production by Escherichia coli

“…Among the mutant strains, E. coli FB-04(pta1) ∆pykF-ptsHN12S achieved a 38.0% increased conversion rate compared with the parent strain [22]. This phenomenon of a higher product yield upon reduced rates of sugar uptake has also been previously described [23,24].…”

Seed Pretreatment for Increased Hydrogen Production Using Mixed-Culture Systems with Advantages over Pure-Culture Systems

“…The maximum molar yield of hydrogen will be around 4 mol from 1 mol of glucose with complete conversion to acetate as the end-product [115]. The highest hydrogen yield reported was around 2.8 mol/mol of glucose in case of mixed-culture systems [116] and was around (4.5 and 5.77 mol/mol of glucose) using an engineered Thermotoga maritima (Tma100 and Tma200) strain [117]. The gene inactivation of the lactate pathway, insertion of a transporter subunit and the strain construction method together improved the fermentation efficiency, and energy management with an increase in hydrogen production.…”

“…The gene inactivation of the lactate pathway, insertion of a transporter subunit and the strain construction method together improved the fermentation efficiency, and energy management with an increase in hydrogen production. The fermentation parameters need to be optimized to block the unwanted pathway(s), minimize by-product formation, to improve mass and energy balance between substrate and product, in presence of hydrogen producing microorganisms to reach maximum molar hydrogen yield [117]. The gene engineering of unwanted-, inactivation-and blocking pathways can be carried out on the hydrogen-producing pure-culture [117], obtained only after carrying out a mixed-culture pre-treatment step.…”

Economic Viability and Environmental Efficiency Analysis of Hydrogen Production Processes for the Decarbonization of Energy Systems

“…There used to be a ceiling for hydrogen production (4 mol H 2 /mol glucose). Recently, strains have been developed showing hydrogen production can be up to 8 mol H 2 /mol glucose [59].…”