To redirect NAD(P)H consumed by dehydrogenases to NiFe-hydrogenase, the genes encoding for lactate dehydrogenase (ddh) and alcohol dehydrogenase (adh) were disrupted in a glucose tolerant mutant of unicellular cyanobacterium Synechocystis sp. strain PCC6803 (GT strain). The cells of GT strain, ddh deficient (Δddh) mutant and both adh and ddh deficient (ΔadhΔddh) mutant were harvested at the late-logarithmic growth phase of photoautotrophic culture in antibiotic-free BG-11 medium. Dark anaerobic hydrogen production in GT strain, Δddh mutant and ΔadhΔddh mutant in nitrate-free HEPES buffer solution without or with glucose has been studied by following the time courses of the number of moles of hydrogen, endogenous and exogenous glucose, lactate, acetate and ethanol per culture volume and the dry cell weight concentration. Dark incubation of cells of Δddh mutant and ΔadhΔddh mutant in HEPES buffer solution without glucose resulted 1.16-fold and 1.15-fold increases in the initial hydrogen production rates over GT strain, respectively, while 1.1-fold and 0.85-fold increases in the number of moles of hydrogen per culture volume at 96 h over GT strain, respectively. When hydrogen production experiments were performed in the glucose-added HEPES buffer solution, dark incubation of cells of Δddh mutant and ΔadhΔddh mutant resulted 1.4-fold and 1.6-fold increases in the initial hydrogen production rates over GT strain without glucose run, respectively, while 1.5-fold and 1.3-fold increases in the number of moles of hydrogen per culture volume at 96 h over GT strain without glucose run, respectively.
Fructose is a potential additive that elevates a supply of electrons to hydrogenase for hydrogen production in cyanobacteria. A series of dark anaerobic hydrogen production experiments was performed to evaluate the validity of this assumption, in which fructose from 0 to 110 mmol/L was added to HEPES buffer solution on which a glucose tolerant mutant of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (GT strain) was incubated. Despite the reported knowledge that fructose was an inhibitor for photoheterotrophic growth of Synechocystis cells, GT strain assimilated fructose and represented a limited heterotrophic growth on fructose in HEPES buffer solution under dark anaerobic condition. The initial hydrogen production rate that was 0.025 mmol/L h in run without fructose increased to 0.0917 mmol/L h in run with 60-83 mmol/L fructose. The associated increase in the initial amount of endogenous glucose from 0.22 mmol/L in run without fructose to 0.36 mmol/L in run with 50 mmol/L fructose was observed. Fructose released the complete suppression of hydrogen production by nitrate. This work presents the first experimental evidence that cells of GT strain are able to assimilate fructose for cell growth in dark anaerobic condition. Our results show that hydrogen production in Synechocystis sp. strain can be significantly elevated by a proper addition of fructose to dark anaerobic HEPES buffer solution.
The effects of fructose on the dark anaerobic hydrogen production of the genetically modified mutants from glucose tolerant derivative of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (GT strain) in nitrate-free buffer solution has been studied by following the time courses of the amounts of hydrogen, fructose, dry cell weight, lactate and acetate per culture volume and the amount of endogenous glucose per dry cell weight. Considering that hydrogen production on bidirectional NiFe-hydrogenase competes NAD(P)H with lactate dehydrogenase and alcohol dehydrogenase, mutants which lack lactate dehydrogenase (Δddh mutant) and both lactate dehydrogenase and alcohol dehydrogenase (ΔadhΔddh mutant), were utilized. These genetic modifications have a high potential to redirect electrons to NiFe-hydrogenase for hydrogen generation. A supply of additive to increase electron supply has found to be a successful method to activate hydrogen production. Fructose was selected as an additive due to our previous observation that hydrogen production in GT strain was highly increased in the presence of 50 μmol mL -1 fructose. Initial hydrogen production rate of ΔadhΔddh mutant in the presence of 50 μmol mL -1 fructose was increased to 0.129 μmol mL -1 h -1 that was 5.11 times that of GT strain. This increase was associated with the initial increase in glucose accumulation rate. Hydrogen production by ΔadhΔddh mutant in the presence of 50 μmol mL -1 fructose was quite stable for at least 120 h. Fructose-mediated hydrogen production resulted in an 11-fold increase in the moles of hydrogen per culture volume at 120 h over controls.
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