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pflB, and the genes of the moa, nik and hyp operons required for the biosynthesis
of the molybdopterin and Ni-Fe cofactors of FHL (Table 1) [15], [19], [27], [28].
The effects on hydrogen yield of multi-copy expression of the global regulators
fnr, arcAB and lhfAB, as well as ModE (the secondary transcriptional activator
of the fhl regulon), and the tRNA for selenocysteine incorporation (selC), were
examined in wild-type W3110 and ∆focA (ZF1) strains.
increased Hydrogen Yields by Gene-inactivated e. coli
strains
The rate of hydrogen production and of growth by wild-type strain W3110, and
four strains containing specific chromosomal gene disruptions were examined (Fig-
ures 2A and 2B). The maximum rate of hydrogen generation occurred between 9
and 16 hours following inoculation of M9 medium under the experimental con-
ditions described. Strains ZF3 (W3110 narL) and ZF4 (W3110 ppc) exhibited
the highest rates of hydrogen production, and had accumulated about 20% to
30% more hydrogen in the culture headspace than W3110 in 25 hours (Fig-
ure 2A). Strain ZF1 (W3110 focA) exhibited a slightly slower rate of hydrogen
generation than W3110 and accumulated 33% less hydrogen by the end of the
fermentation experiment. Strains ZF2 (W3110 hybC) and W3110 had compa-
rable rates of hydrogen formation to each other. The slower rate of hydrogen gen-
eration observed in the later stages of fermentation correlated with an increased
accumulation of biomass, possibly resulting from the hydrogen oxidizing activity
of uptake hydrogenases 1, 2, and/or 4, generating protons and electrons for cel-
lular ATP synthesis, or by inhibition of hydrogenase 3 by the evolved hydrogen
that accumulated in culture headspace [8], [26]. The complete consumption of
glucose accompanied by reduced glycolytic flux to pyruvate and subsequently to
formate could account for the simultaneous reductions in growth and H2 pro-
duction rates. Strains W3110, ZF3 (W3110 narL) and ZF4 (W3110 ppc) ex-
hibited similar generation times and reached similar final cell densities. Strain
ZF2 (W3110 hybC) grew at a slightly slower rate than W3110, while strain ZF1
(W3110 focA) grew at a slower rate than W3110 and reached a 50% lower final
culture density (Figure 2B). The lower growth rates of strains ZF1 and ZF2 might
result from the increased acidification of cellular cytoplasm from the disruption
of formate export, and by the decrease in cellular ATP synthesis by the disrup-
tion of an uptake hydrogenase, respectively. The rate of anaerobic growth and of
hydrogen production by the strains in TYP medium followed a profile similar to
that seen in M9-glucose medium, with the highest rates of hydrogen production
occurring between 4 and 7 hours of fermentation. The culture generation times
were increased by about 2.5-fold in TYP medium, with A600 values reaching
about 6.5 by 10 hours (data not shown).
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