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Klebsiella oxytoca CECT 4460 removes high nitrate loads from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose inK. oxytoca CECT 4460 was considered. To achieve this, we cloned and transferred into this strain theKlebsiella pneumoniae nasB gene, which encodes assimilatory nitrite reductase (Lin et al., J. Bacteriol. 176:2551–2559, 1994). The delivery vector was either the wide-host-range plasmid pUPE2, in which thenasB gene is expressed from theEscherichia coli Plac promoter, or a mini-Tn5 -Km vector, which upon random insertion in the host chromosome allowed expression of thenasB gene from an unidentified chromosomal host promoter. The effect of the increase in the dose of thenasB gene inK. oxytoca CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed thatK. oxytoca CECT 4460 bearing pUPE2 accumulated 88% less nitrite than the wild-type strain, while the recombinant strain bearing theK. pneumoniae nasB gene in the host chromosome showed a 25% lower level of nitrite accumulation in the culture medium than that of the wild type.

Recombinant Klebsiella oxytoca Strains with Improved Efficiency in Removal of High Nitrate Loads