Biosensor for L-lactate determination as an index of E. coli number in crude culture medium

Abstract

In a culture medium E. coli produces L-lactate as a metabolite. L-lactate enzyme sensors were assembled and applied to a crude E. coli growth medium for L-lactate determination. The amount of L-lactate was used as an index of the cell number. In the standard solution, L-lactate oxidase (LOD) sensor gave a linear response from 5 to 300 μM L-lactate. Determination of L-lactate in bacteria cultures of different absorbance ( A) was possible from an absorbance at 600 nm, A 600=0.01 and higher. L-lactate measurements were not modified when the LOD electrode was associated with L-lactate dehydrogenase (LDH) in solution. In contrast, the bi-enzyme sensor consisting of LOD coimmobilized with LDH on the electrode, lowered the detection limit to 30 nM L-lactate by amplification of measurements via coupled reactions and substrate recycling. Responses were obtained in growth medium from A=0.0005 (5×10 5 bacteria ml −1). In comparison, the standard spectrophotometer cell estimation was not reliable below A=0.02. The L-lactate detection limit can be decreased further by incubating bacteria with glucose for 1 to 2 h. Measurements of L-lactate by oxygen consumption could be performed in the presence of bacteria and growth medium components. The recycling enzymatic electrode kept 80% of its initial activity with storage overnight in a buffer, at 4°C, during 2 weeks. Such sensors permit fast and sensitive procedures for L-lactate and E. coli assays. The procedure does not disturb the developing cultures as relatively small sample volumes from 1 to 500 μl and no cells are needed for L-lactate determination.