BIOSENSOR FOR L-LACTATE DETERMINATION AS AN INDEX OF ESCHERICHIA-COLINUMBER IN CRUDE CULTURE-MEDIUM

In a culture medium E. coli produces L-lactate as a metabolite. L-lact ate enzyme sensors were assembled and applied to a crude E. coli growt h 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 o xidase (LOD) sensor gave a linear response from 5 to 300 mu M L-lactat e. Determination of L-lactate in bacteria cultures of different absorb ance (A) was possible from an absorbance at 600 nm, A(600)=0.01 and hi gher. L-lactate measurements were not modified when the LOD electrode was associated with L-lactate dehydrogenase (LDH) in solution. In cont rast. the bi-enzyme sensor consisting of LOD coimmobilized with LDH on the electrode, lowered the detection limit to 30 nM L-lactate by ampl ification of measurements via coupled reactions and substrate recyclin g. Responses were obtained in growth medium from A=0.0005 (5 x 10(5) b acteria ml(-1)). In comparison, the standard spectrophotometer cell es timation 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 performe d in the presence of bacteria and growth medium components. The recycl ing enzymatic electrode kept 80% of its initial activity with storage overnight in a buffer, at 4 degrees C. during 2 weeks. Such sensors pe rmit fast and sensitive procedures for L-lactate and E. Loll assays. T he procedure does not disturb the developing cultures as relatively sm all sample volumes from 1 to 500 mu l and no cells are needed for L-la ctate determination. (C) 1998 Elsevier Science B.V.