Towards an effective biosensor for monitoring AD leachate: a knockout E. coli mutant that cannot catabolise lactate

Development of a biosensor for the convenient measurement of acetate and propionate concentrations in a two-phase anaerobic digestor (AD) requires a bacterium that will be unresponsive to the other organic acids present in the leachate, of which lactate is the most abundant. Successive gene knockouts of E.coli W3110 d-lactate dehydrogenase (dld), l-lactate dehydrogenase (lldD), glycolate oxidase (glcD) and a suspected l-lactate dehdrogenase (ykgF) were performed. The resulting quadruple mutant (IMD Wldgy) was incapable of growth on d- and l-lactate, whereas the wild type grew readily on these substrates. Furthermore, the O2 consumption rates of acetate-grown IMD Wldgy cell suspensions supplied with either acetate (0.1 mM) or a synthetic leachate including acetate (0.1 mM) and dl-lactate (1 mM) were identical (2.79 and 2.70 mg l−1 min−1, respectively). This was in marked contrast to similar experiments with the wild type which gave initial O2 consumption rates of 2.00, 2.36 and 2.97 mg l−1 min−1 when cell suspensions were supplied with acetate (0.1 mM), acetate (0.1 mM) plus d-lactate (1 mM) or acetate (0.1 mM) plus l-lactate (1 mM), respectively. The knockout strain provides a platform for the design of a biosensor that can accessibly monitor acetate and propionate concentrations in AD leachate via O2-uptake measurements.