A Stereospecific Lactic Acid Exclusion Biosensor for Grass Silage Fed Green Biorefinery.

Grass silage fed green biorefineries require high quality grass silage leachates to produce high quality products. Real-time, cost-effective methods to monitor key leachate analytes are needed for small-scale decentralised systems. In this study, Escherichia coli mutants, designated JSP0090 and JSP0094, were created to quantify d- and l-lactic acid concentrations in grass leachate samples using an oxygen probe. The genes encoding either d- or l-lactate dehydrogenase were expressed in an exclusion biosensor strain (JSK0115). This strain required successive gene deletions to prevent interference from sugars, amino acids and organic acids present in grass silage leachates. This strain was incapable of catabolising d-lactic acid, l-lactic acid, acetic acid, propionic acid, formic acid, ethanol, glucose, fructose, l-alanine, VFAs, glycerol, mannitol and succinate. Gene knockouts were achieved using P1 phage lysates and CRISPR Cas 9 methods to target key steps in the catabolism of these compounds, with the exception of succinate. For this metabolite, the di-carboxylic acid transporters YaaH, DctA, YchM, DcuA and DcuB had to be deleted. The effectiveness of the biosensors for selectively measuring d- and l-lactic acid was assessed in Austrian and Irish grass silage leachate. The concentrations measured were comparable to those obtained using a commercial enzyme kit.