Development of a Green Biorefinery Whole Cell Biosensor Array

This research aimed to develop an array of Escherichia coli whole cell biosensors, to be used to detect key performance indicator analytes at-line within biological leachates produced from grass-silage fed green biorefineries. Each biosensor presented within this thesis has been developed to specifically detect a single analyte with the exclusion of all other analytes present in a grass silage fed green biorefinery pressed juice solution by removing catabolic pathways of interfering analytes from a previously developed acetic acid biosensor. The application of this process has resulted in the green biorefinery exclusion strain JSK0115, which does not have a biosensor response for any carbohydrates present in grass silage leachate except for amino acids. This response was around the 1 mV s-1 when supplied with 10% grass silage leachate, which was deemed small enough for the purpose of this study. Attempts were made to develop a biosensor that can detect butyric acid using a precursor of the exclusion strain. This biosensor also had a response for other volatile fatty acid and a poor longevity, more research was needed to develop a butyric acid specific biosensor. However, a biosensor was developed that could be used to detect volatile fatty acids with a chain length of six or higher. This biosensor had a response of around 2 mV s-1 for 72 hours when supplied with hexanoic acid. The exclusion strain was also used to develop two stereospecific lactic acid biosensors, which had a biosensor response around 8.5 mV s-1 and 5.5 mV s-1 for 20 hours when supplied with D- or L-lactic acid, respectively. These biosensors were used to qualify the D- or L-lactic acid content in grass silage leachate. More research is needed to be able to quantify the D- or L-lactic acid in grass silage leachate with these biosensors. This is due to differences between the concentration determined with the biosensor compared to the concentration determined with an enzymatic kit. The method applied to construct the D- and L-lactic acid biosensor can easily be applied to develop an analyte specific biosensor for most key analytes of a grass silage fed green biorefinery.