Abstract

In this article, a different method of investigating bacterial proliferation is suggested. It is based on the detection of enzyme activity as opposed to the traditional physical approaches. It is shown how to monitor microbial ureolytic activity in real time and online using flow analysis (applied to a model experimental biosystem). A solenoid micropumper and microvalve system controlled by an Arduino microcontroller make up the fully automated bioanalytical flow system that was designed. Dedicated flow-through optoelectronic detectors constructed of paired light emitting diodes are used to carry out photometric detection based on the Nessler reaction. With a detection limit below 0.44 U mL-1, a high sensitivity in the linear range of response (up to 200 mV U1 mL), and reasonably high throughput, the developed bioanalytical system enables discrete assaying of microbial urease in a wide range of activity up to 5.4 U mL-1 (9 detection per hour). The suggested differential measurement method allows for the removal of interfering effects from substrate and products of biocatalyzed reactions as well as other components of the medium used for bacterial expansion (i.e., a difference between peaks register for sample with and without external addition of urea is treated as an analytical signal). In order to control the growth of urease-positive bacteria strains (Proteus vulgaris, Klebsiella pneumoniae, and Paracoccus yeei), the developed bioanalytical system was successfully used. This included examining the effects of different microbial cultivation conditions, such as temperature, the makeup of the culture medium, and the quantity of substrate necessary to induce bacterial enzymatic activity. The created bioanalytical flow system can be used to detect the decline phase as well as estimate the parameters of the lag and log phases of microbial growth based on metabolic activity.