Biosensors for monitoring PHB production in bioreactors

Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp, Comamonas spp., in response to stress conditions, i.e. under high carbon and low nitrogen (24:1) sources. PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Bioreactors are provided with biosensors to monitor physio-chemical parameters, such as temperature probe and pH sensor (linked to pumps to add NaOH or HCl), stirring speed, air flux regulation or micro-bubble dispersion by sparging (BIOSTAT Q Multi-Fermentor Bioreactor System with dissolved oxygen probe), to provide dissolved oxygen, needed for aerobic growth. Turbidity (OD600) and glucose consumption need to be 9 measured, at 0, 4, 8, 12, 24, 48, 72 and 96 h. PHA production need to be evaluated too, since after PHA accumulation bacterial cells are collected for PHA extraction. In order to make the process sustainable and economically convenient, two factors need to be optimised: high optical density of cells, and continuous presence of 5-10% sugars. The bacteria need to reach an OD higher than 25- possibly near 50, the obtain a good ratio of cells/volume, exploiting the maximum volume of bioreactor, without dispersing sugar in a high volume. PHA is detected by staining cells with Nile Blue, and evaluating the fluorescence intensity. Here we propose to apply three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by spectrophotometry or other sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining (with washing step), and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.