The complete NANOBE system was constructed on the basis of the commonly agreed
structure of the system and the functions of the individual modules developed
in form of several stand-alone units: sampling; sample treatment and delivery;
in-situ monitoring of pH, dissolved oxygen (dO2) and carbon dioxide (pCO2);
cell counting, sorting and lysis; ELISA (enzyme linked immunosorbent assay)
based protein analysis; and finally CE-MS to analyse intra- and extracellular
metabolites. The planned full setup occupied an area of 2x2 m2 including the
bioreactor and its peripheries. Most of the required space was occupied by
auxiliary components such as pumps, valves or electrical power supplies. The
virtual system control software (LabView) controlled the individual analysis
and core system modules.
In the NANOBE project the primary example of the application was all along the
production of organic acids in yeast Saccharomyces cerevisiae. Lactic acid
production in genetically modified S. cerevisiae strain was focused on when
testing the whole NANOBE system.
In total, four analysis cycles were carried out with the integrated system at
the very end of the project. The monitored dissolved oxygen concentrations
were in good agreement with the values measured using conventional sensor. The
pH and CO2 sensors worked well. There were some issues related to the optical
sensor attachment that can be easily solved in future. The sample was
successfully transported from the bioreactor to the ELISA and MS modules in
two cycles out of four. One out of the two successful system operation cycles
resulted in successful data readout in both the ELISA and MS modules. The
results from the ELISA analysis suggested that the dilution was not totally
reliable. The CE-MS analysis results were qualitatively in good agreement with
data from the manually-taken sample using the same Microsaic mass spectrometer.
The main achievements of the project were:
• Two new analysis tools (micro mass spectrometer already in the market and
automated platform for unattended ELISA tests at industrialised prototyping
• New innovative methods and devices developed for dead-volume free µL-scaled
sampling and sample handling. The 20 µl sample volume is most probably the
smallest sample volume of which so many different analytes can be measured.
• Developed digital microfluidics for handling 1 µL droplets that push the
boundaries of working with really small sample volume so that the initial
sample can be separated for several analytical devices.
• Demonstration of tools for counting and sorting of dead and alive yeast
cells with added cell concentration estimation.
• New tools developed for lysis of the yeast cells.
• New optical sensor technology developed for autoclavable sensors for pH in
the range of 3 to 6
• More robust optical sensors for measuring dissolved CO2
• One patent application.
• 19 scientific journal publications and 15 conference presentations.
Overall, the on-line monitoring tools developed in the NANOBE project can help
to increase the production rate, yield and concentration of the final product
of a fermentation process. These improvements in process monitoring may be
crucial for the economic viability of a new bio-based product.