Perfusion Microfermentor Integrated into a Fiber Optic Quasi-Elastic Light Scattering Sensor for Fast Screening of Microbial Growth Parameters

This research presents a microfermentor integrated into an optical fiber sensor based on quasi-elastic light scattering (QELS) to monitor and swiftly identify cellular growth kinetic parameters. The system uses a 1310 nm laser light that is guided through single-mode silica optical fibers to the interior of perfusion chambers, which are separated by polycarbonate membranes (470 nm pores) from microchannels, where a culture medium flows in a constant concentration. The system contains four layers, a superior and an inferior layer made of glass, and two intermediate poly(dimethylsiloxane) layers that contain the microchannels and the perfusion chambers, forming a reversible microfluidic device that requires only the sealing of the fibers to the inferior glass cover. The QELS autocorrelation decay rates of the optical signals were correlated to the cells counting in a microscope, and the application of this microsystem to the monitoring of alcoholic fermentation of Saccharomyces cerevisiae resulted in the kinetic parameters of KM = 4.1 g/L and μm = 0.49 h−1. These results agree with both the data reported in the literature and with the control batch test, showing that it is a reliable and efficient biological monitoring system.

. Neubauer chamber containing 0.143 C, where C is the cell concentration on the yeastpeptone-dextrose (YPD) medium saturated on yeast cells. It is possible to notice tridimensional agglomerations.
Due to the relatively high concentration, it is possible to notice tridimensional agglomerations of cells, what makes Figure S1 inadequate for the cell counting. Then, the medium must be further diluted before the evaluation of the concentration of cells. If a sample with volume V of saturated medium is diluted in water, then the concentration C2 of the diluted medium after mixing (total volume V2) will be related to the concentration C and to the sample volume V by Equation (S1), which is a direct consequence of the conservation of mass (the mass must be the same at the beginning and at the end of the dilution process).
The Neubauer chamber is divided in square regions with defined volumes. The squares present different areas, in order for making it possible to evaluate different types of cells, but present the same height, 0.1 mm. For the evaluation of yeast cells, it is possible to use the squares with 16 subdivisions and volumes of 4 × 10 −6 mL.
The experimental concentration will be given by the number of cells divided by the volume used for the analysis. If the dilution is not very high, then the distribution of cells inside the chamber is approximately homogeneous and it is possible to take the average value obtained for individual squares as the concentration. On the other hand, if the concentration is too low, the distance between individual cells is very high and the distribution inside the chamber is not homogeneous, with squares containing no cells. In this case, it is necessary to sum different squares and to divide the counting by the total volume of analysis.
It is interesting to note that the results can be improved by adding a small amount of methylene blue dye solution (5 mg of methylene blue/ L of water) as a cell viability analyzer: the dead cells are permeable to this dye and are seen as dark objects on the microscope. It is then possible to count only the viable cells [1,2].
For the calibration of the sensor, the five squares of the main diagonal of the chamber were used, totalizing a volume of 2 × 10 −5 mL. 500 μL of the methylene blue solution were added to 800 μL of a suspension with concentration C2 = 0.125 C, and 1 μL of this solution was mixed to 99 μL of milli-Q water. Then, this final suspension (concentration of 7.69 × 10 −4 C) was analyzed in the Neubauer chamber using an objective lens with magnification of 10×, as shown on Figure S2, where the counted cells and the analyzed squares are highlighted in red. The cell counting resulted in concentration of cells on the YPD saturated medium of C = 3.25 × 10 8 cells/mL. It is important to notice that the Neubauer chamber manufactures guarantee an uncertainty of 20-30% for this procedure (i.e., uncertainty of 9.75 × 10 7 cells/mL) [3].
Then, suspensions progressively diluted, with concentrations of C, 0.650 C, 0.500 C, 0.300 C, 0.200 C, 0.167 C, 0.143 C and 0.125 C were evaluated with the FOQELS, resulting in the calibration curve.

S2. Batch Fermentation Analysis
Saccharomyces cerevisiae ATCC 7754 cells were inoculated in 25 mL of YPD medium previously sterilized using a microbiological handle, and the system was kept under 33 °C and 100 rpm rotation for 10 h. Every hour, a sample was collected from the fermentation broth and introduced into the Neubauer chamber, being posteriorly analyzed in the microscope. In this experiment, the cells were homogeneously distributed inside the chamber, so the average concentration inside each one of the 5 squares of the main diagonal of the Neubauer chamber was took as the real concentration for a given time and the standard error (s/5 0.5 , where s is the standard deviation) was took as the uncertainty.
The first sample of 1 μL was not diluted; the samples of 10 μL corresponding to the times of 1 h to 6 h were mixed with 90 μL of milli-Q water (dilution of 10 times) before the analyses; the other samples, of 1 μL, were mixed with 99 μL of milli-Q water (dilution of 100 times) before the analyses.
Defining N as the number of cells per mL of analyzed volume, the kinetic curve can be obtained ( Figure S3). It is possible to notice a sigmoidal behavior, with an initial latency followed by a fast exponential growth and a final baseline. Figure S3 also shows the cells inside the chamber for three different times (images obtained using microscope lens with magnification of 10×). A first important consideration is the presence of a latency phase of approximately 5 h before the fermentation reaches the exponential growth, which is related to the protein complexes of the microorganisms and to their adaption to the fermentation environment [1,2]. Due to this result, the fermentation broth was kept under 33 °C for 5 h before the analysis of the other experiments, favoring the observation of the cell growth and the detection by the optical system. Another remarkable fact is that the exponential growth phase is very fast, with ~2 hours of duration.
Since the fermentation broth used in this experiment consists of a complex medium with great excess of substrates, the microbial growth can be evaluated in terms of a sigmoidal model, Equation (S2) [4]. The parameter Nm represents the maximum concentration of cells, the final baseline, and μm is the maximum specific growth rate.
Equation (S2) can be manipulated for the separation of variables, and then it can be integrated, leading to Equation (S3) [4], where N0 = N(t = 0 h).
The fitting of experimental data to Equation (S3) allows the estimation of μm without the precise knowledge of the substrate concentration. Assuming N0 = N(t = 0h) = 9.5 x 10 6 cells.mL -1 , and Nm equals to the average value of the points of the final plateau obtained on Figure S3, Nm = 1.36 x 10 8 cells.mL -1 , the regression results in the maximum specific growth rate μm = 0.50 h -1 and a corresponding adjusted R² of 0.859. The comparison between the results obtained by the logistic fitting and the experimental data is shown on Figure S4. The estimated value of μm is in accordance with the reported in literature for this microorganism (Atala et al. reported μm = 0.42 h -1 for S. cerevisiae fermentation under 33 °C using a sugar substrate that offers more difficult to process than either the dextrose of YPD medium or the sucrose, naturally leading to a lower value of maximum specific rate [5], while Sonnleitner and Käppeli [6] and Amillastre et al. [7] reported 0.50 h −1 ). It is also important to take in account again that the manufacturers of Neubauer chambers guarantee a great uncertainty for this procedure, in the order of 20-30% of the results [3], and that the exponential growth phase was very fast, so few points could be collected during this period.

S3. Comparison Between Experiments
The results comparing the batch experiment, the test using the microfermentor with sucrose concentration of 30 g/L and the optical fiber sensor, and the observations of the microfermentor on the microscope, without the optical fibers, are shown on Figure S5 for the first 4 h of each experiment.