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bioRxiv · 10.1101/2024.06.26.600471

Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses

Martins, C. T.·Jacobus, A. P.·de Luna, R. C.·Barbin, D. F.·Bolini, H.·Gombert, A. K.
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Abstract

In scenarios where yeast and bacterial cells coexist, e.g. the food and bioethanol industries, it would be of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared ([~]107, yeast cells/mL or [~]109, bacterial cells/mL) and mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries. The following methods were used: bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter counter (at 1:1 and 100:1 ratios). By subjecting the same mixed cell suspension to each technique, we observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (P>0.05). In contrast, the Coulter counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (P<0.05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasnt possible due to an observed overlap between yeast cell debris and bacterial cells. The results from this work indicate that each method has limitations, advantages, and disadvantages, meaning that the best option will always depend on the application. We present a comparison of the techniques, in terms of time-to-results, cost of analysis and equipment, range of detectable cell/particle diameters, adequacy for simultaneous enumeration, and general pros and cons. Graphical AbstractThis study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/600471v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@875599org.highwire.dtl.DTLVardef@104b849org.highwire.dtl.DTLVardef@ded39borg.highwire.dtl.DTLVardef@11692b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Martins, C. T., Jacobus, A. P., de Luna, R. C., Barbin, D. F., Bolini, H., Gombert, A. K.. 2024-06-27. Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses. https://doi.org/10.1101/2024.06.26.600471

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