Which methods for viable yeast cell quantification can be used in lignocellulosic fermentation processes

Conference poster, 2016

Cell concentration is a primary characteristic of fermentation processes. The total cell concentration in a particle-free liquid medium can be easily assessed by cell counts, optical density or dry weight. The quantification of viable cells is not as straightforward. Viable cells can be defined as culturable, metabolically active and intact cells. Culturable cells can be assessed by colony-forming unit (CFU) assay. Metabolically active and intact cells have been quantified by e.g. qPCR, dielectric spectroscopy probes, and flow cytometry using various dyes. All these methods work well for applications in clear liquid media, but have not been validated in 2nd generation bioprocesses using lignocellulosic materials. In this study we evaluate the applicability of several methods for quantitative assessment of both total and viable cell concentrations in lignocellulosic media. In order to mimic typical conditions of lignocellulosic fermentations, we used a central composite design of experiments with known cell numbers, water insoluble solids content (WIS) and osmolality as factors. For the osmolality, we used sorbitol and NaCl to differentiate hyperosmotic conditions at different ion strengths and conductivities. The cell concentrations were determined using cell enumeration in a hemocytometer (with and without methylene blue staining), plating and enumeration of CFU, qPCR on extracted DNA and RNA, and on-line permittivity using a capacitance probe. These methods have the potential to be less affected by impurities and water insoluble solids in lignocellulosic media than e.g. dry weight and turbidity. The number and viability of cells used to create the test conditions of the experimental design were first determined from the seed culture on defined mineral medium. Considering all experimental points and some validation points within the design space, all the selected methods were used for measuring total and viable number of cells. With these data we built a quantitative model to fit all interaction effects and curvature, and to calibrate the qPCR and permittivity results to the number of total and culturable cell counts. Data of qPCR on DNA were fitted to total cell numbers, WIS level and osmolality. The permittivity measured by the dielectric probe was fitted to CFUs, WIS level, osmolality and measured conductivity. Parameter optimization resulted in statistically significant models with good predictive capacity. The results showed that cell counts and CFU were not sensitive to WIS and osmolarity levels. Therefore they can be used as reference methods in lignocellulose-based media. Furthermore, using the selected methodologies in simultaneous saccharification and fermentation (SSF) process of pre-treated wheat straw showed consistent results in total and viable cell numbers. Development of reliable and validated total and viable cell quantification methods will contribute to wellmonitored lignocellulosic fermentation processes both for research and industry in bio-based production.