Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase

Background: Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requires
cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity
with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching
efficient and robust fermentations.
Results: In this study, we assessed responses of isogenic yeast cell populations in different physiological states to
combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited
significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate
growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different
buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and
non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic
inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not
the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive
oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations.
Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high
frequency of cells with low pHi
and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to
tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance
to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory
medium that were uniformly sensitive to the inhibitors at low pH.
Conclusions: ESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH,
and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH.
Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments
which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at
low pH. Furthermore, flow cytometry analysis of pHi
and ROS level distributions in ESP cultures revealed responses
that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population
distribution responses as described herein may be applied to predict the outcome of environmental perturbations
and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.