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

Narayanan, Venkatachalam LU ; Schelin, Jenny LU ; Gorwa-Grauslund, Marie F LU ; van Niel, Ed LU and Carlquist, Magnus LU (2017) In Biotechnology for Biofuels 10.
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Carbon starvation, Stress tolerance, Acetic acid, Vanillin, Furfural, Reactive oxygen species, Intracellular pH,
in
Biotechnology for Biofuels
volume
10
pages
15 pages
publisher
BioMed Central
external identifiers
  • scopus:85020694140
  • wos:000401619900001
ISSN
1754-6834
DOI
10.1186/s13068-017-0794-0
language
English
LU publication?
yes
id
0890a461-f97a-4c40-aa0b-8b9b48c51d82
date added to LUP
2017-05-07 20:01:39
date last changed
2017-09-18 13:34:18
@article{0890a461-f97a-4c40-aa0b-8b9b48c51d82,
  abstract     = {Background: Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requires<br/>cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity<br/>with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching<br/>efficient and robust fermentations.<br/>Results: In this study, we assessed responses of isogenic yeast cell populations in different physiological states to<br/>combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited<br/>significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate<br/>growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different<br/>buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and<br/>non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic<br/>inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not<br/>the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive<br/>oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations.<br/>Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high<br/>frequency of cells with low pHi<br/>and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to<br/>tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance<br/>to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory<br/>medium that were uniformly sensitive to the inhibitors at low pH.<br/>Conclusions: ESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH,<br/>and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH.<br/>Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments<br/>which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at<br/>low pH. Furthermore, flow cytometry analysis of pHi<br/>and ROS level distributions in ESP cultures revealed responses<br/>that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population<br/>distribution responses as described herein may be applied to predict the outcome of environmental perturbations<br/>and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.},
  articleno    = {114},
  author       = {Narayanan, Venkatachalam and Schelin, Jenny and Gorwa-Grauslund, Marie F and van Niel, Ed and Carlquist, Magnus},
  issn         = {1754-6834},
  keyword      = {Carbon starvation, Stress tolerance, Acetic acid, Vanillin, Furfural, Reactive oxygen species, Intracellular pH,},
  language     = {eng},
  month        = {05},
  pages        = {15},
  publisher    = {BioMed Central},
  series       = {Biotechnology for Biofuels},
  title        = {Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase},
  url          = {http://dx.doi.org/10.1186/s13068-017-0794-0},
  volume       = {10},
  year         = {2017},
}