Abstract
Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative to fossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination and a combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield and productivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt to low pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation and adaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strain TMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosic inhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitor cocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 h to yield 0.45 ± 0.01 g ethanol g glucose−1) and portray significant detoxification of inhibitors at pH 3.7, when compared to non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to grow and ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust population was obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was not stable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With further research, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanol plants to prevent bacterial contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0234-8) contains supplementary material, which is available to authorized users.
Highlights
Increasing concerns over the need for sustainable and scalable fuels as a means to curb global warming has led to focus on bioethanol production from renewable biomass, such as agricultural and industrial residues (Limayem and Ricke 2012)
Yeast cells are treated with dilute sulphuric acid (H2SO4) at pH between pH 1.8 and 2.5 for 1–2 h (Basso et al 2011), which results in reduction in intracellular pH (Beales 2004), yeast viability and low ethanol yield (De Melo et al 2010)
Robust phenotype through short‐term adaptation and role of individual inhibitors at low pH To test the influence of short-term adaptation on tolerance to low pH and individual inhibitors, Brewer’s yeast strain (TMB3500) cells short-term adapted with the inhibitor cocktail (IC) at pH 5.0 and nonadapted cells were inoculated in defined media at pH 3.7 with acetic acid and individual inhibitors
Summary
Increasing concerns over the need for sustainable and scalable fuels as a means to curb global warming has led to focus on bioethanol production from renewable biomass, such as agricultural and industrial residues (Limayem and Ricke 2012). Several attempts have been made to study and control bacterial contamination in lignocellulosic ethanol production including: (1) adding NaCl and ethanol to wood hydrolysate (Albers et al 2011), (2) high solid loading in simultaneous saccharification and fermentation (SSF) (Ishola et al 2013), (3) usage of an antibiotic like gentamicin and biomass autoclaving (Serate et al 2015), and (4) usage of bacteriophages (Worley-Morse et al 2015) These strategies encounter challenges including: (1) additional cost and need for extensive fine tuning and testing of concentrations of NaCl and ethanol (Albers et al 2011), (2) loss of cell viability due to mechanical stress caused by solid particles in high cell loading (Ishola et al 2013), (3) cost and environmental challenges posed by gentamicin, energy expenditure and formation of inhibitors due to autoclaving (Serate et al 2015), and (4) rise of bacteriophageinsensitive mutants and possibilities of gene transfer from bacteriophages to yeast (Worley-Morse et al 2015). It might be efficient to develop S. cerevisiae strains tolerant to lower pH induced by inorganic acids
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