Abstract:The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains perf… Show more
“…3) in comparison to an inoculum size of 0.5 or 1 gdw L −1 , where no growth was observed even after 130 h of incubation. The short-term adapted biomass at higher concentration might possess the required volumetric reductase activity to efficiently detoxify HMF and furfural to their corresponding alcohols (Modig et al 2008) reaching inhibitor threshold levels for allowing growth at the low pH in presence of acetic acid.Fig. 3Effect of initial cell density on growth and inhibitor tolerance of strain TMB3500 at pH 3.7 with the IC after short-term adaptation.…”
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.
“…3) in comparison to an inoculum size of 0.5 or 1 gdw L −1 , where no growth was observed even after 130 h of incubation. The short-term adapted biomass at higher concentration might possess the required volumetric reductase activity to efficiently detoxify HMF and furfural to their corresponding alcohols (Modig et al 2008) reaching inhibitor threshold levels for allowing growth at the low pH in presence of acetic acid.Fig. 3Effect of initial cell density on growth and inhibitor tolerance of strain TMB3500 at pH 3.7 with the IC after short-term adaptation.…”
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.
“…6). Additionally, tolerance toward furaldehydes is clearly strain dependent and obtained by the reduction of these compounds to less-toxic alcohols (6,9,39,40). It is conceivable that yeast strains with greater tolerance of furaldehydes would require higher concentrations of furfural and HMF to cause the formation of P-bodies and/or SGs.…”
Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.
“…The effects of inhibitory compounds in a fermentation process were shown as longer lag-phase, slower growth, lower cell density and decreased ethanol productivity [9,10]. To be able to use hydrolysate for biofuel production on an industrial level, these effects need to be reduced.…”
To compare the composition and performance of various lignocellulosic biomass hydrolysates as fermentation media, 8 hydrolysates were generated from a grass-like and a wood biomass. The hydrolysate preparation methods used were 1) dilute acid, 2) mild alkaline, 3) alkaline/peracetic acid, and 4) concentrated acid. These hydrolysates were fermented at 30°C, pH 5.0 using Saccharomyces cerevisiae CEN.PK113-7D as model strain. The growth in different hydrolysates varied in the aspects of lag-phase, growth rate, glucose consumption rate and ethanol production rate. Subsequently, 11 potential inhibitory compounds as described in literature were selected for further analysis. The concentrations of these compounds were determined in the time-samples of the 8 fermentations, using a novel analytical method, ethyl-chloroformate derivatization-GC-MS. Some of these compounds, e.g. furfural, decreased during the fermentation process, while others, such as formic and benzoic acid, remained almost constant. Inhibitory effect analysis of individual compound revealed that most of these compounds exhibit little effect at the concentrations detected in hydrolysates. Only furfural and benzoic acid clearly affected the growth of the model yeast: furfural elongated the lag-phase, while benzoic acid reduced the growth rate and biomass yield.
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