Abstract
BackgroundOne of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation.ResultsA long-term adaptation in repetitive batch cultures in shake flasks using a cocktail of 12 different inhibitors and a long-term chemostat adaptation using spruce hydrolysate were used as evolutionary engineering strategies to improve the inhibitor tolerance in the metabolically engineered xylose utilizing Saccharomyces cerevisiae strain, TMB3400. The yeast was evolved for a period of 429 and 97 generations in repetitive batch cultures and chemostat cultivation, respectively. During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h-1 to 0.33 h-1 and the time of lag phase was decreased from 48 h to 24 h. In the chemostat adaptation, after 97 generations, the specific conversion rates of HMF and furfural were found to be 3.5 and 4 folds higher respectively, compared to rates after three generations. Two evolved strains (RK60-5, RKU90-3) and one evolved strain (KE1-17) were isolated from evolutionary engineering in repetitive batches and chemostat cultivation, respectively. The strains displayed significantly improved growth performance over TMB3400 when cultivated in spruce hydrolysate under anaerobic conditions, the evolved strains exhibited 25 to 38% increase in specific consumption rate of sugars and 32 to 50% increased specific ethanol productivity compared to TMB3400. The evolved strains RK60-5 and RKU90-3 were unable to consume xylose under anaerobic conditions, whereas, KE1-17 was found to consume xylose at similar rates as TMB3400.ConclusionUsing evolutionary engineering strategies in batch and chemostat cultivations we have generated three evolved strains that show significantly better tolerance to inhibitors in spruce hydrolysate and displayed a shorter time for overall fermentation of sugars compared to the parental strain.
Highlights
One of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation
Evolutionary engineering in repetitive batch cultures with inhibitor cocktail A cocktail of 12 different inhibitors spanning three different inhibitor categories: furans, weak acids and phenolics (Table 1) were used to evolve S. cerevisiae TMB3400 in repetitive batch cultures aiming at enhancing its inhibitor tolerance
The potential of evolutionary engineering is more vivid in improving a complex phenotypic trait that requires multi-gene modifications such as inhibitor tolerance
Summary
One of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation. Phenolics include a wide variety of aromatic alcohols, aldehydes and acids, some of them notably include catechol, coniferyl alcohol, coniferyl aldehyde, vanillin, syringaldehyde, hydroquinone, cinnamic acid, p-coumaric acid and these compounds have been shown to limit the growth of S. cerevisiae and ethanol formation [13], the mode of action of these weak acids and phenolics on microbial physiology still remains unclear due to their molecular heterogeneity and lack of qualitative and quantitative analyses of high accuracy Several detoxification methods such as alkali treatment, sulfite treatment, evaporation, anion exchange and treatment with laccase have been used to remove or decrease the level of inhibitory compounds in lignocellulosic hydrolysate leading to improved fermentability, these methods resulted in loss of fermentable sugars [14]. The aims of the present work were to compare the fermentative performance of evolved strains with that of parental strain using spruce hydrolysate and to compare the outcome of two modes of evolutionary engineering
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
