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Tomas-Pejo E.,Chalmers University of Technology | Bonander N.,Taurus Energy AB | Olsson L.,Chalmers University of Technology
Biofuels, Bioproducts and Biorefining | Year: 2014

There have been intense efforts in the scientific community and industry to engineer S. cerevisiae strains to co-consume xylose and glucose. In this context, this paper describes a process for introducing a barcode into an industrial xylose fermenting yeast strains to ensure quick and reliable identification of patented industrial strains. Furthermore, this work presents a novel procedure for screening and selecting the clones that reflects the characteristics of the industrial process. The key points of efficient selection of high-performing clones are elaborated and discussed. In spite of the unforeseen effects on yeast physiology after the introduction of a new sequence into the genome, we obtained barcoded clones with the same xylose consumption rates and ethanol yields as the cells without the barcode. © 2014 Society of Chemical Industry and John Wiley & Sons, Ltd.


Koppram R.,Chalmers University of Technology | Albers E.,Chalmers University of Technology | Albers E.,Taurus Energy AB | Olsson L.,Chalmers University of Technology
Biotechnology for Biofuels | Year: 2012

Background: 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. Results: A 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. Conclusion: Using 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. © 2012 Koppram et al.; licensee BioMed Central Ltd.


Koppram R.,Chalmers University of Technology | Nielsen F.,Lund University | Albers E.,Chalmers University of Technology | Albers E.,Taurus Energy AB | And 5 more authors.
Biotechnology for Biofuels | Year: 2013

Background: While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising process for bioconversion of lignocellulosic materials to ethanol, there are still relatively little demo-plant data and operating experiences reported in the literature. In the current work, we designed a SSCF process and scaled up from lab to demo scale reaching 4% (w/v) ethanol using xylose rich corncobs. Results: Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluated for their fermentation performance in hydrolysates of steam pretreated corncobs. Two strains, RHD-15 and KE6-12 with highest ethanol yield and lowest xylitol yield, respectively were further screened in SSCF using the whole slurry from pretreatment. Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the preferred strain since it produced 26% lower xylitol from consumed xylose compared to RHD-15. Model SSCF experiments with glucose or hydrolysate feed in combination with prefermentation resulted in 79% of xylose consumption and more than 75% of the theoretical ethanol yield on available glucose and xylose in lab and PDU scales. The results suggest that for an efficient xylose conversion to ethanol controlled release of glucose from enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout the SSCF. Fed-batch SSCF in PDU with addition of enzymes at three different time points facilitated controlled release of glucose and hence co-consumption of glucose and xylose was observed yielding 76% of the theoretical ethanol yield on available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF in combination with prefermentation and a feed of substrate and enzymes 47 and 40 g l§ssup§-1§esup§ of ethanol corresponding to 68% and 58% of the theoretical ethanol yield on available glucose and xylose were produced at 10.5% WIS in PDU and demo scale, respectively. The strain KE6-12 was able to completely consume xylose within 76 h during the fermentation of hydrolysate in a 10 m§ssup§3§esup§ demo scale bioreactor. Conclusions: The potential of SSCF is improved in combination with prefermentation and a feed of substrate and enzymes. It was possible to successfully reproduce the fed-batch SSCF at demo scale producing 4% (w/v) ethanol which is the minimum economical requirement for efficient lignocellulosic bioethanol production process. © 2013 Koppram et al; licensee BioMed Central Ltd.

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