Designing simultaneous saccharification and co-fermentation of lignocellulose for improved xylose conversion

Kim Olofsson

Designing simultaneous saccharification and co-fermentation of lignocellulose for improved xylose conversion

Kim Olofsson

Avdelningen för kemiteknik

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

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Popular Abstract in Swedish
Global uppvärmning, säkerhetspolitik och framtida minskad tillgång på fossila bränslen har de senaste åren skyndat på sökandet efter nya, förnyelse-bara drivmedel. Användningen av bioetanol har ökat avsevärt och är idag ett av de vanligaste biobränslena i världen.

Idag framställs bioetanol vanligen genom jäsning av socker från socker-rör eller från stärkelse (från till exempel majs eller vete), av vanlig bagerijäst, Saccharomyces cerevisiae. Detta brukar ofta benämnas första generationens bioetanol. Användningen av dessa råvaror kan dock konkurrera med använd-ning till livsmedel och djurfoder. För att tillgodose framtida behov måste därför råmaterial bestående av lignocellulosa användas, till exempel rest-produkter från skogsbruk och jordbruk. Denna tillverkningsprocess brukar kallas andra generationens bioetanol.

En av de stora utmaningarna är att denna råvara innehåller pentoser, framförallt xylos, vilka jästen Saccharomyces cerevisiae, inte kan jäsa. Genom genteknik kan dock modifierade jäststammar konstrueras med förmåga att ta upp och jäsa exempelvis xylos. Dock är jäsningen inte optimerad för xylos, vilket gör jäsningen mycket långsammare än för andra sockerarter som till exempel glukos.

Utbytet är en av de viktigaste parametrarna när det gäller att utveckla en ekonomiskt hållbar industriell process för produktion av andra genera-tionens bioetanol. Arbetet i denna avhandling har syftat till att utveckla en SSCF-process (simultaneous saccharification and co-fermentation) där jästen så effektivt som möjligt tar upp och fermenterar såväl xylos som glukos, som frigörs i processen då enzymer (cellulaser) bryter ner lignocellulosa till olika socker. Genom att experimentellt undersöka bland annat temperatur-påverkan, enzym- och jästkinetik, samt kombination av olika feed-strategier har en effektivare SSCF-process kunnat tas fram. Vidare har nya, genetiskt förbättrade, xylosjäsande jäststammar undersökts i SSCF. Både genom för-bättrad processteknik och förbättrade jäststammar har detta arbete har lett fram till avsevärd ökning av xylosomvandling i SSCF, i vissa fall med upp till det dubbla, vilket i sin tur ökat det totala etanolutbytet.

Utbytet är en av de viktigaste parametrarna när det gäller att utveckla
en ekonomiskt hållbar industriell process för produktion av andra generationens
bioetanol. Arbetet i denna avhandling har syftat till att utveckla en
SSCF-process (simultaneous saccharification and co-fermentation) där jästen
så effektivt som möjligt tar upp och fermenterar såväl xylos som glukos, som
frigörs i processen då enzymer (cellulaser) bryter ner lignocellulosa till olika
socker. Genom att experimentellt undersöka bland annat temperaturpåverkan,
enzym- och jästkinetik, samt kombination av olika feed-strategier
har en effektivare SSCF-process kunnat tas fram. Vidare har nya, genetiskt
förbättrade, xylosjäsande jäststammar undersökts i SSCF. Både genom förbättrad
processteknik och förbättrade jäststammar har detta arbete har lett
fram till avsevärd ökning av xylosomvandling i SSCF, i vissa fall med upp till
det dubbla, vilket i sin tur ökat det totala etanolutbytet.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution	Avdelningen för kemiteknik
Handledare	Lidén, Gunnar, handledare
Tilldelningsdatum	2011 maj 13
ISBN (tryckt)	978-91-7422-270-8
Status	Published - 2011

Bibliografisk information

Defence details

Date: 2011-05-13
Time: 10:30
Place: Lecture hall K:B, Kemicentrum, Getingevägen 60, Lund University Faculty of Engineering

External reviewer(s)

Name: van Zyl, Willem H.
Title: Prof.
Affiliation: Department of Microbiology, University of Stellenbosch, South Africa

---

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title = "Designing simultaneous saccharification and co-fermentation of lignocellulose for improved xylose conversion",

abstract = "Fuel ethanol from lignocellulose is one sustainable alternative to the fossil fuels of today. All sugars in the material must be utilized in order to achieve high overall ethanol yields. Baker{\textquoteright}s yeast, Saccharomyces cerevisiae, has been engineered to ferment the pentose sugar xylose from lignocellulose to ethanol. However, ethanol production from xylose is slow and often incomplete. In this work simultaneous saccharification and co-fermentation (SSCF) of xylose and glucose has been investigated with the purpose of improving xylose conversion and ethanol yields in non-detoxified lignocellulosic hydrolyzates. There are two main approaches to improve xylose conversion, which both have been investigated in this work. One way is to enhance the performance of the yeast by designing the process. The other way is to improve the yeast itself by genetic and/or evolutionary engineering. It was found that through careful design of different feeding strategies the xylose fermentation in SSCF could be significantly increased. Fed-batch, prefermentation, controlled enzyme feeding as well as combined enzyme and substrate feeding, had all positive effects on the xylose conversion. Depending on feed strategy and process conditions, this also resulted in a significant increase of the ethanol yield. Moreover, by designing the SSCF process, it was possible to increase the final solids content in the SSCF and still obtain a relatively high ethanol yield on total sugars, which is crucial for the process economy in commercial scale. The effect of improved xylose transport capacity in the yeast was investigated by expression of the glucose/xylose facilitator Gxf1 from Candida intermedia, in strains of S. cerevisiae which were assessed in SSCF. The improved transport proved to increase xylose uptake, but had only a minor effect on the ethanol yield. The enzyme xylose reductase (XR) was implicated to control xylose fermentation in SSCF of pretreated lignocellulose. When a mutated XR (mXR), with higher activity and altered co-factor preference, was integrated in S. cerevisiae the xylose uptake was significantly improved, resulting in a higher ethanol yield in comparison to when the native Pichia stipitis XR was used. Gxf1 had, however, only little influence when the combined effect of Gxf1 and mXR was studied in SSCF, which indicated that the initial xylose catabolism was still rate limiting.",

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N2 - Fuel ethanol from lignocellulose is one sustainable alternative to the fossil fuels of today. All sugars in the material must be utilized in order to achieve high overall ethanol yields. Baker’s yeast, Saccharomyces cerevisiae, has been engineered to ferment the pentose sugar xylose from lignocellulose to ethanol. However, ethanol production from xylose is slow and often incomplete. In this work simultaneous saccharification and co-fermentation (SSCF) of xylose and glucose has been investigated with the purpose of improving xylose conversion and ethanol yields in non-detoxified lignocellulosic hydrolyzates. There are two main approaches to improve xylose conversion, which both have been investigated in this work. One way is to enhance the performance of the yeast by designing the process. The other way is to improve the yeast itself by genetic and/or evolutionary engineering. It was found that through careful design of different feeding strategies the xylose fermentation in SSCF could be significantly increased. Fed-batch, prefermentation, controlled enzyme feeding as well as combined enzyme and substrate feeding, had all positive effects on the xylose conversion. Depending on feed strategy and process conditions, this also resulted in a significant increase of the ethanol yield. Moreover, by designing the SSCF process, it was possible to increase the final solids content in the SSCF and still obtain a relatively high ethanol yield on total sugars, which is crucial for the process economy in commercial scale. The effect of improved xylose transport capacity in the yeast was investigated by expression of the glucose/xylose facilitator Gxf1 from Candida intermedia, in strains of S. cerevisiae which were assessed in SSCF. The improved transport proved to increase xylose uptake, but had only a minor effect on the ethanol yield. The enzyme xylose reductase (XR) was implicated to control xylose fermentation in SSCF of pretreated lignocellulose. When a mutated XR (mXR), with higher activity and altered co-factor preference, was integrated in S. cerevisiae the xylose uptake was significantly improved, resulting in a higher ethanol yield in comparison to when the native Pichia stipitis XR was used. Gxf1 had, however, only little influence when the combined effect of Gxf1 and mXR was studied in SSCF, which indicated that the initial xylose catabolism was still rate limiting.

AB - Fuel ethanol from lignocellulose is one sustainable alternative to the fossil fuels of today. All sugars in the material must be utilized in order to achieve high overall ethanol yields. Baker’s yeast, Saccharomyces cerevisiae, has been engineered to ferment the pentose sugar xylose from lignocellulose to ethanol. However, ethanol production from xylose is slow and often incomplete. In this work simultaneous saccharification and co-fermentation (SSCF) of xylose and glucose has been investigated with the purpose of improving xylose conversion and ethanol yields in non-detoxified lignocellulosic hydrolyzates. There are two main approaches to improve xylose conversion, which both have been investigated in this work. One way is to enhance the performance of the yeast by designing the process. The other way is to improve the yeast itself by genetic and/or evolutionary engineering. It was found that through careful design of different feeding strategies the xylose fermentation in SSCF could be significantly increased. Fed-batch, prefermentation, controlled enzyme feeding as well as combined enzyme and substrate feeding, had all positive effects on the xylose conversion. Depending on feed strategy and process conditions, this also resulted in a significant increase of the ethanol yield. Moreover, by designing the SSCF process, it was possible to increase the final solids content in the SSCF and still obtain a relatively high ethanol yield on total sugars, which is crucial for the process economy in commercial scale. The effect of improved xylose transport capacity in the yeast was investigated by expression of the glucose/xylose facilitator Gxf1 from Candida intermedia, in strains of S. cerevisiae which were assessed in SSCF. The improved transport proved to increase xylose uptake, but had only a minor effect on the ethanol yield. The enzyme xylose reductase (XR) was implicated to control xylose fermentation in SSCF of pretreated lignocellulose. When a mutated XR (mXR), with higher activity and altered co-factor preference, was integrated in S. cerevisiae the xylose uptake was significantly improved, resulting in a higher ethanol yield in comparison to when the native Pichia stipitis XR was used. Gxf1 had, however, only little influence when the combined effect of Gxf1 and mXR was studied in SSCF, which indicated that the initial xylose catabolism was still rate limiting.

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KW - lignocellulose

KW - simultaneous saccharification and co-fermentation

KW - xylose fermentation

M3 - Doctoral Thesis (compilation)

SN - 978-91-7422-270-8

ER -

Designing simultaneous saccharification and co-fermentation of lignocellulose for improved xylose conversion

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