Pentose utilizing S.

cerevisiae in biorefineries

Brbel Hahn-Hgerdal Lund University

Helsinki 2006

My colleagues
Marie F Gorwa-Grauslund marie-francoise.gorwa@tmb.lth.se Guido Zacchi guido.zacchi@chemeng.lth.se Gunnar Lidn gunnar.liden@chemeng.lth.se Mats Galbe mats.galbe@chemeng.lth.se

Folke Tjerneld folke.tjerneld@biokem.lu.se

Henrik Stlbrand henrik.stalbrand@biokem.lu.se

LU Ethanol Program is/has been financially supported by

Swedish National Energy Administration (STEM) Swedish National Science Research Council (VR) Swedish Foundation for International Co-Operation in Research and Higher Education (STINT) Nordic Energy Research Programme EC BIO-CT95-0107 Yeast Mixed Sugar Metabolism EC QLK3-1999-00080 BIO-HUG http://www.nile-bioethanol.org/ EC Marie Curie Training Site QCIM EC Marie Curie EST BIONEL EC Marie Curie Fellowships

The greatest research challenge of the 21st century.... ....provide energy for
....

reduce global warming!

transport heating/ cooling electricity

....

provide carbon for

chemicals & materials

How to replace this......

.....with this!

CO2

The "driving force" for developing bio-refineries.......

The Economist, 1996.... ......10 years later

>

=> $ 80/barrel

12th European Congress on Biotechnology, 2005: Biotechnology for the sustainable production of fuels is likely to affect the lives of more people than any other application of biotechnology

Biotechnology in process industry

LU process concept
Chemicals & Materials

Raw material

Pretreatment

Enzymatic hydrolysis

Fermentation

Distillation

Ethanol

SSF

Lignin

Why S. cerevisiae?
E. coli
Filamentous fungi

rec. genes

Pentoses rec. genes

P. stipitis

Ethanol

Z. mobilis

S. cerevisiae

S. cerevisiae is oldest, largest and adapted to the industrial context

Process integration 20% reduced production cost

ngpannefreningen (1994) Report P23332-1, NUTEK, Stockholm, Sweden Wooley et al. (1999) NREL/TP-530-26157 Hahn-Hgerdal et al. (2006) TRENDS in Biotechnol (in press)

Bench-marking pentose-utilizing yeast

Water economy Inhibitor tolerance Product yield Productivity

The raw material

Wheat straw
Marie Linde, LU

Steam pretreated wheat straw

40

30 Conc. (g/L) 20

Ethanol

Xylose
10

Glucose
0 0 20 40 60 80 100

Time (h) SSF, Fed-Batch, 11% Fiber content

hgren et al. (2006) J Biotechnol doi:10.1016/j.jbiotec.2006.05.001

50

Energy demand in distillation Batch SSF, BY

40

Energy (MJ/kg ethanol)

30

Batch SSF, TMB3400

20

Fedbatch SSF, TMB3400

10

0 0 2 4 6 8 10 12

Ethanol conc. in feed (wt-%)

Zacchi and Axelsson (1989) Biotechnol Bioeng 34:223-233

Bench-marking pentose-utilizing yeast

Water economy Inhibitor tolerance Product yield Productivity

CH2OH O OH OH HO OH
CH2OH O OH OH HO
CH2OH O HO
OH

glucose

Hydrolysate
Hemicellulose (20%) Cellulose (42%)

CH2OH O OH OH HO OH

glucose

OH

mannose galactose

OH OH

HO

CH3 OH

O OH

rhamnose

OH

Extractives Lignin (21%)

O OH OH HO OH

xylose arabinose
FURALDEHYDES
HO

O OH HOH2C

OH

OH

ALIPHATIC ACIDS acetic acid

CH3COOH HCOOH

PHENOLICS
R

CH2

CHO

R OH

HMF
O CHO

formic acid
CH3COCH2CH2COOH

furfural

levulinic acid

WOOD RESIN Terpenoids etc.

FERMENTATION INHIBITORS

Strain development and engineering

Yeast isolates Glucose Xylose Arabinose Ethanol +++ -

TMB yeasts
Ethanol +++ ++ +

In situ detoxification
Batch
Measurement
CER (mmol/h) 80 70 60 50 40 30 20 10 0 0 5 10 15 Time (h) 20 25 30

CFU= 88% CFU= 0%

Controlled Fed-batch
1000 900 800 CER (mmol/h)

Control
Modelling (on-line)

700 600 500 400 300 200 100 0 0 5 10 15

C FU = 100%

C FU = 100% 20 25
Time (h)

30

35

40

Rudolf et al (2004). Appl Biochem Biotechnol 113-116:601-617

Gene chip

mRNA

TMB3000 TMB3000 + HMF CBS8066 CBS8066 + HMF

Reductase/dehydrogenase genes Analyses Scanning & quantification

ADH6 strain in spruce hydrolysate

Control
30

+Hydrolysate
0,9 0,8

ADH6
+Hydrolysate
30 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 30 40 50

(g/l) (% x 5)

EtOH
20

0,7 0,6 0,5 0,4

HMF
20

(g/l)

10

0,3

10

CO2
0 0 10 20 30 40 50

0,2 0,1 0

0 0 10 20

Modig et al. (2006) 28th Biotech Conf Fuels Chem

Bench-marking pentose-utilizing yeast

Water economy Inhibitor tolerance Product yield Productivity

Product yield

mixed sugar utilisation

Glucose Mannose Galactose Xylose Arabinose Saccharomyces cerevisiae Ethanol

....in a very harsh environment!!!

Relative amounts of pentoses

Substrate Corn stover Corn cob hulls Grass Wheat bran Glucan 35% 36% 37% 25%
Percentage of dry weight

Xylan 19% 20% 16% 19%

Arabinan 3% 14% 5% 15%

Hayn et al. (1993) In Bioconversion of forest and agricultural plant residues. p 33-72

Arabinose + xylose co-fermentation

Bacterial arabinose pathway
L-arabinose D-xylose NAD(P)H

Fungal xylose pathway

AraA
L-ribulose

XR
D-xylitol ATP

NAD(P)+ NAD+

AraB

XDH

L-ribulose-5-P

NADH D-xylulose ATP

AraD

XK
D-xylulose-5-P

Ethanol

Karhumaa et al. (2006) Microbial Cell Factories 5:18

Strain construction
Xyl
+AraBAD

Ara + Xyl
+AraA

Ara + Xyl TMB 3063

TMB 3063

TMB3400
8

TMB3061

OD

TMB 3061

TMB 3400
0 0 40 80 120

Aerobic growth on arabinose

Karhumaa et al. (2006) Microbial Cell Factories 5:18

Bench-marking pentose-utilizing yeast

Water economy Inhibitor tolerance Product yield Productivity

Improved ethanol productivity:

high XR and XDH

Xylose 1 1
Xylitol Glucose Xylulose

Xylitol 0.59 out 0.27 0.41 0.73

ox PPP

Low XR +XDH High XR +XDH

nonnon-ox PPP

0.03 Glycerol 0.14 Ethanol 0.28 0.36

Karhumaa et al. (2006) Appl Microb Biotechnol (in press)

Conclusion: The industrial context defines the benchmarks

Molecular biology Process design Strain engineering

Fermentation technology

Enzyme technology