C5 fermentation; strain

engineering for high level

xylitol (and xylonate)

production

Merja Penttilä VTT Technical Research Centre of Finland

Name - Date

CIMV ORGANOSOLV PROCESS: C5 SYRUP

2

C6 cellulose

pulp C5

hemicellulose

syrup

lignin

Biotechnical products

Increased value of the fraction in a multiproduct biorefinery

Name - Date

PRODUCT OPTIONS FROM XYLOSE

3

C5 syrup

Dry matter (%) 48

Total Xyl (%) 55

Total Glu (%) 4

Lignin (%) 10

Protein (%) 44

Ash (%) 75

Glucose

Xylose

Arabinose

Ethanol Itaconic acid

Isopropanol Lactic acid, etc

A ”traditional” case:

Pentose fermentation

to ethanol from mixed

sugar solutions

MICROBE

Yeast

The CIMV C5 syrup is

rich in xylose

Name - Date

PRODUCT OPTIONS FROM XYLOSE

4

Xylose

Xylitol

A single step

reduction MICROBE

Yeast

Xylose

Xylonate

MICROBE

Yeast

A single step

oxidation

AIM: Specific conversion of the xylose-rich C5 syrup to a higher-value product

with good yields and productivities

XR XYD

Name - Date

● A natural sweetener with caries reducing

properties. Sweet as sucrose, 33% less calories.

● A renewable component for polyesters

(with TA/sebacic acid, alkyd resins, explosives)

● Today produced chemically from biomass

streams (e.g. sulphite spent liquor) with nickel

catalyst at high temperatures

XYLITOL

5

Xylose

Xylitol

A single step

reduction MICROBE

Yeast

XR

Name - Date

● Various yeast hosts engineered

- baker’s yeast Saccharomyces

- Candida/Pichia

● Industrially relevant productivities

of xylitol on pure xylose

● Titer > 160 g/l

● Rate > 2 g/l/h

● Yield > 0.9 g/g xylose

● Efficient production strains

available

XYLITOL PRODUCTION

6

0

50

100

150

0 50 100 150

Xylit

ol (g

l-1

)

Time (h)

Strain 2 - 3 copy

Strain 2 - 1 copy

B67002 multi-copy

B67002 2 copy

B67002 1 copy

B67002

Increases in xylitol production

through production host development

Production on pure xylose

Strain 2, P.kudriavzevii

Name - Date

● Formic acid concentration in the C5 syrup is high (50-100g/l; needs removal)

● Total xylose is ~ 200 g/l, but remains partly polymeric

C5 SYRUP CHALLENGES

7

0

20

40

60

80

100

120

0 50 100 150 200

Xylit

ol, r

esid

ua

l xylo

se

, fo

rma

te, a

ce

tate

(g L

-1)

Time (h)

70% v/v CIMV3 C5 in feed

Xylitol

Xylose

Formate

Acetate

Fed-batch culture

Feed of 70% CIMV C5 fraction

Titer > 100 g/l xylitol,

Rate > 0.82 g/l/h

Yield 0.89 g/g xylose

• Engineering yeast tolerance

towards formic acid improves

fermentation performance

Name - Date

● Alternative to gluconic acid

● Production from lignocellulose sugar xylose

● Chelator, dispersant, clarifying agent

● Cement retardant

● Polyester, -amide or hydrogel modifier

● 1,2,4 –butanetriol precursor

XYLONATE

8

Xylose

Xylonate

MICROBE

Yeast A single step

oxidation

XYD

Name - Date

XYLONATE PRODUCTION

9

0

50

100

150

200

0 50 100 150 200

D-X

ylo

na

te (g

/L)

Time (h)

pH 5.5 (165 g/l)

pH 5.5 (90 g/l)

pH 3 (155 g/l)

pH 3 (50 g/l)

0

50

100

150

Strain 1 Strain 2 Strain 3 Strain 4 Strain 5 Strain 6 Strain 7

D-X

ylo

na

te (

g l

-1)

strain

enzyme

condition

1

1

1

4

3

4

1

2

1

1

3

1

3

3

2

4

3

3

2

1

1

● Various yeast hosts engineered

- baker’s yeast Saccharomyces

- Candida/Pichia

● Industrially relevant productivities

of xylonate on pure xylose

● Titer > 160 g/l

● Rate > 1.4 g/l/h

● Yield > 0.9 g/g xylose

● Good production at low pH (pH 3)

● Efficient production strains available

Increases in xylonate production

through production host development

Production on pure xylose (amount of xylose provided in brackets)

Yeast strain

Name - Date

SUSTAINABILITY OF PRODUCT CHAINS

Distribution

ProcessingProcessing Processing

Pretreatment

Fractionation (Organosolv)

C5 C6 Lignin

Residues

Heat / power

generation

Use and end of life

Distribution Distribution

Fuel

Material

inputs

BIOCORE

biorefinery

Further

conversion

in chemical

industry

Processing

Fertiliser

Distribution of products

…

…Ethanol

… …

Itaconic acidXylitol

Ethanol

Lignin powder

Biomass

Power

Water

-5 -4 -3 -2 -1 0 1 2

Xylitol / IA

               Xylitol / ethanol

Ethanol / IA

SHF ethanol

t CO2 eq. / t biomass (dry)

Advantages Disadvantages Climate change

-15 -10 -5 0 5 10

Xylitol / IA

               Xylitol / ethanol

Ethanol / IA

SHF ethanol

kg SO2 eq. / t biomass (dry)

Advantages Disadvantages Acidification

● Xylitol is a favourable product based

on sustainability calculations

Name - Date

● Xylitol (and xylonate) provide biotech products

obtainable specifically from the hemicellulose C5

fractions of biorefineries

● Biotechnical production offers industrially relevant

productivities

● Yeasts are good and robust hosts for production

● CIMV hemicellulose syrup can be used in production

– process optimisation and host strain engineering to

improve tolerance towards toxic components

● Xylitol (and xylonate) provide good options to

increase the sustainability and economy of

multiproduct biorefineries

CONCLUSIONS

11

Name - Date

● Thank you all Biocore partners for a nice collaboration!

● The European Commission is kindly acknowledged for funding this

FP7 project under contract no FP7-241566 (2010-2014)

THANK YOU !