DSM enzymes and process conditions for cellulosic ethanol

© 2015 DSM. All rights reserved1

DSM enzymes and process conditions for cellulosic ethanol

Herman PelDSM Biotechnology CenterDelft, The Netherlands

[email protected]

© 2015 DSM. All rights reserved2 Page 2

Chemical synthesis & biotechnology

Energy, chemistry & polymer technology

Biotechnology

Hoffman La Roche’s Vitamins (1930s)

DSM (1902)

Gist-Brocades (1869)

Vitamins Omega’s

CarotenoidsPremixes for food & feed

EnzymesMinerals

Cultures & YeastsNutraceuticals

PharmaceuticalsCellulosic bioethanolBiomedical materials

Bio-plasticsHigh Performance PlasticsPolyamides and precursors

Resins for coatings and composites

Functional MaterialsSolar – advanced surfaces

Life Sciences

Materials Sciences

Building on an impressive history


1000 30002000

Oil

cons

umpt

ion

Living off the landA brief

momentin history

Living off the land

At DSM sustainability is not just a buzz word


In the end we need to biorefine

Source: http://www.biocore-europe.org/page.php?optim=biocore-in-brief


Overview large scale bio-ethanol plant

700-1000 short tons / day 20-30 M gallons / yearGlucose, xylose


It is real! POET-DSM JV Project Liberty

• First commercial scale cellulosic ethanol plant of POET-DSM JV• Starting capacity 75 million liter Ethanol per year from corn

residues• Shared infrastructure with existing grain ethanol plant in

Emmetsburg Iowa• Local corn residues• Power for both plants

• Construction completedSeptember 2014

• Startup in progress

http://poetdsm.com/liberty


Feedstock / net wrap removal



Fermentation / Solid-liquid separation



The substrate is complex

38% Glucan

21% Xylan

18% Lignin

23% Other

Variable…Viscous…

Sand & Stones…


Xylose

Glucose

Acid pretreatment

Multi-enzyme hydrolysis A

cce

ssib

ilit

y

In 3 steps from Corn Residues to Ethanol

YeastFermentation

Ethanol

Glucose

Xylose

Glucan hydrolysis

Xylan hydrolysis


Main cellulases

LPMO


Removal of acetyl esters from xylo-oligosaccharides

Acetyl xylan esteraseAXH-m

Arabinofuranosidase active on mono substituted arabinoxylan

AXH-d3

Arabinofuranosidaseactive on the 3 position of double substituted arabinoxylan

Ferulic acid esterase

Removal of ferulic acid

Glucuronidase

Removal of glucuronic acid from xylo-oligosaccharides

Galactosidase

Removal of galactose from side chains

HemicellulasesEndo-xylanase

β-xylosidase


Requirements for enzyme hydrolysisEnzyme production process:• Low cost• Flexible raw materials

Hydrolysis process:• High sugar yield• Fast liquefaction• Low contamination

High yield – low cost requires integral approach


DSM thermostable enzyme cocktail for biofuel productionEnzyme cocktail build around enzymes derived from thermophilic filamentous fungus Rasamsonia emersonii

Enzyme development target: • Increase enzyme performance

Enzyme cocktail improvement strategies:• Classical strain improvement• Enzyme screening• Protein engineering


Natural selection of improved cocktailsClassical Strain Improvement (CSI)

Culture supernatant:Protein composition

analysis

Add to suspension of pretreatedwheat straw

Incubation:pH 4.5

time = 20 hrsTemp 65oC

SugarAnalysis

Cellmutagenesis Growth


CSI and Process Optimization

Parent0

0.2

0.4

0.6

0.8

1.0

Strain 1 Strain 2

Rela

tive

cel

lula

seac

tivi

ty

4x

2x

8-fold increase inrelative cellulaseactivity


CSI effects enzyme cocktail compositionStrong changes in amount and ratio of main cellulases

BG0

4

8

12

16

CBH

Rela

tive

pro

tein

con

tent

EG

ParentStrain 1


Enzyme ScreeningRecombinant produced single enzymes

• Spiking to base mix

• Compose artificial mixes

• Identify weak links

• Identify enzymes with

highest impact on

cellulose degradation

BG

CBHEG/LPMO

Others

26%42%

13%

Classes of enzymes tested

18%


Enzyme Mix OptimizationExperimental set-up

• Enzyme mix varied in 4 cellulases• Statistical mixture design:

– 55 incubations– same total protein dose– variation in ranges:

• BG 0.04-0.12• CBH 0.10-0.60• EG 0.18-0.68

• Conditions wheat straw assay:– pH 4.5– Temperature 65°C– Time 20h 0

10

20

30

10

Glucose released

(mM)

20 30 40 50


Optimized Enzyme Cocktail


Enzyme cocktail is active above 60°C

*

High performance in broad temperature range

Active at acidic pH

Improved control of contamination in hydrolysis


The effect of scale on oxygen supply

• LPMOs require oxygen for their oxidative action on crystalline cellulose

• Production scale: hardly any transfer of oxygen from head space due to unfavorable surface area to volume ratio!

• IP filed on oxygen/air supply and control on production scale

• Lab scale: good transfer of oxygen from head space due to favorable surface area to volume ratio!


The effect of scale on oxygen supply

Oxygen supply beneficial

Lab Pilot Production

Minimal oxygen level for LPMO

Reactor volume

Oxy

gen

tran

sfer

fro

m h

ead

spac

e


Example: the effect of oxygen at pilot scale

0

10

20

30

40

50

60

70

80

90

0 20 40 60 80 100 120 140

Glucose con

version (%

)

Hydrolysis time (h)

Pilot scale20 % NREL pCS

With air

Without air

Controlled aeration speeds up glucan conversion


Fast feedstock liquefaction

0 24 48 72 96 120

Hydrolysis time [h]

viscosity

glucose

Glu

cose

conv

ersi

on (%

)

Rel

ativ

e vi

scos

ity

Fast viscosity reduction 20% acid-pretreated wheat straw, 62*C

100 %

20 %

40 %

60 %

80 %

0 %

100 %

20 %

40 %

60 %

80 %

0 %


DSM enzymes for biofuel production

DSM thermostable enzymes

• Fast liquefaction allows high dry matter content up to 25%

• Thermostability allows for less energy use for cooling

• Thermostability gives improved control of contaminations

CAPEX savings due to lower required capacity of process equipment

OPEX savings due to reduced energy costs

Reduced antibiotic costsIncreased revenues because less sugars are lost

Cost savings!


On-Site Manufacturing of DSM enzymes

• The OSM unit is co-located with a lignocellulosic ethanol plant

• OSM makes optimal use of the existing infrastructure like utility systems, waste water treatment and rail/road connections

• OSM will use carbon sources directly available on site

• The OSM product is a whole broth enzyme and has not gone through the usual recovery step (filtration, concentration, formulation & packaging)

The main attributes of the OSM unit


Global commercialization

Examples of public partnerships on various continents


Acknowledgements

Partnership betwen


Thank you !!

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DSM enzymes and process conditions for cellulosic ethanol

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