The LignoRef project;
- A national research initiative to enhance biorefinery
process developments in Norway Nasjonalt Seminar Industriell Bioteknologi, Oslo, 06.06.2013
Karin Øyaas, Kai Toven1, Ingvild A. Johnsen1, Swarnima Agnihotri2, Størker Moe2, Al MacKenzie3,
Vincent Eijsink3, Nils Dyrset4, Roman Netzer4, Bjarte Holmelid5, Tanja Barth5, Ingvar Eide6.
1Paper
and Fibre Research Institute (PFI),
2Norwegian University of Science and Technology (NTNU),
3Norwegian University of Life Sciences (UMB),
4SINTEF Materials and Chemistry,
5University of Bergen (UoB),
6STATOIL Research Centre
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
LignoRef project
 Competence building (KMB) type project
 Period: 2009 – 2013
 Budget: 22,5 mNOK (70% public – RCN, 30% industry)
 Project partners (value-chain set-up):
 R&D:
o Universities: Norwegian University of Science and Technology (NTNU),
Norwegian University of Life Sciences (UMB), University of Bergen (UoB)
o R&D Institutes: SINTEF Materials and Chemistry, Paper and Fibre Research
Institute (PFI,project owner and coordinator)
 Industry:
o Forest owners: Allskog
o Biomass converters/biorefineries: Borregaard, Weyland, Xynergo/Norske Skog
o Technology providers: Cambi
o Energy producers: Statoil, Hafslund
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
LignoRef - Objectives
 LignoRef = Lignocellulosics as a basis for second generation biofuels and the
future biorefinery”
 Main objective:
→ Develop fundamental knowledge about central processes for cost
effective conversion of lignocellulosic materials into 2G biofuels and
value-added products.
 Central processes studied
 Pretreatment / decrystalisation and separation of biomass
• Biochemical and thermochemical conversion routes
 Enzymatic hydrolysis of biomass carbohydrates (cellulose, hemicelluloses)
 Fermentation of carbohydrates
 Thermochemical conversion of process by-products (e.g. lignin)
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
The LignoRef project - Layout
Research focus: Biofuels and chemicals from lignocellulose
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
R&D partners
 Trondheim:
 PFI
 Norwegian Univ. of Science
and Technology (NTNU)
 SINTEF
 Bergen:
 University of Bergen
 Ås:
 Univ. of Life Sciences
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
The lignocellulosic raw material
Biofuels / Biochemicals /
Biomaterials
Extractives 2-5%
Lignin 15-30%
Cellulose 35-50%
Fibre products /
Biofuels / Biochemicals /
Biomaterials
Hemicelluloses 25-30%
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
LignoRef – Inital definition of challenges
 Inhomogeneity of raw material → robust/flexible pretreatment processes
 Low energy density and high moisture content → high dry matter
concentration
 Physical inaccessibility; Cell wall barrier; Cellulose crystallinity → effective
biomass fragmentation to obtain high yields of fermentable sugars and good
separation of components
 Inhibitor formation during pretreatment → reduce inhibitor formation
 Lignin separation efficiency → improve enzyme accessibility → avoid inhibition
 High enzyme costs → improve enzyme efficiency
 Ineffective utilization of carbohydrates → C5-fermenting organisms
 Need to improve overall process economy → effective utilization of process
by-products (e.g. lignin, hemicelluloses); → biorefinery mindset
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP1: Pre-treatment
PFI / NTNU / UMB
 Optimizing pretreatment for cost- and energy effective
conversion;
 Pretreatment for biochemical conversion;
• Maximise yield of fermentable sugars
• Minimize formation of inhibitors – fate of hemicellulose
• Raw material flexibility
• Methods studied: Pre-extraction of hemicelluloses; Organosolv pulping;
Sulphite based pulping; Steam pretreatment
 Pretreatment for thermochemical conversion;
• Energy densification by torrefaction
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP1: Pre-treatment
PFI / NTNU / UMB
 Sulfite pretreatment
 “low temperature sulfonation” process
→ effective lignin dissolution (>75%),
lignosulfonates as by-product
→ high enzymatic saccharification yields
→ good control of hemicellulose degradation
 Organosolv (ethanol/water) pretreatment
 Up to 70% of spruce lignin could be removed; more effective
dissolution for bagasse than for spruce at lower temperatures.
 High enzymatic saccharification yield; up to 100 %.
Saccharification of spruce less sensitive to lignin content.
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP1: Pre-treatment
PFI / NTNU / UMB
 Steam pretreatment of wheat straw:
 → high glucose yields, but inhibitor formation remains a
challenge
 Hemicellulose pre-extraction:
 Hot water extraction suitable.
 Temp/time conditions aiming at high DP determined for bagasse
and spruce
 Torrefaction of spruce
 Significant increase in energy density.
 Suited for energy dense pellets and feed in entrained flow
gasifiers (after milling)
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP2: New enzymatic processes
UMB (Norw. Univ. of Life Sciences)
 Optimize enzyme mixtures and process conditions for
maximized hydrolysis yield
 Enzyme development:
• Accessory proteins (CBP21 and GH61-like)
• Contribution of hemicellulases and acetyl xylan esterases
• Optimization of enzyme mixtures/process conditions
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP2: New enzymatic processes
UMB (Norw. Univ. of Life Sciences)
 Use of helper proteins (CBM33s, GH61s) to better
degrade biomass
 Strengthen the effect of more traditional enzyme mixtures used in
cellulose hydrolysis.
 Search for novel biomass degrading enzymes focusing on
hemicellulose degradation
 E.g. genes involved in biomass degradation in the gut of Svalbard
reindeer identified and characterized
 Studies aimed at developing effective enzymatic
conversion protocols undertaken
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP3: Strain development and fermentation
SINTEF
 Develop new yeast strains capable of effective C5 and
C6 sugar fermentation;
 New strains for use in bioethanol production;
• Maximise ethanol yield, production rate, and tolerance to ethanol
and inhibitors
 Microbial products other than ethanol
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP3: Strain development and fermentation
SINTEF
 Strain developments based on S. cerevisiae
 This strain has excellent glucose fermentation capability, high
ethanol tolerance and resistant to inhibitors 
 Cannot utilize xylose 
 Development of S. cerevisiae mutants able to utilize
xylose:
 Genes for xylose utilization from the yeast Pichia stipitis
introduced into the chromosome of industrial S. cerevisiae strain
 Resulting mutants can convert xylose efficiently to cell biomass
and recombinant proteins
 Potential host for further genetic engineering.
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP4: Thermo chemical processing
Univ. of Bergen
 Conversion of lignin to fuel components and value-added
products;
 Mechanistic studies to identify major reaction pathways and ratelimiting steps
 Catalyst testing to reduce reaction time and/or pressure
 Optimisation of reaction conditions and product composition
 Evaluation of optimal product composition
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP4: Thermo chemical processing
Univ. of Bergen
 Solvolytic approach; Lignin-to-liquid (LtL) process further
refined (raw material demands, reaction conditions)
 Analysis of fragmentation patterns has shown:
 Different lignins behave rather similar during solvolysis, i.e the
LtL process is robust. Detailed composition of products may vary
somewhat.
 Mechanistic studies → basis for the further optimizations
 Model compound studies: Central reaction pathways identified.
 Solvent system studies (e.g. formic acid, paraformaldehyde,
water): Water identified as an alternative "green" solvent.
 Catalyst screenings → effective lignin depolymerisation, oxygen
removal and hydrogen incorporation at low temp. (< 360 ̊C)
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP5: Evaluation of bio-fuel quality
Statoil
 Analyses/characterisation of bio-fuels/bio-oils
 Chemical composition
 Fuel properties – needs for upgrading
 Health and environmental effects
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
SP5: Bio-oil quality
Statoil
 Comparisons of bio-oils to petro oils.
 Methods established:
• Standardized methods for analysing conventional fuels
• New methods for analysing components not normally found in petrobased products
 Needs for upgrading of bio-oils to transport fuel quality
determined using
 Mass spectrometry based “fingerprinting” techniques and chemo
metrics central in this work
 HSE studies of bio-oils ongoing
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
Other project activities
 Initial standardisation/calibration of analytical methods among
partners
 Literature surveys describing the possible co-production of
chemical/biotechnological products and their value-adding
effect
 Continuous exchange of samples between partners
 Implementation of joint demo trial in the latter phase of the
project involving:
Sulphite pretreatment (PFI) – Enzymatic hydrolysis (UMB) –
Fermentation (SINTEF) – Solvolysis of by-products (UoB)
 Researcher education – 3 postdocs and 1 PhD
 Disseminations
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
Conclusions
 The LignoRef project has gathered central Norwegian
players along the value chain from biomass to
bioproduct.
 Fundamental knowledge about central processes
involved in the development of cost-effective conversion
of lignocelluloses has been established.
 Pretreatment and separation
 Enzymatic hydrolysis
 Fermentation and thermochemical conversion processes
 The project has promoted national collaboration and
progress in the biorefinery area.
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute
Acknowledgement
 We gratefully acknowledge The Research Council of
Norway (grant no. 190965/S60), Statoil ASA, Borregaard
AS, Allskog BA, Cambi AS, Xynergo AS/Norske Skog,
Hafslund ASA and Weyland AS for financial support.
 My co-authors:
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PFI: Ingvild A. Johnsen, Kai Toven
NTNU: Størker Moe, Swarnima Agnihotri (postdoc)
UMB: Vincent Eijsink, Al MacKenzie (postdoc)
UoB: Tanja Barth, Bjarte Holmelid (postdoc), Mikel Oregui (PhD)
Statoil: Ingvar Eide
Papir- og fiberinstituttet AS
Paper and Fibre Research Institute