advanced biofuels from wood using the biotech route presentations/wentzel.pdf · 2017. 2. 15. ·...
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ADVANCED BIOFUELS FROM WOOD USING THE BIOTECH ROUTEDr. Alexander WentzelSenior ResearcherSINTEF Materials and Chemistry, Dept. Biotechnology and Nanomedicine
Norwegian Centre for Sustainable Bio-based Fuels and Energy (Bio4Fuels) - Kick-off SeminarNMBU, Ås, Norway - 2017-02-10
SINTEF Materials and ChemistryDepartment of Biotechnology and Nanomedicine
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Mass spectrometryKolbjørn Zahlsen
BiotechnologyHåvard Sletta
Vice president researchTrond E. Ellingsen
Polymer Particles and Surface Chemistry
Heidi Johnsen
Pro
ject
s an
d in
fras
tru
ctu
re
63 employees + PhD students, post-docs
Nat
ion
al a
nd
inte
rnat
ion
al
rese
arch
gro
up
s an
d
ind
ust
ry
2016:> 70 running projects
Industry
Researcher-driven
Locally and project-wise closely integrated with other SINTEF departments and NTNU
Biotechnology R&D at SINTEF
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Bacteria
Yeast
Fungi
ANTIBIOTICS
SUGAR
PRODUCT
ENZYMES
LIPIDS
PIGMENTS
ORGANIC
ACIDS
BIO-POLYMERS
FUELS
LYSINE
GLUTAMIC ACID
Current tool-box
• General microbiology
• Molecular Biology
• Bioprocess engineering
• Fermentation technology
• Systems Biology
• Synthetic Biology
• Metabolic engineering
• Recomb. gene expression
• MS analytics
• High throughput screening
• Functional Metagenomics
• Polymer/nanoparticles
• Enzyme immobilization
Current markets
• Biorefinery / Biofuels
• Biopolymers
• Biopharmaceuticals
• Industrial enzymes
• Bio-processes and up-scaling
• Methylotrophy
• Fish vaccines
• Oil reservoir microbiology
• Marine Bioprospecting
• Microbial production strains
• Food and Feed
• Medical (bio-)technology
• Clean water
Technology platforms
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Advanced MS analyticsLC, GC, MALDISQ, QQQ, QTOF, FT-ICR, ICP, FFF
Molecular biology
Cytometry, cell biology
Fermentation facilities48x 1-3 ml RoboLector16x 0.5-1 L DASGIP16x 1-3 L Applicon50 L, 300 L pilot plant
High throughput technologyScreening, cultivation,downscaling, HT-MS
NorBioLab
Biotechnology for Biorefineries
• Enzymatic hydrolysis of polysaccharides to fermentable sugars
• Microbial conversion of sugars to specific chemicals (e.g. ethanol, n-butanol, acetone,
diols, lipids, amino acids, protein, high value products); also syngas/CO2 as feedstock
• Products: fuels, platform chemicals, commodity chemicals, feed, food additives, pharmaceuticals, etc.
• Platform organisms: yeasts, bacteria, fungi
• Genetic tools for desired modifications (products, productivity, tolerance), metabolic engineering (e.g.
CRISPR/Cas technology), 'omics technologies (Systems Biology), Synthetic Biology
• Anaerobic digestion of residuals to biogas, fertilizer
• Enzymatic conversion of aromatic fraction/lignin to aromatic chemicals, bioplastics
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SP3WP3.4
Up-sides of the biotech route:
• Defined products; limited refining efforts after fermentation and product recovery
• Large spectrum of possible products; entire pyramid addressable
• Only commercial large scale biofuel produced based on the biotech route, including from cellulosic material
Special challenge using wood as a feedstock:
• Density of the material; specialized preprocessing and fractionation technology needed to make carbohydrates accessible for fermentation (e.g. BALI process of Borregaard allowing multiple product streams)
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Biotechnology for Biorefineries
Challenges of the biotech route:
• Multiple step procedure prior to fermentation (pretreatment, fractionation, enzymatic hydrolysis)
• Often low initial productivities in product formation
• Challenge of complete utilization of sugars (C6/C5)
• Sensitivity to inhibitors from pretreatment and high product concentrations
• Production in the aqueous phase; energy demands for product recovery
• Potential strain and process robustness issues; genetic stability, contaminants, feedstock flexibility
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Biotechnology for Biorefineries
Suggested workarounds
Strain selection and development
• Strain selection (consider use of thermophilic cell factory platforms)
• Implement and metabolically optimize formation of the desired product (Metabolic engineering, Synthetic Biology)
• Increase tolerance to products produced and inhibitors from feedstock preprocessing (Adaptive evolution, metabolic engineering)
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Process development
• High temperature processes for overall improved reaction rates, facilitated product removal, lower contamination risks
• Optimize for formation of product vs. side products (e.g. cell mass, CO2)
• Integrate aspects of biomass processing/hydrolysis into fermentation (SHF/SFF/CBP), enzyme recycling, high dry matter processes
• Reduce inhibitor formation by optimized biomass preprocessing
• Implement efficient product removal and process integration
Thermophilic strain selection
• Choose/select for microorganisms that are naturally able to degrade lignocellulosic
material at elevated temperature. The diversity of enzymes produced by these
organisms reflects the complexity and heterogeneity of lignocellulosic material.
• The microorganisms themselves or their enzymes are potential candidates to be
included in CBP platforms (thermophilic microbial cell factories).
• Known thermophilic species able to degrade lignocellulosic material include
Clostridium thermocellum, Thermoanaerobacter mathranii and Acidothermus
cellulyticus
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• Thermophilic species able to ferment C5 and C6 sugars to higher alcohols and
esters are Clostridium thermobutyricum, Clostridium pathermopalmarium, and
species within the genera Thermoanaerobacterium and Thermobacterium.
• The ability for e.g. butanol/butyric acid producing bacteria to utilize lignocellulose is
yet quite limited. Co-culturing with organisms able to hydrolyze cellulose is an
option; e.g. co-culturing of Clostridium thermocellum with Clostridium
thermobutyricum is one bioreactor.
• Combining desired metabolic traits can be targeted in subsequent strain
engineering strategies.
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Thermophilic strain selection
Biotech tools for strain development
• Adaptive evolution
• Metabolic engineering
• Systems Biology
• Synthetic Biology
• Aims: improved tolerance, improved productivity, new products
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Adaptive laboratory evolution
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Adaptive evolution results from the generation
and propagation of advantageous mutations
through positive selection. (Wikipedia)
ALE aims (examples):• Increased use of nutrients, growth• Increased fitness to environmental stress (Temp., UV, osmotic,
products, toxic chemicals, etc.)
Dragosits & Mattanovich (2013) Microbial Cell Factories 12(1):64
http://www.hngn.com/articles/68528/20150211/darwins-finches-gene-behind-icon-of-adaptive-evolution-revealed-for-the-first-time.htm
Metabolic engineering
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Metabolic engineering is the practice
of optimizing genetic and regulatory
processes within cells to increase the
cells' production of a certain substance. (Wikipedia)
Can be guided by Systems Biology
understanding of cell function.
https://www.sciencemag.org/
http://sustainablepulse.com/
MetEng aims (examples):• Channelling of carbon to specific products• New products by linking new genes to metabolism• Optimizing import of substrates, export of products• Implementing findings from ALE
http://www.sysbio.de/
Systems Biology
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Systems biology is a holistic
approach to understanding
biology.
It aims at system-level
understanding of biology, and to
understand biological units as a
system. This means an
examination of the structure and
dynamics of cellular and
organismal function, rather than
the characteristics of isolated
parts of a cell or organism. (Wikipedia)
Modelling of cellular metabolic networks
Synthetic Biology
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Synthetic biology is an interdisciplinary branch of
biology, combining disciplines such as biotechnology,
evolutionary biology, molecular biology, systems
biology, biophysics, computer engineering, and
genetic engineering. (Wikipedia)
A definition: "Designing and constructing biological
devices, biological systems, and biological machines
for useful purposes."
http://synbiology.co.uk/
Bottom-up: create synthetic life
Top-down: engineer new traits (e.g. synthetic pathways thatgenerate desired products) in established microbial chassis
SysBio TD aims (examples):• New products and substrate uses by
introducing entire new pathways• Changing of overall cell properties (e.g.
whole cell biocatalysis)
Nielsen J, Keasling JD. (2011) Nat Biotechnol. 29(8):693-5.
Synthetic Biology
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(Barrett et al., 2006: Curr Opinion Biotechnol 17:488-492)Systems biology = analyticalSynthetic biology = operational → Implement module that produces the product of choice.
• Henry Ford, Ford Motor Company (1925):
"The fuel of the future is going to come
from fruit like that sumac out by the road,
or from apples, weeds, sawdust – almost
anything."
• Well-established commercial
biotechnological 1st and 2nd generation
production (yeast fermentation,
distillation, dehydration) from high sugar
content crops (corn, sugar cane) and
lignocellulosics (switchgrass, Miscanthus,
poplar, corn stover, wheat straw)
Bioethanol
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From: RFA (2016): Fuelling a High Octane Future - 2016 Ethanol Industry Outlook
• Challenges: hygroscopic (transportability, spectrum of use), 1/3 lower energy density
than gasoline, motor adjustment necessary for high E fuels
• Platform for catalytic conversion into other fuel and chemical products, e.g. alcohol to
ethylene, jet synthetic paraffinic kerosene (ATJ-SPK)
• Other biofuel compounds directly addressable by microorganisms (native, engineered)
• Higher alcohols: isopropanol, n-butanol (Clostridium spp., E. coli, Yeast)
• Higher alcohols and alkanes (Vibrio spp., E. coli)
• Other hydrocarbons, e.g. limonene (E. coli, yeast, cyanobacteria, plants)
• Lipids (Yarrowia spp., oleagineous fungi, cyanobacteria, microalgae, E. coli)
• Esters (by enzymatic esterification of org. acids and alcohols)
Bioethanol - other biofuel compounds
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Jet A-1 compatibles chemicals
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"Limonene fulfilled all the requirements of an alternative aviation fuel, though butyl butyrate and ethyl octanoatewere acceptable except for the reduced energy density."
Limonene
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Max. 1,35 g/L in engineered E. coli BL21(DE3)
Max. 1.48 mg/L in engineered S. cerevisiae
Max. 4 mg/L in wild-type Synechococcus sp.
Butyl butyrate
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http://www.tudelft.nl/fileadmin/UD/MenC/Support/Internet/TU_Website/TU_Delft_portal/Samenwerken/Patenten_vitrine/Patenten_IPMS_pdf/OCT-11-005_Biofuel.pdf
C. acetobutylicum fed-batch fermentation with continuous extraction into hexadecane
Yield: 5 g/L BuB
• Commodity chemical (fruity flavour, similar to pineapple)• Potential drop-in biodiesel/jet fuel
Butyl butyrate
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ENERGIX/IndNor: EcoLodgeEfficient Production of Butyl Butyrate from Lignocellulose Derived Sugars
• Separate optimization of n-butanol and butyric acid fermentation
• Integrated intermediate product removal• Enzymatic esterification and BuB extraction
→ Smart process design for improved production yields
Process integration
• Integration of unit operations like production, removal and recovery of volatile
alcohols will increase recovery efficiency and productivity.
• Consolidated Bioprocessing (CBP) may be integrated with one or more in situ
product removal techniques like gas stripping, pervaporation and adsorption to
prevent product toxicity and increase yield and productivity.23
Xue, C. et al (2016) Biotechnology and Bioenginering, Vol 113, No 1
• Key bottleneck in butanol fermentations is
product toxicity (low yield and productivity).
• Integrated processes are the choice of
operation.
Placement of biotech value chains in the FME
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Fermentation (WP3.4)(thermophilic strains, strain
engineering/SynBio, SHF, SSF, CBP, integrated and intensified
processes)
Improved pretreatmentand fractionation (WP2.1)
Anaerobic digestion to methane; gas
purification (WP2.3)
Gasification to syngas; gas purification (WP2.4)
Process modelling and process design (WP4.1)
Techno-economics (WP4.2)
LCA (WP1.3)
Process integration and intensification, up-
scaling (WP4.3)
Advanced analytics for compositional analysis of feedstock
and process fractions, including detection of potential inhibitors for the biocatalytic processes (WP??)
New and improved enzyme cocktails for saccharification (WP2.2)
Product evaluation (WP4.4)
Softwood lignocellulosic biomass
Biofuels(higher alcohols, esters,
hydrocarbons, lipids)
Integration of WP3.4 with other modules and value chains
Chemo-catalytic conversion (WP3.4)
Enzyme technology (WP3.4)
Thermal liquefaction (WP3.2)
cell mass
syngas
methane
enzymes
Enzymes, genes
cell mass
Value added side products
Technology for a better society