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

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63 employees + PhD students, post-docs

Nat

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al

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gro

up

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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