biocatalytic solar fuels for sustaiblinablemobility in … · biocatalytic solar fuels for...
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Biocatalytic solar fuels fori bl bili i Esustainable mobility in Europe
EU algae roadmap conferenceHilke Heinke (project coordinator)Hilke Heinke (project coordinator)
09.06.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Outline
Overall objective: Promote the development of high quality low impact transportation fuelsquality, low impact transportation fuels
Challenges: Photobiocatalyst development ‐ SynthesisChallenges: Photobiocatalyst development Synthesis of fuel compounds ‐ Excretion to medium – Direct, continuous separation – Fuel blending – Engine tests
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
continuous separation Fuel blending Engine tests
Target molecules
Butanol: Drop in fuel for gasloine and possible diesel enginesButanol: Drop in fuel for gasloine and possible diesel enginesincluded in gasoline norm
Octanol: flash point according to diesel normOctanol: flash point according to diesel normless hygroscopic than ethanol and butanolresearch fuel
Bisabolen: after hydrotreatment drop in diesel fuelCZ 52, CFPP ‐25°C
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
ApproachWP 2 Biocatalyst development 2 cyanobacterial strains 1 microalgae Target fuels butanol, medium chain alcohols and
Patrik Jones (ICL)
alkanes, sesquiterpenes. Best strain(s) selected and jointly improved in the
last 18 month of the project.WP 3 Production upscaling ( )WP 3 Production upscaling Final volume 5m ³ Outdoor production fuel compounds Continuous fuel separation
Olaf Kruse
WP 4 Fuel blending Analyse raw products Blend on-spec fuelsWP 5 Engine tests IPP
DMAPP
GPP
IPP
MEPpathway
(UniBi)WP 5 Engine tests Analyse raw products Blend on-spec fuelsWP6 Assessment
FPPSesquiterpenefuel precursors
BS
FSPeter Lindblad
(UU)
LCA Economic RisksWP7 Business case (UU)
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP7 Business case Business case development
Project facts
Budget 6 million EUR Funding 6 million EURDuration 48 Months Start May 2015yEC-DG Research Priority Area Renewable energiesContract No. 640720
Coordinator Hilke Heinke Contact [email protected] Simon Kühner Contact [email protected] www.photofuel.euInternet www.photofuel.eu
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Project plan
Management & Dissemination
M2
Biocatalystdevelopment Biocatalyst selection
M3
M4
Cultivation / fuel production
Fuel production at… 120 L
5000 LM4
Fuel blendingEngine tests
5000 L
M1
AssessmentAssessment criteria agreed
Business case
*Mil t30.03.2016 Photofuel – EU‐BCE 2016 Amsterdam
EU‐H2020 grant No. 640720
2016 2017 2018 2019*Milestone
WP2: Biocatalyst development
Objectives and approach: • Direct, sustainable and efficient conversion of solar
energy, CO2 and water to liquid fuel compounds• Implement fuel excretion to the liquid culture, turning
cells to biocatalysts• 3 groups, 3 microbes (cyanobacteria and microalgae)
different pathways, different products (medium chain l h l lk )alcohols, alkanes)
• Select best cell factories for joint development in last 18 h / /d l b dmonth, target 100 mg/L/day in lab system and 34
mg/L/day in outdoor PBR (WP3)
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP2: Biocatalyst development
Patrik Jones (ICL)
IPP
GPPMEP
h
Olaf Kruse (UniBi)
DMAPP
GPP
IPP
BS
pathway
FPPSesquiterpenefuel precursors
BS
FSPeter Lindblad
(UU)
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
(UU)
Biocatalyst development (UniBi)
Bielefeld UniversityBielefeld University Bisabolene synthesis in the green algae
Chl d i h d iiChlamydomonas reinhardtii Kyle Lauersen, Julian Wichmann, Olaf Kruse
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (UniBi)
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (UniBi)
Bisabolene synthaseBisabolene synthasein C. reinhardtii•Abies grandis bisabolene synthase (AgBs) 2451 bp (817 aa)
• For C. reinhardtii expression: intronaddition = 3321 bpp
•Converts FPP Bisabolene C15H24
Hi h ifi it•High specificity ~100% (E)‐α‐bisabolene as product
(vs PcPs ~34% patchoulol synth ) ʺAbi di R ó 6ʺ b C i
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
(vs. PcPs ~34% patchoulol synth.) ʺAbies grandis Rogów 6ʺ by Crusier ‐Own work. Licensed under CC BY‐SA 3.0 via Commons
Biocatalyst development (UniBi)
Strategy
Abies grandis bisabolene synthase = AgBs fused to mVenus (YFP) reporter
Constructs transformed and mutants screened for fluorescence.
More fluorescence more synthase more productMore fluorescence = more synthase = more product
AgBs
i i i i i2i i i
Farnesyl pyrophosphate (FPP)
i i i i2
Bisabolene synthase (AgBs) – YFP fusion
i i
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (UniBi)
15.815.8
14.7 15.1 15.1 15.5 17.316.014.2 14.4 16.1 17.416.5 18.017.816.6 17.1 18.1
243 µM standard17.816.6 17.1 18.1
15.8
15.8
standard
Simply identifiable in
80
100
ce
17.8 17.817.414.4 15.7 15.915.114.1 18.814.7 18.416.715.515.0 16.1 18.216.4 17.216.8 AgBs strain
Simply identifiable in dodecane by GC MS
0
20
40
60
80
Rel
ativ
e A
bund
an
14.8 17.614.514.2 15.0 16.716.415.515.3 17.216.8 18.415.9 16.1 18.517.3 18.717.8 18.0 Parental
Fluorescence positive clones produce(E) α bisabolene
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5Time (min)
0
strain(E)‐α‐bisabolene
Biocatalyst development (UU)
Uppsala UniversityUppsala University 1‐butanol and isobutanol in cyanobacteria
Pi Li db R i Mi X f Li P Li dbl dPia Lindberg, Rui Miao, Xufeng Liu, Peter Lindblad
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (UU)
Pathways to desired products in Synechocystis sp. PCC 6803:Glucose
Glycolysis
PyruvateLactate ddhslr1556
2-Ketoisovalerate IsobutyraldehydeilvDilvCalsS
L-Valinekivd yqhd/yjgB
Isobutanolsll1981 sll1363 slr0452 slr1192/slr0942
leuABCD L-leucine
cimA3.7 leuCD2-Ketovalerate 1-Butanol
leuB2-Ketobutyrate
leuABCD
L-Threonine ilvAslr2072
C: sll1444 B: slr1517B: slr1517
C: sll1470D: sll1444
A: sll1564/slr0186slr1192/slr0942
cimACya
kivd yqhd/yjgB
sll1564
Acetyl-CoA
Fatt
accBCDAAcetate
pta
acs
phaAsll0542
slr1993sll1299 slr2132 Acetoacetyl-CoA
Malonyl-CoAnphT7
Acetyl-ACPButyryl-ACP
D: sll1470
phaA
sll1564
ackA
Fatty acid biosynthesis
Acetyl -Pslr2132
phaBslr1994
Acetoacetyl CoA
phaB
phaE
(R)-3-Hydroxybutyryl-CoA
slr1829phaJ
tes4
B t i id
fadB
fadB Red: nativeFatty acid
PHB
ter/ccrButyryl-CoA
pduP/mhpF
phaE
phaC
slr1829
slr1830
pButyric acid
CAR
B t ld h d
Crotonyl-CoA
1 B lyqhd/yjgB
tes7Red: native Synechocystis genesGreen: heterologouslyexpressed genes
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Butyraldehyde 1-Butanolslr1192/slr0942
yq yjg
: Knock‐outp g
Biocatalyst development (UU)
t
Example of early results: psbA2 promoter constructs for isobutanol production, in Synechocystis 6803
pyruvate
alsS (sll1981)
2-acetolactateCO2
pyruvate
ilvC (sll1363)
2 acetolactate
ilvD (slr0452)
NADPH
Isobutanol production at day 62-ketoisovalerate
kivd (Lactococcus lactis)
/alsS (sll1981)CO2
Isobutanol production at day 6
isobutyraldehyde
/alsS (sll1981)
1 yqhd (E coli)ADH:1. yqhd (E. coli)2. yjgB (E. coli)3. slr11924. slr0942
NADPH
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
isobutanol
Biocatalyst development (UU)
Effect of different promoters and growth conditions:PpsbA2 vs. PtrcBCD promoters driving expression of genetic constructs in Synechocystis 6803. Using different promoters and growth conditions results in different levels of isobutanol produced.
PpsbA2GC‐analysis of growth medium:
PtrcBCDPtrcBCD
Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (Imperial)
Imperial CollegeImperial CollegeMedium‐chain alcohols and alkanes in cyanobacteria
P ik J Di k Gh h I YPatrik Jones, Dipankar Ghosh, Ian Yunus
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (Imperial)
T2.1 Enhanced availability of key metabolic intermediates
starch Fatty alcohol Fatty alkanelight H2O
G6P Butanal Fatty aldehydeAhr ADO/PetF/Fpr
Photosynthesis
ATPNADPHAhr
Calvinl
G6P
G3P BuCoA FA
y yTes/CAR CAR
CO cycle G3P
Pyr AcCoA
BuCoA
FA-ACP
FAAtoB/Hbd/Crt/Ter
(NhpT7) Tes
CO2
CoATPPPyr AcCoA FA-ACP
CoA,NAD(P)H?
ACP
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (Imperial)
T2.2 Implement synthetic pathways to target products
starch Fatty alcohol Fatty alkanelight H2O
G6P Butanal Fatty aldehydeAhr ADO/PetF/Fpr
Photosynthesis
ATPNADPHAhr
Calvinl
G6P
G3P BuCoA FA
y yTes/CAR CAR
CO cycle G3P
Pyr AcCoA
BuCoA
FA-ACP
FAAtoB/Hbd/Crt/Ter
(NhpT7) Tes
CO2
CoATPPPyr AcCoA FA-ACP
CoA,NAD(P)H?
ACP
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Biocatalyst development (Imperial)
Early findingsy g• Computational approach to predict enzyme substrate specificty appears promisingsubstrate specificty appears promising
• All target products require active transport for cellular effluxcellular efflux
• Complex (>5 parts, >5kb) engineering is challenging, requires evaluation of approach and new strategies
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP3: Upscaling & Production
Objectives and approach: O ti i lti ti d ti it• Optimise cultivation, assess productivity, scale up 1L –> 120L –> 5000L, batch tocontinuous lab to outdoorcontinuous, lab to outdoor
• Develop continuous cultivation and product separation system up to pilot UniFi GWP®‐IIproduct separation system up to pilotscale
• References: Lipid‐strains, B. brauniiReferences: Lipid strains, B. braunii• Fuel compounds for upgrading and testing• Data for LCA‐ and economic assessmentData for LCA and economic assessment A4F GMM‐PBR
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Upscaling & Production (UniFi)
University of FlorenceUniversity of FlorenceTest of wild‐type strains of biocatalysts and biofuel
f i B b ii dreference strains Botryococcus braunii and Nannochloropsis oceanica
Natascia Biondi, Mario Tredici
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Upscaling & Production (UniFi)
WILD‐TYPE STRAINS TO BE TESTED
• Chlamydomonas reinhardtii CC124• Chlamydomonas reinhardtii CC400•Chlamydomonas reinhardtii CC1690
hl d h d• Chlamydomonas reinhardtii CC1883• Synechocystis PCC 6803• Synechococcus PCC 7002ONGOING ACTIVITY: LABORATORY CULTURESyONGOING ACTIVITY: LABORATORY CULTURES
• Chlamydomonas culture medium optimisation for outdoor cultivation, particularly concerning:
Cultures of Chlamydomonas CC124in 300‐mL bubbled tubes (left) and 5‐L Annular Column (right) at UNIFI
‐ the buffer and its concentration‐ the nitrogen source‐ the carbon sourceNEXT STEPS the carbon source
• ALL STRAINS : ‐ effect of temperature , pH and light intensity on growth at laboratory scale
td lti ti t ll l t fi th lti ti t l
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
‐ outdooor cultivation at small‐scale to refine the cultivation protocol
Upscaling & Production (UniFi)
REFERENCE STRAINS TO BE TESTED
• Botryococcus braunii UTEX572
Culture of Botryococcus UTEX 572in a 2‐L Erlenmeyer flask at UNIFI
Botryococcus braunii UTEX572• Botryococcus braunii SAG30.81• Nannochloropsis oceanica F&M‐M24
ONGOING ACTIVITY: LABORATORY CULTURES• Botryococcus culture medium optimisation :
‐ nutrient balance‐ vitamins
• Botryococcus: effect of bubbling and light intensity• Botryococcus: effect of bubbling and light intensity
NEXT STEPS
• Botryococcus:• Botryococcus: ‐ tests for optimisation on a race B strain‐ outdoor culture of the best strain at small‐scale to refine the cultivation protocol
Culture in a GWP®‐II photobioreactor
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
• Botryococcus and Nannochloropsis : cultivation at pilot scale
Upscaling & Production (A4F)
PILOT PLANT GREENHOUSE COMPLIANT WITH GMM CULTIVATION ‐ 270 M2
STRAINS TO BE TESTED
GMM strainsGMM strains• Chlamydomonas reinhardtii CC124• Chlamydomonas reinhardtii CC400 Reference strains:
• Synechocystis PCC 6803• Synechococcus PCC 7002
• Botryococcus braunii SAG30.81• Nannochloropsis oceanica F&M‐M24
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Upscaling & Production (A4F)
PHOTOFUEL CULTIVATION SCALE‐UP AT PILOT PLANT
Cultivation at 120 L in flat panelCultivation at 120 L in flat panel
Cultivation at 5000 L in tubularphotobioreactor
OTHER AVAILABLE PRODUCTION TECHNOLOGIES AT A4F
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
CRW (Cascade RaceWay)GWP (Green Wall Panels) Conventional raceway
WP4: Characterisation, fuel upgrading
Objectives and approach: • Characterise raw fuel components (from WP3)• Purify/upgrade to blend components• Purify/upgrade to blend components• Develop fuel blending matrix (with WP5)• Blend fuels for WP5S h d l d f M 2017• Scheduled for May 2017
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP5: Engine testing
Objectives and approach: • Engine performance with Photofuel‐blends• Develop future fuel blending matrix (w WP4)Develop future fuel blending matrix (w. WP4)• Gasoline and diesel trials in single cylinder testbench/ full engine/ vehicle for car and truck
• Scheduled for November 2016Scheduled for November 2016
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP6: LCA‐, cost‐, risk‐assessment
Objectives and approach: • Decision‐support for sustainable development byDecision support for sustainable development byenvironmental (LCA) and economic assessment
• Diagnosis of technology risk perception of stakeholdersand actors
• KIT: Risk assessment and LCA until Photofuel rawproductproduct
• IFPEN: LCA for downstream processes and costassessmentassessment
• VW, VOLVO: Support
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
LCA‐design (KIT+IFPEN)
Life Cycle Assessment (LCA) of fuel in a transportation contextSystem boundaries
Take into account the whole life cycle stages of fuel/energy production: from primary
Well to Wheel (WtW) = Well to Tank (WtT) + Tank to Wheel (TtW)
Take into account the whole life cycle stages of fuel/energy production: from primary material extraction to fuel use
In Photofuel project: biofuel from cultivation of microalgae/cyanobacteria to fuel use, through harvesting and biomass transformation into energythrough harvesting and biomass transformation into energy
Vehicle’s life cycle excluded
Well to Wheel (WtW)
Fuel/Energy Life Cycle (Well to Tank)Fuel/Energy use
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Fuel/Energy Life Cycle (Well to Tank) (Tank to Wheel)
LCA design Photofuel (KIT and IFPEN)
WP3
Anaerobic digestion, hydrothermal treatment
Algal PurgeRecycling of water, nutrients
Energy (MJ)
(i.e. biogas from algae)
KIT
IFPEN
CO2
UniBiUpscaling in lab to high density
CultivationInoculum HarvestingWP2Continuous or batch microalgae growth Fuel
Mechanical, chemicalseparation of Fuel precursors
WP4
UniBiChlamydomonasreinhardtii
high density mother culture
M t i l i t
microalgae growth and excretion of
Fuel UpgradingBisabolene
separation of Fuel precursors
M t i l i tWP5
UUSynechocystissp.PCC 6803
Engine test
Material inputsOperating supplies• Nutrients incl. CO2/C‐source• Water (medium, cleaning)• Chemicals (pesticides, cleaning)
Material inputsOperating supplies• I.e. cyclohexane (solvent)• Water (cleaning)• Chemicals (cleaning)
Butanol
Imperial
(p , g)Infrastructure• Reactor system (PBR, pipes etc.)• Equipment (Pumps, light bulbs etc.)Energy inputs• Pumping mixing cooling
Synechocystis
( g)Infrastructure• Buildings/maintenance • Equipment (e.g. centrifuge)Energy inputs• Recycling (nutrients water)
Energy (MJ)
(Algae based fuel blend)
Alkanes
• Pumping, mixing, cooling• CO2 supply const./air flow mixingOther• Land (overhead)
• Recycling (nutrients, water)• Precursor transport to refineryOther inputs• Land
( g f )
NannochloropsisB t
WP3: References
Lipids, hydrocarbons
22.04.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Botryocuccus Lipids, hydrocarbons
WP7: Business case development
Objectives and approach: • Develop a sound business case based on Photofuel experimental‐ and assessment‐Photofuel experimental and assessmentresults
( )• Target: Larger pilot plant (TRL6)• Outlook: Demo‐/first commercial plant (TRL7/8)Outlook: Demo /first commercial plant (TRL7/8)• Scheduled for July 2018
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP1: Management&dissemination
Objectives and approach: • Coordination and project management• Coordination and project management• IPR management• Share the results on conferences workshops• Share the results on conferences, workshops and publicationsC t ib t t hi i i t l d• Contribute to achieving environmental and climate‐change targets of the European Union
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
WP1 ‐ dissemination
Key events: W k h bi l d l f di f l• Workshop on biocatalyst development for direct fuel production. CeBiTec Industrial Biotechnology, Bielefeld September 24 27 2017Bielefeld September 24‐27 2017
• Workshop on large scale solar fuel production, Li b 2018Lisboa 2018
• Workshop on risk‐, economic and environmental t (t b d )assessment (t.b.d.)
• Workshop for European stakeholders from policy and i d t k lt (t b d )industry on key results (t.b.d.)
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under
Thank you!
grant agreement No 640720.
30.03.2016 Photofuel – EU‐BCE 2016 AmsterdamEU‐H2020 grant No. 640720
Thank you!