biocatalytic solar fuels for sustainable mobility in...
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Biocatalytic solar fuels for sustainable mobility in Europe
EU algae roadmap conference
Simon Kühner (project manager)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Outline
Overall objective: Promote the development of high quality, low impact transportation fuels
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Challenges: Photobiocatalyst development - Synthesis of fuel compounds - Excretion to medium – Direct, continuous separation – Fuel blending – Engine tests
IPP
DMAPP
GPP
IPP
FPP
Sesquiterpenefuel precursors
BS
FS
MEPpathway
Patrik Jones (ICL)
Peter Lindblad(UU)
Olaf Kruse (UniBi)
Approach
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
WP 2 Biocatalyst development
2 cyanobacterial strains 1 microalgae
Target fuels butanol, medium chain alcohols and
alkanes, sesquiterpenes.
Best strain(s) selected and jointly improved in the
last 18 month of the project.
WP 3 Production upscaling
Final volume 5m ³
Outdoor production fuel compounds
Continuous fuel separation
WP 4 Fuel blending
Analyse raw products
Blend on-spec fuels
WP 5 Engine tests
Analyse raw products
Blend on-spec fuels
WP6 Assessment
LCA
Economic
Risks
WP7 Business case
Business case development
Project facts
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Budget 6 million EUR Funding 6 million EUR
Duration 48 Months Start May 2015
EC-DG Research Priority Area Renewable energies
Contract No. 640720
Coordinator Hilke Heinke Contact [email protected]
Manager Simon Kühner Contact [email protected]
Project plan
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
2016 2017 2018 2019
Biocatalyst development
Cultivation / fuel production
Fuel blending
Engine tests
Assessment
Business case
Management & Dissemination
M2
M3
M4
M1
Biocatalyst selection
Fuel production at… 120 L
5000 L
Assessment criteria agreed
*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 alcohols, alkanes)
• Select best cell factories for joint development in last 18 month, target 100 mg/L/day in lab system and 34 mg/L/day in outdoor PBR (WP3)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
WP2: Biocatalyst development
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
IPP
DMAPP
GPP
IPP
FPP
Sesquiterpenefuel precursors
BS
FS
MEPpathway
Patrik Jones (ICL)
Peter Lindblad(UU)
Olaf Kruse (UniBi)
Bielefeld University Bisabolene synthesis in the green algae
Chlamydomonas reinhardii Kyle Lauersen, Julian Wichmann, Olaf Kruse
Biocatalyst development (UniBi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Biocatalyst development (UniBi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Bisabolene synthase
in C. reinhardii
• Abies grandis bisabolene synthase (AgBs)
• 2451 bp (817 aa)
• For C. reinhardtii expression: intron addition = 3321 bp
• Converts FPP Bisabolene C15H24
• High specificity ~100% (E)-α-bisabolene as product (PcPs ~34% patchoulol)
"Abies grandis Rogów 6" by Crusier - Own work. Licensed under CC BY-SA 3.0 via Commons
Knowledge gained from Patchouli-PcPs expression directly translates to strategy for Bisabolene synthase
Abies grandis bisabolene synthase = AgBs
Constructs designed, transformed, and screened as with the PcPs workflow
Biocatalyst development (UniBi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Strategy i i i i i2
i i i i i2
Bisabolene synthase (AgBs) – YFP fusion
i i i
AgBs
Farnesyl pyrophosphate (FPP)
Biocatalyst development (UniBi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
20151020_jw_bisabolene_39_900-2 27/10/2015 11:44:00 PM
RT: 14.0 - 18.8
14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
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 18.8
15.8
15.8
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
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
NL:2.50E6
m/z= 90.50-91.50 MS 20151020_jw_bisabolene_39_900-2
NL:2.50E6
m/z= 90.50-91.50 MS 20151102_bis_pat_test_17_bis_sd1_day7_3
NL:2.50E6
m/z= 90.50-91.50 MS uvm4
243 µM standard
AgBs strain
Parental strain
Simply identifiable in
dodecane by GC MS
Fluorescence positive clones produce
(E)-α-bisabolene
Biocatalyst development (UU)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Uppsala University 1-butanol and isobutanol in cyanobacteria
Pia Lindberg, Rui Miao, Xufeng Liu, Peter Lindblad
Biocatalyst development (UU)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Pathways to desired products in Synechocystis sp. PCC 6803:
Pyruvate
Acetyl-CoA
Lactateddh
Fatty
acid
biosynthesis
Fatty acid
accBCDAAcetate
Acetyl -P
pta
acs
phaA
Glucose
Glycolysis
slr1556
sll0542
slr1993sll1299
slr2132
2-Ketoisovalerate IsobutyraldehydeilvDilvCalsS
L-Valine
kivdyqhd/yjgB
Isobutanolsll1981
PHB
phaBslr1994
Acetoacetyl-CoA
ter/ccr
phaB
Butyryl-CoA
Malonyl-CoA
nphT7
pduP/mhpF
phaE
(R)-3-Hydroxybutyryl-CoA
phaC
slr1829
slr1830
cimA3.7 leuCD
2-Ketovalerate 1-Butanol
phaJ
sll1363 slr0452
Acetyl-ACP
Butyryl-ACP
tes4
Butyric acid
CAR
Butyraldehyde
leuB2-Ketobutyrate
leuABCD
L-ThreonineilvA
slr2072
C: sll1444D: sll1470
B: slr1517B: slr1517
C: sll1470D: sll1444
A: sll1564/slr0186slr1192/slr0942
slr1192/slr0942
leuABCD L-leucine
cimACya
kivd yqhd/yjgB
phaA
sll1564
Crotonyl-CoA
1-Butanolslr1192/slr0942
yqhd/yjgB
fadB
fadB
tes7
ackA
: Knock-out
Red: native Synechocystis genes Green: heterologously expressed genes
Biocatalyst development (UU)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
pyruvate
2-ketoisovalerate
isobutyraldehyde
ilvC (sll1363)
alsS (sll1981)
isobutanol
2-acetolactate
ilvD (slr0452)
kivd (Lactococcus lactis)
/alsS (sll1981)
1. yqhd (E. coli)
2. yjgB (E. coli)
ADH:
3. slr1192
4. slr0942
NADPH
NADPH
CO2
CO2
pyruvate
Isobutanol production at day 6
Example of early results: psbA2 promoter constructs for isobutanol production, in Synechocystis 6803
Biocatalyst development (UU)
Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
PpsbA2
PtrcBCD
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.
GC-analysis of growth medium:
Biocatalyst development (Imperial)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Imperial College Medium-chain alcohols and alkanes in cyanobacteria
Patrik Jones, Dipankar Ghosh, Ian Yunus
Biocatalyst development (Imperial)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Calvin
cycle
G6P
G3P
starch
Pyr AcCoA
BuCoA
Butanal
Fatty alcohol
FA-ACP
FA
Fatty aldehyde
Fatty alkane
AtoB/Hbd/Crt/Ter
(NhpT7)
Tes/CAR
Ahr
Tes
CAR
ADO/PetF/Fpr
CO2
Photosynthesis
ATPNADPH
light H2O
Ahr
CoA,
NAD(P)H?
ACP
CoATPP
T2.1 Enhanced availability of key metabolic intermediates
Biocatalyst development (Imperial)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Calvin
cycle
G6P
G3P
starch
Pyr AcCoA
BuCoA
Butanal
Fatty alcohol
FA-ACP
FA
Fatty aldehyde
Fatty alkane
AtoB/Hbd/Crt/Ter
(NhpT7)
Tes/CAR
Ahr
Tes
CAR
ADO/PetF/Fpr
CO2
Photosynthesis
ATPNADPH
light H2O
Ahr
CoA,
NAD(P)H?
ACP
CoATPP
T2.2 Implement synthetic pathways to target products
Biocatalyst development (Imperial)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Early findings
• Computational approach to predict enzyme substrate specificty appears promising
• All target products require active transport for cellular efflux
• Complex (>5 parts, >5kb) engineering is challenging, requires evaluation of approach and new strategies
WP3: Upscaling & Production
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach:
• Optimise cultivation, assess productivity, scale up 1L –> 120L –> 5000L, batch to continuous, lab to outdoor
• Develop continuous cultivation and product separation system up to pilot scale
• References: Lipid-strains, B. braunii
• Fuel compounds for upgrading and testing
• Data for LCA- and economic assessment A4F GMM-PBR
UniFi GWP®-II
Upscaling & Production (UniFi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
University of Florence Test of wild-type strains of biocatalysts and biofuel
reference strains Botryococcus braunii and Nannochloropsis oceanica
Natascia Biondi, Mario Tredici
Upscaling & Production (UniFi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
WILD-TYPE STRAINS TO BE TESTED
• Chlamydomonas reinhardtii CC124
• Chlamydomonas reinhardtii CC400
• Synechocystis PCC 6803
• Synechococcus PCC 7002
Cultures of Chlamydomonas CC124 in 300-mL bubbled tubes (left) and 5-L Annular Column (right) at UNIFI
ONGOING ACTIVITY: LABORATORY CULTURES
• Chlamydomonas culture medium optimisation for outdoor cultivation, particularly concerning:
- the buffer and its concentration
- the nitrogen source
- the carbon source NEXT STEPS
• ALL STRAINS :
- effect of temperature , pH and light intensity on growth at laboratory scale
- outdooor cultivation at small-scale to refine the cultivation protocol
Upscaling & Production (UniFi)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
REFERENCE STRAINS TO BE TESTED
• Botryococcus braunii UTEX572
• Botryococcus braunii SAG30.81
• Nannochloropsis oceanica F&M-M24
Culture of Botryococcus UTEX 572 in a 2-L Erlenmeyer flask at UNIFI
ONGOING ACTIVITY: LABORATORY CULTURES
• Botryococcus culture medium optimisation :
- nutrient balance
- vitamins
• Botryococcus: effect of bubbling and light intensity
NEXT STEPS
• Botryococcus:
- tests for optimisation on a race B strain
- outdoor culture of the best strain at small-scale to refine the cultivation protocol
• Botryococcus and Nannochloropsis : cultivation at pilot scale
WP4: Characterisation, fuel upgrading
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach:
• Characterise raw fuel components (from WP3)
• Purify/upgrade to blend components
• Develop fuel blending matrix (with WP5)
• Blend fuels for WP5
• Scheduled for May 2017
WP5: Engine testing
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach:
• Engine performance with Photofuel-blends
• Develop future fuel blending matrix (w. WP4)
• Gasoline and diesel trials in single cylinder test bench/ full engine/ vehicle for car and truck
• Scheduled for August 2017
WP6: LCA-, cost-, risk-assessment
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach: • Decision-support for sustainable development by
environmental (LCA) and economic assessment • Diagnosis of technology risk perception of stakeholders
and actors • KIT: Risk assessment and LCA until Photofuel raw
product • IFPEN: LCA for downstream processes and cost
assessment • VW, VOLVO: Support
LCA-design (KIT+IFPEN)
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Well to Wheel (WtT + TtW) LCA, incl. vehicle’s life cycle System boundaries
Fuel / Energy Life Cycle (Well to Tank)
Fuel /
Energy use
(Tank to
Wheel)
Horizontal axis: take into account the whole life cycle
stages of fuel/energy production: from primary material
extraction to fuel use
In Photofuel project: biofuel!
Cooperation with demo projects sought!
WP7: Business case development
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach:
• Develop a sound business case based on Photofuel experimental- and assessment-results
• Target: Larger pilot plant (TRL6)
• Outlook: Demo-/first commercial plant (TRL7/8)
• Scheduled for July 2018
WP1: Management&dissemination
30.03.2016 Photofuel – EU algae industry roadmap conference EU-H2020 grant No. 640720
Objectives and approach: • Coordination and project management • IPR management • Share the results on conferences, workshops
and publications • Contribute to achieving environmental and
climate-change targets of the European Union
WP1 - dissemination
Key events:
• Workshop on biocatalyst development for direct fuel production. CeBiTec Industrial Biotechnology, Bielefeld September 24-27 2017
• Workshop on large scale solar fuel production,
Lisboa 2018
• Workshop on risk-, economic and environmental assessment (t.b.d.)
• Workshop for European stakeholders from policy and industry on key results (t.b.d.)
Collaboration with other projects sought
30.03.2016 Photofuel – EU algae industry roadmap conference
EU-H2020 grant No. 640720