Biocatalytic solar fuels for sustaiblinablemobility in … · Biocatalytic solar fuels for sustaiblinablemobility in Europe ... addition = 3321 bpp ... NADPH ATP Ahr Calvin l G3P

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


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


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)


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


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)


WP2: Biocatalyst development

Patrik Jones (ICL)

IPP

GPPMEP

h

Olaf Kruse (UniBi)

DMAPP

GPP

IPP

BS

pathway

FPPSesquiterpenefuel precursors

BS

FSPeter Lindblad

(UU)


(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





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


(vs. PcPs ~34% patchoulol synth.) ʺAbies grandis Rogów 6ʺ by Crusier ‐Own work. Licensed under CC BY‐SA 3.0 via Commons


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



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


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



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


Butyraldehyde 1-Butanolslr1192/slr0942

yq yjg

: Knock‐outp g


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


isobutanol


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



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



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



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


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


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



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


‐ outdooor cultivation at small‐scale to refine the cultivation protocol


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


• 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


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


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


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


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


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


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


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


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


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.)


This project has received funding from the European Union’s Horizon 2020 research and innovation programme under

Thank you!

grant agreement No 640720.


Thank you!

Documents

Biocatalytic solar fuels for sustaiblinablemobility in … · Biocatalytic solar fuels for sustaiblinablemobility in Europe ... addition = 3321 bpp ... NADPH ATP Ahr Calvin l G3P