biocatalytic solar fuels for sustainable mobility in...

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


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


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


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


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)


WP2: Biocatalyst development


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)




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



Strategy i i i i i2

i i i i i2

Bisabolene synthase (AgBs) – YFP fusion

i i i

AgBs

Farnesyl pyrophosphate (FPP)



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)


Uppsala University 1-butanol and isobutanol in cyanobacteria

Pia Lindberg, Rui Miao, Xufeng Liu, Peter Lindblad



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



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


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)


Imperial College Medium-chain alcohols and alkanes in cyanobacteria

Patrik Jones, Dipankar Ghosh, Ian Yunus



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



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



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



• 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)


University of Florence Test of wild-type strains of biocatalysts and biofuel

reference strains Botryococcus braunii and Nannochloropsis oceanica

Natascia Biondi, Mario Tredici



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



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



• 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



• 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


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)


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



• 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


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


Thank you!

This project has received funding from the European Union’s

Horizon 2020 research and innovation programme under grant agreement No 640720.

biocatalytic solar fuels for sustainable mobility in...

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