Thursday, May 10, 2012 Miraikan, Tokyo, JAPAN

Prof. Dr. Yoshihiro Shiraiwa (Faculty of Life and Environmental Sciences,

University of Tsukuba, Japan)

CREST Project: Microalgae for Bioenergy Production

Norway-Japan Marine Seminar 2012

Arctic Ice Meltdown

Oceanic Acidification

Arctic Ice Area

Algal Fuel Headed For The Racetrack Algae Could 'Supply Entire

World with Aviation Fuel' Japan Airlines conducted test flights with jets using biofuels.

First flight of algae-fuelled jet

>90-minute flight

>a 50-50 blend of biofuel and normal aircraft fuel.

8 January 2009

Algae, Jatropha Tapped To Power Continental Airlines’ First Biofuel Test Flight

Topics on Algal Fuel

Algeoleum

Creation of Basic Technology for Improved Bioenergy Production through Functional Analysis and Regulation of Algae and

Other Aquatic Microorganisms Tadashi Matsunaga (President, Tokyo University of Agriculture and Technology)

2010 -

Year Started: Research Supervisor:

Aims: Creating new basic and innovative technologies for bioenergy production using algae and other aquatic microorganisms by promoting high lipid, carbohydrate s, hydrocarbons, and high growth capability.

Focus on: Improvement in the efficiency of energy production through the elucidation of the physiological functions and metabolic pathways of algae and other aquatic microorganisms, which are effective bioenergy producers, using advanced scientific technologies from the fields of genomics, proteomics, metabolomics, and cell analysis.

Year Started : 2010 Research Director

Affiliation Research Project

Haruyuki Atomi

Professor, Kyoto Univ.

Enhancing and fusing archaeal metabolism: a new approach towards bioenergy production

Shigeru Okada

Associate Professor, Univ. of Tokyo

Characterization of hydrocarbon biosynthesis and secretion mechanisms by the green microalga, Botryococcus braunii to control biofuel production

Shigeyuki Kawano

Professor, Univ. of Tokyo

Establishment of innovative technology to create new microalgal strains increasing biofuel production by polyploidization and heavy-ion beam irradiation

Yoshihiro Shiraiwa

Professor, Univ. of Tsukuba

Research on the metabolic pathway of alkenones in marine haptophte algae and the development of new algal oil production technology

Koji Sode Professor, Tokyo Univ. of Agriculture & Technology

The Cyanofactory

Year Started : 2011 Research Director

Affiliation Research Project

Mitsuyoshi Ueda

Professor, Kyoto Univ. Focused biotechnologies suitable for complete utilization of marine macroalgae

Hiroyuki Ohta Professor, Tokyo Inst. of Technology

Strategic Construction of Algal Lipid Production System Utilizing Plant Vegetative Organs as a Model

Tatsuo Omata Professor, Nagoya Univ.

Development of an efficient system for free fatty acid production using cyanobacterial mutants affected in nitrate assimilation.

Toru Hisabori Professor, Tokyo Inst. of Technology

Research on the efficient biosynthesis of nitrogenous substances using artificially optimized nitrogen fixing cyanobacteria

Shin-ya Miygaishima

Associate Professor, National Inst. of Genetics

Creation of heat and acid tolerant algae toward high biomass production

Thursday, May 10, 2012 Miraikan

Prof. Dr. Yoshihiro Shiraiwa (Faculty of Life and Environmental Sciences)

Emiliania bloom Oslo

Norway

Denmark

Marine microalgae have contributed to change in global environment and such change has been the motive force for the evolution of microalgae during three billion years. Marine coccolithophorids (Haptophytes), unicellular calcifying algae, had contributed the production of petroleum and lime stones at the Cretaceous era. They are still producing a huge bloom in the present ocean. In this talk, I will focus on the potential of "coccolithophorids" for CO2 fixation and bio-oil production.

Photosynthesis

Calcification

Compatible Solute

Carbon Sequestration by Coccolithophorids

Ca

Se I

～8 Mt CaCO3/bloom

～115kt CaCO3/d

Acrylic acid

8

Emiliania huxleyi (Emily)

http://home.hiroshima-u.ac.jp/er/Class/UGR08_9.html

Distribution of Carbonate-rocks like limestone

White Cliff, Dover, IK http://en.wikipedia.org/wiki/Petroleum

Proven world oil reservoirs, 2009

▲100 Ma （Ron Blakey, NAU Geology）

Structure of Coccolith in Emiliania huxleyi

V/R Model of Crystal Formation of Coccolith

1 um l

Young et al. (1992, 1999) Intracellular Coccolith Production

Kayano et al. (2011)

EBSD Analysis

Kayano et al. (2011) Mar. Biotechnol

11

CaCO3 Crystals Produced by In vitro Calcification

Emiliania CP Regulates Crystal Morphology

SEM & EBSD analysis

The Crystal => Calcite

Pyrolysis

Dried Algal

powder

Solar Energy

Heating

Stored In Glass Ample Under N2 gas

Liquid Hydrocarbons

Gaseous Hydrocarbons

Non-oxygenic conditions

Liquid Hydrocarbons Produced by Pyrolysis

of E.huxleyi

C31

C20

C12

Wu, Shiraiwa et al. (1999) Mar Biotechnol 1: 346-352

Liquid hydrocarbons

Gasoline C6-C12

Jet fuel C10-C15

Diesel C10-C20

13

Hydrocarbon GasesProduced by Pyrolysis of E.huxleyi

Wu, Dai, Shiraiwa et al. (1999) J Applied Phycol 11: 137-142

Hydrocarbon gases

14

C37:2 Akenone

trans Keto-group

Fatty acid

Long-chain ketone

cis

C18:2 Linolenic acid

Emiliania huxleyi

16

1

2

3

4+5

6 7

8 9 10

1 37:3 Me 2 37:2 Me 3 36:2 FAME (methyl alkenoate) 4 36:2 FAEE (ethyl alkenoate) 5 38:3 Et

6 38:3 Me 7 38:2 Et 8 38:2 Me 9 39:3 Et 10 39:2 Et

Alkenones (Long-chain Ketones) C37-39

Speculation of Metabolic Path for Alkenone Biosynthesis

Expand

Much Alkenones, but no TGA

Fatty Acids

Membrane lipids

Elongation

trans-unsaturation

keto-group formation Fatty acid biosynthesis

Chlt ER, Cyt

?

Long-chain Lipids

Alkenones

25℃→15℃

C38:2Et

C37:3

C38:3 Et

C37:2 C38:3Me

C39:2 C39:3

Temperature-dependent Alkenone Biosynthesis in E. huxleyi NIES 837

18

NIBA WU Light

20℃

15℃

15℃ →Dark

(5 days)

10 µm

19

CO2 C3 Cycle Fatty Acid Synthesis cis-type

Alkenone Precursors trans-type

K37:2

K37:3

?

2 double bonds

3 double bonds ? ?

Case A

Degradation

?

C3 Cycle Fatty Acid Synthesis cis-type

Alkenone Precursors trans-type

K37:2

K37:3

?

2 double bonds

3 double bonds ? ?

Case B

?

Degradation

CO2

Equipment for Metabolome Analysis by 14C isotope

Isotope Center in University of Tsukuba Thermostatic Chamber

Nihonika LP-3P

Wet laboratory

20

Agilent G7100 CE system

PerkinElmer 610-TR Scintillation

counter

Agilent 1260 LC system

ABSciex 5500 ION TRAP/MS/MSn

538 540 542 544 546 548 550 552 554 556 558 560 562 564 566 568 570 572 574 576 578 580 582 584m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.3e6

543.9545

MS Spectrum of Alkenones for E. huxlyei in Positive mode

21 研究総括サイトビジット（2011.12.09)

Metabolome analysis of primary metabolites for lipid biosynthesis by CE/LC-ION TRAP MS/MSn

Glucose

Fructose 1, 6-BP

3-Phosphoglycerate

Phosphoenol-pyruvate

Pyruvate

Acetyl-CoA

Oxalo-acetate

Citrate

Isocitrate

2-Oxoglutarate

Succinate semialdehyde Succinate

Fumarate

Malate

TCA cycle

Fatty acids

DAG TAG

G3P

CoA

Serine Glycine

Cysteine

Tyrosine

Phenyl-alanine

Aspartate

Asparagine

Lysine Homoserine

Threonine

Isoleucine

Methionine

Glutamate

Proline

Glutamine

Histidine Arginine

Alanine

Alkenon C37:2 C37:3 C38:2 C38:3

?

LC-ION TRAP/MS/MSn

CE-ION TRAP/MS/MS

22

23

CO2

Photo- synthetic C3 Cycle

Unknown Biosynthesis

Pathway

Alkenones Alkenes

Metabolic Pathway toward Alkenones/Alkenes

Genetic manipulation & Mutation

Intermediates & New Metabolites

Biofuel & Biorefinery

Shiraiwa’s CREST Project

Acknowledgement

・CREST, JST (FY2011-2015) ・

Shiraiwa’s Lab