Ｉｎｄｕｓｔｒｉａｌ Ｉｎｇｒｅｄｉｅｎｔｓ. Kunimitsu Kaya

Transcription

1 Ｐｏｓｓｉｂｉｌｉｔｙ ｏｆ Ａｌｇａｌ Ｌｉｐｉｄｓ ａｓ Ｉｎｄｕｓｔｒｉａｌ Ｉｎｇｒｅｄｉｅｎｔｓ Kunimitsu Kaya y of Tsukuba University

2 Lipid accumulating microorganisms Green Algae Nile red stained Pseudochoricystis ellipsoidea Botryococcus braunii Euglenoid Oil granules Euglena gracilis Fluorescent image of chlroplast Oil drop Nile red stained Conforcal m image

3 Cost of Algae Production algae Botryococcus cost ( JPYen/kg DW) 80 market price ( JPYen/kg DW)? Chlorella , , Spirulina 160 2,000 7,000 Euglena? 50, ,000

4 Lipid id Composition of Algae Botryococcus * Euglena (light condition) (dark condition) Hydrocarbons %/DW Triglyceride %/DW Glycolipids %/DW Triglyceride 82% 8.2% 205% 20.5% Glycolipids 70.4% 5.9% Phospholipids 20.7% 74.3% Phosphlipids %/DW *The lipid content and composition are dependent on media nutrients and culture conditions.

5 Hydrocarbons of Botryococcus braunii Strain No H.C. content Growth rate Mol.Formula of the ((% in algae g DW)) ( μ μ/day) y) most abundant H H.C. C T: terpene; A: alkene; E: alkane epoxide C34H58 (T) mw=466 C33H64 (A) mw=460 C34H58 (T) mw=466 C31H58 (A) mw=430 C34H58 (T) mw=466 C34H58 (T) mw= C19H38O (E) mw=282 Purity (%) of the H H.C. C

6 Structures of hydrocarbons produced from Botryococcus Race A Race B C27H52 E/Z C27H50 E/Z Botryococcene (C34H58, MW 466) Cox, RE et al. J. Chem Soc.- Chem. Commun. 12, (1973) C34H56, mw 466

7 Hydrocarbon compositions of B. braunii Races Isolate from Japan Rt Mol.% mw Molecular formula Unsatu. Race-A: C31H60 C27H50 C31H Race-B: C34H58 C34H58 C34H Race-Sa:25.94 Race Sa: C18H36O C19H34O 1 3 Race-La: C18H36O C21H42O C39H72O C40H78O 2 GC/MS Column: methylsilicon; Temp: 60ºC(2min)- 5ºC/min -280ºC

8 Major Hydrocarbons of Races of B. buraunii collected from Japanese freshwaters La 2 Sa 19 A 2 B 37 A: C 27 H 50, C 27 H 52,C 29 H 56 B: C 32 H 54, C 34 H 58 La: C 39 H 72 O C 40 H 78 O 39 72, Sa: C 18 H 36,O,C 19 H 34 O

9 Hydrocarbon composition of Botryococcus isolated from a dam composition Rt (min) C18H36O, mw 268 unsaturation 6.4% (weight %) C21H34 C21H34, mw diterpene C33H56, mw triterpens C33H56, mw triterpene C32H54, mw triterpene is i a geometric t i isomer i off 3 5. O compound epoxide

10 Application to fuel Race A C27H52 E/Z C27H50 E/Z Direct Use Diesel oil C t l ti Cracking Catalytic C ki Gasoline (C5 C8)

11 Application to chemical ingredients Polymer y Fuel Chemicals The purities Th iti are over 90%

12 Squalene is a minor component of hydrocarbon fraction of microalgae Squalene is isolated from shark liver oil. The market price of squalene for cosmetics is JPYen16,000/100g. Squalene is used to keep skin moisture, and protect skin surface against oxidants.

13 How to control squalene production in microalgae O CO H3C pyruvate + C O O OP HO HO OP P2C OPP non-mevalonate pathway OP FPP D-glyceraldehyde gy y 3-phosphate OPP OPP IPP DMAPP presqualene diphosophate CH2 OPP Squalene synthase triterpene synthase C30 botryococcene C31- C34 Inhibitor or gene control Cholesterol synthase Inhibitor or gene control cholesterol h l t l

14 Algaenans g ((insoluble biopolymers p y as the main component of colony matrix) Oil Drop Colony matrix

15 Algaenans : the major component of colony matrix insoluble with organic solvents (chloroform/methanol, ethanol/diethy lether)-----stubborn biopolymers partially soluble by the treatment with K/Methanol or trifluoroacetic acid and HCl/methanol) HCl/methanol),but but not all consist of hydrocarbon units, mainly chains, (CH2)n- methylenic

16 Algaenan-A H CHO (CH 2 ) 6 (CH 2 ) 12 (CH 2 ) 7 H CHO n Algaenan-B O H CHO O O CH (CH 2 ) 6 (CH2 ) 12 (CH 2 ) 7 H CHO 3 (CH 2 ) 6 (CH 2 ) 12 (CH 2 ) 7 7 n Algaenan-L H CHO O O CH (CH 2 2) 6 (CH2 ) H CHO 12 (CH 2 ) 7 3 (CH 2 ) 6 (CH 2 ) 12 (CH 2 ) 7 7 n

17 Algaenan forms sponge-like sponge like shape (reticulum structure), and hold a lot of hydrocarbon secreted to out-side of cells. The colony is ensured by hydrophobic cohesion of algaenans. Can we use algaenans as some ingredients? g

18 N-Alkyl l and Alkenyl l phenols of fb. buraunii iirace- A OMe OMe CH 3 -(CH 2 ) 7 -CH=CH-(CH 2 )y- CH 3 -(CH 2 ) 7 -CH=CH-(CH 2 )y- HO OMe OMe OMe OMe CH 3 -(CH 2 ) 24 - CH 3 -(CH 2 2) 7 -CH=CH-(CH 2 2)y- OMe OMe Y= 15-21, odd number The phenolic moieties protect the aliphatic chains against degradation by The phenolic moieties protect the aliphatic chains against degradation by Bacteria and fungi.

19 Productions oduct o s of o hydrocarbon yd oca bo aand d sslime e exopolysaccarides by Botryococcus braunii strain Biomass (g/l) Hydrocarbon (% of w/w) Exopolusaccarides g/l LB SAG UC58 SI Radial secretion of polysaccharides, stained with methyl violet Exopolysaccarides p y (violet color )

20 Sugar composition of the slime exopolysaccarides CH 2 O CH 3 O CO O α-d-galactose D α-d-fucose D α-d-galacturonic D acid I f t i UC58 th b h d t f th li l id In a case of strain UC58, the carbohydrate of the slime exopolysaccarides is mainly composed of α-d-glucose, α-d-fucose and α-d-galacturonic acid. Fucose may be related with apoptosis of cancer cells

21 Content and composition of slime exopolysaccarides of UT009 and 010 Sugar composition (mol ratio) Total* (Fuc / Gal / G-uron.A / Unknown compound) 009 Exocarbohydrate 15mg/100mL Cell debris (upper 5μm) 11 mg 1/2/1/1 1/2/1/1 010 E Exocarbohydrate b h d t 1 1.5g/L 5 /L 1/3/2** Fuc: αα D-fucose; fucose; Gal: αα D-galactose; galactose; G G-uron. uron A: αα D-galcturonic galcturonic acid acid. * Excretion of slime exopolysaccarides is dependent on culture conditions **not containing unknown compound.

22 Summary 1) Many microalgae accumulate hydrocarbons or triglycerides. 2) In culture costs, Botryococcus is the cheapest. The Hydrocarbon (HC) content reaches about 50% in Botryococcus dry cells. 3) The major HC are terpene, alkene and alkane epoxide, these chemical species are dependent on strains. Especially, many Japanese strains produce alkane epoxides. The purities of terpens in HC are over 90%. 4) Alkene and alkane epoxide can be utilized as fuel. Terpenes can be utilized as ingredients of polymer, detergents and other medicinal and industrial chemicals. 5) Squalene is an expensive ingredient of cosmetics, but a minor component of H.C. of microalgae. If we use inhibitors or gene modification of key enzymes of squalene biosynthesis, we obtain a large amount of squalene 6) Algaenans are insoluble biopolymers consisted of HC. 7) Slime exopolysaccarides is a major by-product of Botryococcus, and is composed of α-d-glucose, α-d-fucose and α-d-galacturonic acid. Also, these can be utilized as industrial ingredients. When the lipids and by-products of microalgae will be utilized in various industries, the cost of biofuel produced by microalgae will be reduced. As the results, bioleum (bio + oleum: oil of bioresources) will become an alternative ti resource of petroleum.

23 Thank you for your attention This study is supported by JST-CREST (Algal Oil Project)