Ｐｏｓｓｉｂｉｌｉｔｙ ｏｆ Ａｌｇａｌ Ｌｉｐｉｄｓ ａｓＩｎｄｕｓｔｒｉａｌ Ｉｎｇｒｅｄｉｅｎｔｓ

Kunimitsu Kayay of Tsukuba
UniversityLipid accumulating microorganisms

Green Algae
Nile red stained

Pseudochoricystis ellipsoidea
Botryococcus braunii
Euglenoid

Oil granules

Euglena gracilis
Fluorescent image ofchlroplast

Oil drop

Nile red stained
Conforcal m image

Cost of Algae Production
algae

cost
( JPYen/kg DW)

market price( JPYen/kg DW)

Botryococcus

80?

Chlorella

159

2
2,000
000 – 10,000
10 000Spirulina

160

2,000 – 7,000

Euglena?

50,000 – 100,000

Li id C
LipidCompositioniti off AlAlgae
Botryococcus *Euglena (light condition)

Hydrocarbonsy-- 5-75%/DW

Triglyceride

8 2%
8.2%

20
20.5%
5%

Triglyceride-- 1-3%/DW

Glycolipids

70.4%

5.9%

Glycolipids-- 3-5%/DW

Phospholipids

20.7%

74.3%

Phosphlipids-- 1-2%/DW(dark condition)*The lipid content and composition are dependent on media nutrients andculture conditions.

Hydrocarbons of Botryococcus braunii
Strain No

H.C. content Growth rate Mol.Formula of the((% in algaeg DW)) ( μμ/day)y)most abundant H
H.C.
C001

40.8

0.078

002

21.1

0.187

003

42.3

0.066

004

45.3

0.158

005

25.1

0.33

006

45.7

0.2

007

25.3

0.088

T: terpene; A: alkene; E: alkane epoxide

C34H58 (T)mw=466
C33H64 (A)mw=460C34H58 (T)mw=466
C31H58 (A)mw=430C34H58 (T)mw=466
C34H58 (T)mw=466466
C19H38O (E)mw=282

Purity (%) ofthe H
H.C.
C
92
54
97
56
94
95
51

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, 284-285 (1973)

C34H56, mw 466

Hydrocarbon compositions of B. braunii Races
Isolate from Japan
RtMol.%

mw

Molecular formula

Unsatu.

Race-A: 40.32
40.78
41 34
41.34

40
456

432
374
430

C31H60C27H50C31H58233

Race-B: 41.29
43 18
43.18
43.52

3950
11466
466
466

C34H58
C34H58
C34H5866
6Race-Sa:25.94
Race
Sa:25.94
27.61

73
27268
278

C18H36O
C19H34O

13

Race-La:25.92
10268
C18H36O
131.935310
C21H42O
144.4842557
C39H72O
449.0444575
C40H78O
2GC/MS Column: methylsilicon; Temp: 60ºC(2min)- 5ºC/min -280ºC

Major Hydrocarbons of Races of B.
B buraunii
collected from Japanese freshwaters
A2Sa
19B37

La

2

A: C27H50, C27H52 ,C29H56
B: C32H54, C34H58
La: C39H72O, C40H78O
Sa: C18H36,O,C19H34O

Hydrocarbon composition of Botryococcus isolated from a dam
compositionRt
1. 18.01(min)C18H36O, mw 268

unsaturation
6.4% (weight %)12 20.32
2.
20 32

C21H34
C21H34,mw 286

19 1
19.1

5diterpene

3. 27.19

C33H56,mw 452

20.2

6triterpens

4. 27.30

C33H56,mw 452

26.0

6triterpene

5. 27.57

C32H54,mw 438

28.3

6triterpene

1
1.
2.

3.
4 isi a geometrict i isomeri
off 3
5.

O

compoundepoxide

Application to fuelRace A
C27H52

E/Z

C27H50

E/Z

Direct Use
Diesel oil

C t l ti Cracking
CatalyticCkiGasoline (C5 –C8)

Application to chemical ingredientsPolymeryFuel

Chemicals

The puritiesTh
iti areover 90%

Squalene is a minor component of hydrocarbon fraction of microalgae
Squalene is isolated from shark liver oil. The market priceof squalene for cosmetics is JPYen16,000/100g.

Squalene is used to keep skin moisture, and protect skin surface against oxidants.

How to control squalene production in microalgae
OCOOH
H3C

OH

pyruvate+OHC

OO

OP
OH

HOHOOP

OH

OH

POH2C

OH
OPP

non-mevalonate pathway

OP
OH

FPP

D-glyceraldehydegyy
3-phosphate

OPP

OPP

IPP

DMAPP

presqualene diphosophate
CH2 OPPSqualene synthasetriterpene synthase

C30
botryococcene
C31- C34

Inhibitor orgene control

Cholesterol synthase

Inhibitor or gene control

cholesterolh l t lAlgaenansg((insoluble biopolymers
p yas the maincomponent of colony matrix)

Oil Drop

Colony matrix

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

Algaenan-AHCHO(CH2)6(CH2)7(CH2)12HCHOnOAlgaenan-BOHCHO(CH2)6CH(CH2)12(CH2)7O(CH2)63H(CH2)12(CH2)7CHO
7nAlgaenan-LO
HCHO((CH2)6CH(CH2)12(CH2)73O(CH2)6H(CH2)12(CH2 )7CHO
7n

Algaenan forms sponge-likesponge like shape (reticulum structure), and holda 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N Alk l andN-Alkyld Alkenyl
Alkl phenols
hl off B.
B buraunii
bii RaceR
AOMe

CH3 -(CH2 )7-CH=CH-(CH2)yHO

CH3 -(CH2)24-OMe

CH3-(CH2)7-CH=CH-(CH2)y-OH

OMe

OMeOMeOMe

OH

CH3 -(CH( 2 )7-CH=CH-(CH( 2)y)y-OMe

Y= 15-21, odd number
The phenolic moieties protect the aliphatic chains against degradation byBacteria and fungi.

OMe

Productionsoduct o s oof hydrocarbon
yd oca bo anda d slimes e
exopolysaccarides by Botryococcus brauniistrain

Biomass (g/L)

LB 5722.0‐3.6

20‐35

1‐2

SAG30.81

1.5‐2.2

40‐50

0.5‐2UC58
SI30

Hydrocarbon (% of w/w)

Exopolusaccarides g/L

4‐4 5
4‐4.5
10

Radial secretionof polysaccharides,
stained with methyl
violet10‐28

Exopolysaccarides
p y(violet color )Sugar composition of the slime exopolysaccarides
COOH

CH3

CH2OH
O
OH

O

OH

OH

OH

OH

O

OH

OH

OH

OH
OH

OH

α-D -Galactose
αGalactose

α D -fucose
αfucoseOH
α D-Galacturonic
αGalacturonic acidIIn a case off straint i UC58,
UC58 ththe carbohydrateb h d t off ththe slimeliexopolysaccarides
lid
is mainly composed of α-D-glucose, α-D-fucose and α-D-galacturonic acid.
Fucose may be related with apoptosis of cancer cells

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
Exocarbohydrateb h d t11.5g/L
5 /L

1/3/2**

Fuc: αα D-fucose;
fucose; Gal: αα D-galactose;galactose; GG-uron.
uron A: αα D-galcturonic
galcturonic acidacid.
* Excretion of slime exopolysaccarides is dependent on culture conditions**not containing unknown compound.

Summary1) Many microalgae accumulate hydrocarbons or triglycerides
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,
terpene alkene and alkane epoxide,
epoxide these chemical
species are dependent on strains. Especially, many Japanese strainsproduce alkane epoxides. The purities of terpens in HC are over 90%.
4) Alkene and alkane epoxide can be utilized as fuel. Terpenes can beutilized as ingredients of polymer, detergents and other medicinal andindustrial chemicals.
5)) Squalene
qis an expensive
pingredientgof cosmetics,, but a minor component
pof H.C. of microalgae. If we use inhibitors or gene modification of key
enzymes of squalene biosynthesis, we obtain a large amount of squalene6) Algaenans are insoluble biopolymers consisted of HC.
7) Slime exopolysaccarides is a major by-product of Botryococcus, and iscomposed 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,
bi lbioleum(bi + oleum:(biol
oilil off bioresources)bi) will
ill b
become an alternativelt
ti resource offpetroleum.

Thank you for your attention

This study is supported by JST-CREST (Algal Oil Project)