(Wff. uqun &Jhfl)

-u-

-fl-

Thesis Title

Thesis Credits

Candidate

Supervisors

Degree of Study

Division

Academic Year

Synthesis of Stabilized Essential Fatty Acid by Yeasts and

Chemical Method

1 2

Mr. Paitoon Suksatavorapan

Assoc. Prof. Dr. Kanit Krisanangkura

Asst. Prof. Narumon Jeyashoke

Master of Science

Biotechnology

1 9 9 4

Abstract

The goal of this study was to synthesize relatively stable essential fatty acid of n-3

and n-6 series by chemical and biological method.

The first attempt was to elongate alpha-linolenic acid to eicosatrienoic acid or fatty

acids of longer chain length with Rhodotorula glutinis and yeast strain 572. The yeast grew

rapidly at 30°C. in medium with or without linseed soap which contained alpha-linolenic

acid about 60% of total fatty acid. Analysis of fatty acids from yeasts by gas

chromatography showed that the yeast could assimilate alpha-linolenic acid into the cell but

did not elongate. Assimilation of fatty acid as well as growth of yeast decreased as

temperature decreasing. It was concluded that the yeasts, R. gUnis and yeast strain 572,

could not convert linseed oil alpha-linolenic acid to any fatty acid of longer chain length.

Attempt biohydrogenate fish oil anaerobically with Sacc~omyces cerevisiae was

mlsuccessful. Lipid isolated from yeast treated with fish oil did not contain any

hydrogenated fatty acid of eicosapentaenoic acid or docosahexaenoic acid, suggesting that

the yeast was not able to biohydrogenate at this condition.

Hydrazine reduction of alpha-linolenic acid of linseed oil was very promising.

Analysis of reduction products by gas chromatography and mass spectrometry indicated that

the product contained one double bond at the n-6 (o&a&c-12-enoic acid) and n-3 (octadec-

1%enoic acid) position. The optimum molar ratio of fatty acid to hydrazine was 1 to 8 in

ethanol and amount of potassium ferricyanide was 0.2 gram per 5 millilitre. When water

was used as hydrogenation solvent, yields of octa&c-12-enoic acid and octadec-15-enoic

acid were increased from 14.1% and 6.4% to 15.4% and 8.7% respectively, but there was

no selectivity on the double bond position. At pH 9.9, rate of reaction was faster and easily

controlled. Reaction rates were highest at temperature 70 to 90°C but the maximum yields

were equal to that obtained at 30°C .

Keywords : Biohydrogenation / Essential fatty acid / Hydrazine reduction /

Linolenic acid / Linseed oil / Synthesis / Yeast

1.2.1.1 1.2.1.1 IUiuoil43laIUiuoil43la

1.2.1.2 1.2.1.2 %lllhlJmml%lllhlJmml

1.2.1.3 1.2.1.3 oy?bhR"ihoy?bhR"ih

U

9

Q

II

Q

Q

iu

1

1

2

2

3

3

3

4

4

4

4

5

7

7

11

11

15

16

2 0

2 0

2 0

-%-

1 . 3 . 3 . 1 1 . 3 . 3 . 1 er1nhJer1nhJ

1 . 3 . 3 . 2 1 . 3 . 3 . 2 Wf~n"hWf~n"h

1 . 3 . 3 . 3 1 . 3 . 3 . 3 ihhlt51ihhlt51

2 12 1

2 12 1

2 22 2

2 22 2

2 52 5

2 52 5

2 52 5

2 82 8

2 82 8

2 82 8

3 03 0

3 03 0

3 03 0

3 63 6

3 63 6

3 83 8

4 04 0

4 14 1

4 34 3

4 44 4

4 54 5

4 54 5

4 74 7

4 94 9

5 05 0

5 05 0

5 05 0

5 05 0

5 15 1

-All-

51

51

51

52

52

52

52

53

53

5 4

55

55

56

57

57

65

71

75

79

8 4

87

9 0

93

96

HiL

OllSld Hlb

1 . 1

1.2

1 . 3

1.4

1 . 5

1.6

1.7

1 . 8

1.9

3.1

3.2

3.3

3.4

3.5

3.6

6

26

26

27

29

37

37

39

40

61

66

66

7 4

99

1 . 1

1.2

1 . 3

1.4

1 . 5

1 . 6

1.7

1.8

1 . 9

1.10

1.11

3 . 13 . 1

3 . 23 . 2

3 . 33 . 3

3 . 43 . 4

3 . 63 . 6

3 . 73 . 7

3 . 83 . 8

99

1 01 0

1 21 2

1 31 3

1 41 4

1 51 5

1 61 6

1 81 8

1 91 9

1 91 9

2424

5 85 8

5 95 9

6 06 0

6 36 3

64

6 76 7

68

6 96 9

70

73

85

86

8 9

9 1

9 2

9 4

9 5

9 7

n. =

un. =

UQ. =

R. =

Oobl. =

AA =

ALA =

DHA =

DHGLA =

EPA =

G C =

GC-MS =

GM =

PUFA =

m/z, m/e =

nr'unr'u

ikl6nfuikl6nfu

4 4 *LL*LLaJQQR913aJQQR913

hshs

EMllWlCihJffEMllWlCihJff

Arachidonic Acid

alpha-Liuolenic Acid

Docosah~xaenoic Acid

di-homo-gamma-Linolenic Acid

Eicosapentaenoic Acid

Gas Chromatography

Gas Chromatography and Mass Spectrometry

gamma-Linolenic Acid

Polyunsaturated Fatty Acid

Mass/Charge Ratios

-2--2-

1.2 YE&

1.2.1 m (Lipids) [ 8, 9, 10 I

-3-

-4-

1.2.2 fQd!Uh (Fatty acids)

-5

a1.2.3 fl7rlgun(lrofIT&Wl* (Nomenclature)

0 - terminus CH,--CH,CH,-CH,--CH,--CHz--CH,COOH Ca&oxgl-

terminus

A numbering 10 9 8 7 6 5 4 3 2 1

normnumbering 1 2 3 4 5 6 7 8 9 10

Letter designation o o-1 s Y P cx

-6-

12:o

140

1 6 : 01 6 : 0

l&O

2o:o2o:o

24LO24LO

1611 A9;n-7

18:l A9;n-9

1812 A9,12;n-6

18:3 A9,12,15;n-3

20:420:4 fi,8,11,14;11-6fi,8,11,14;11-6

LNlliC

MyriStiC

Palmitic

stearic

AlWhidiC

Lignoceric

Palmitoleic

Oleic

Linoleic

Lholenic

AWAidOIliC

Dodecanoic 4 4 . 24 4 . 2

Tetradecanoic 5 3 . 95 3 . 9

Hexadecanoic 6 3 . 16 3 . 1

Octadecanoic 6 9 . 66 9 . 6

Eicosanoic 7 6 . 57 6 . 5

Tetracosanoic 8 6 . 08 6 . 0

Hexadecenoic -- 0 . 5 0 . 5

Octadecenoic 1 3 . 41 3 . 4

Ocbdecadienoic -- 5 . 0 5 . 0

Octadecatfienoic - 11.0

Eicosatetraenoic - 49.5

-7-

1.2.4 flWI!¶lfiU#Ub (Essential fatty acids)

ti1.2.5 fi.mT4lflllwHftl~luY lh (Fatty acid synthesis)

-8-

Acetyl CoA + HC%- + ATP + h&my1 CoA + A D P + P i

-9-

MITOCHONDRIA

CITRATE ______ . a - - - - - _ _ > CITRATE

CoASH 1

ACETYL CoA

OXALOACETATE

ADP Pi

ATP

PYRUVATE c--------.

CYTOPLASM

2 ACETYL CoA

OXALOACETATE

MALATE

NADP +

NADPH

y.Ji 1.1 nlsrfl0~~6~%~6BU~c11d60nlsrfl0~~6~%~6BU~c11d60 oofloln~aJl~nou~~4uoofloln~aJl~nou~~4u

1 : citrate synthetase, 2 : citrate lyase, 3 : malate dehydrogenase,

4 : malic 5 :enzyme, pyruvate carboxylase [ 9 ]

-lO-

ATP 1 A D P + P i

ACETYL CoA

3-KETOACYL - ACP

D-3-HYDROXY - ACP

H2O 48

A2,3-trans-ENOL - ACP

NADPH

NADP +-3

7

ACYL - ACP (04)

L- MALONYL CoA, 2 NADPH

ACYL - ACP (C6)

L- MALONYL CoA, 2 NADPH

ACYL - ACP (03)

L- MALONYL CoA, 2 NADPH

ACYL - ACP (ClO)+titi

PALMITATE (Cld)

Jdi 1.2 r-IlYfSjl~Yl,,*,Y,~~u'uUr-IlYfSjl~Yl,,*,Y,~~u'uU 1: acetyl CoA carboxylase, 2: acxztyl-

transferase, 3: malonyl transferase, 4: synthetase, 5: reductase,

6: hydratase!, 7: reductase [ 91

-ll-

-1%

00

II

-0OC - *Cl-$ - C - SCOA Malonyl CoA

9 - (CH,)1, - C -SCoA

Palmitoyl CoA

-L CO,, CoASH

0 0

I I

CH, - (Cl-I&,, - C - ‘CH2 - C - SCoA

NADPH

ILN A D P +

H 0

I I

CH3 - (CH,),, - C - ‘CHZ - C - SCoA

I

OH

L 40

0

I

3 - (Cl-& - Cl-I = ‘CH - C - SCoA

NADPH

I”

NADP +

0

I

a-~,,-(a-i,),,-CH/CH,-C-SCoA Steamy1 CoA

-13--13-

t

; CH,(CHJ,- Cl3 = CH - (CIiJ,- : - SCoA

Phitoboyl - CoA

-14-

.n-6 term

C l&O a-, C18:l A9 -b cm2 A9,12 __, c m 3 A9,12,15

StWliC linoleic 01 - linolenic

I I

C18:3 A 6,9,12 C20:3 A11,14,17

C20:2 A8,ll C20:3 A8,11,14 C20:4 A8,11,14,17

1 di-hano- y l/ok& (DHLA) 1

C20:3 A5,8,11 C20:4 A5,8,11,14 C205 L&8,11,14,17

eicosatrienoic arachidonic (AA) eicosapentaenoic (EPA)

C225 A7,10,13,16,19

docosapentaenoic

C22:6 A4,7,10,13,16,19

docaahexaenoic (DHA)

-15-

GLYCEROL - 3 - PHOSPHATE

2 FATTY ACYL CoA

2 CoASH

PHOSPHATIDATE CDP - DIACYLGLYCEROL

DIGLYCERIDE

FATTY ACYL CoA

CoASH

CAMP

TRIGLYCERIDE PHOSPHOGLYCERIDE

-16-

CYTOPLASM lbWlDC.HONDRIA

------- > ACETYL CoA

CoASH AMP + PPi I ~-OXIDATION

FATI-Y ACYL CoA FA’ITY ACYL CoA

.- CAR&It 8

b CoASH

----> FATTY ACYL CARNITiNE

Jdd 1.7 ~l~~l~~~~~tl’ul(i7~Ut~~~Ul~~~ 1: fatty acyl CoA synthetase

2,3: fat&y acyl CoA camitine trausferase [ 9 ]

-17-

-1%

FATTY ACYL

1A 2,3-tram-E!NOYL CoA

L3-HYDROXYACYL CoA

NAD+ 7

NADH 1 3

3-KETOACYL CoA

CoASH

ACETYL CoA 1 4

FATTY ACYL CoA (n-2)

1

Lc FADH 2 + NADH + ACJZTYL CoA

FATTY ACYL CoA (n-4)

FADH 2 + NADH + ACETYL CoA

FATTY ACYL CoA (n-6)

FADH 1 + NADH + ACE!TYL CoA

FATTY ACYL CoA (n-8)

4

4

FADH,+ NADH+ACETYL CoA

ACE!TYL CoA

Jdd 1.8 nYtu?UnlY~(sl-88n~l~~UnYtu?UnlY~(sl-88n~l~~U 1: fatty acyl CoA dehydrogenase, 2: enolhydratase

3: L-3-hydroxyl CoA &hydrogenase, 4: 3 - ketothiolase [ 9 ]

-19-

ODD-CHAIN FATTY ACID

~-o~A~ON

I%n ACETYL CoA

PROPIONYL CoA

ADP

METHYL MALONYL CoA

2 ISUCCWYL COA

&I-cis-EBJOYL CoA

I 1

A2,3-trans-EBJOYL CoA

Ifl - OXIDATION

A2.3-cis-ENOYL CoA

D3-HYDROXYACYL CoA

L-3-HYDROXYACYL CoA

Ifl - OXIDATION

-2o-

1.2.8.1 l&i%lh!Il&i%lh!I k&%K (Hydrolysis)

CH,OCOR CHZOH

ICHOCOR + H,O --heat+ CHOH + 3 RCOOH

I ICH,OCOR CH20H

Triglyceride Water Glycerol 3 Free fatty acids

r4 w1.2.8.2 m (E!shification)

- 2 1 -- 2 1 -

0 0

I I

R - C - O H + H O R , - R - C - O R , + Hz0

CI$OCOR

I

CH,OH

I

CH20COR

Alkaliue catalyst ICHOCOR + CHOH ______, CHOCOR

I ICH,OCOR C%OH CH20H

COCOR

I+ CHOH

dH20HdH20H

Triglyceride Glycerol Diglyceride Monoglyceride

1.2.8.4 -- ae (Hydrogenation)

-22-

CH3(CH2)4 CH = CHCH2CH = CH (CH,),COOH - CH3<CH2> ,,COOHPt or Ni

Liuoleic acid Steak acid

1.2.8.5 l&???Jl~V&I,,&l%~U (Polymerization)

R + R - (RI2

Polymerization Reaction

1.2.8.6 v (Oxidation) [ 15 ]

-23-

R’ + A r - O H _____, RH + Ar-0.

active radical hindered phenol inactive radical

I R=

neutral molecule

till?idlf Butylatecl Hydroxyanisole (BHA) Butylated Hydroxytoluene (BHT) Propyl

Gallate (PG) AAQZ Tert-Butyl Hydroquiuone (TBHQ) l!%J&J

-24-

CH,(CHJ), - CE&- CH = CH - CH,- (C~,COOCH,

101 9

1 - H

-‘CH-CH=CH-C%- -CT-+CH=CH-‘CH-

11 10 9. 8 11 10 9 8

Jf-C!H=CH-°CH-C~- -q-eCH--CHP-

11 10 9 8 11 10 9 8

-*CH -CH=CH-C3$- + 0, -

-CH,-CH=CH- +

newintactfatty acid mokcukI

1’

11

-CH -I

CH=CH-CCH,-

0 - 0.

-CH-CH=CH---

I0 - d

-H

-‘a-I -CH-CH- + -CH-CH=CH-CX&-I

new radical O - O H

Third:10 9

ketone t-40 - cIqq,cH = CH - CH - a-L&~:,

Ib--OH

*

CH,(CHJ,CH - CH - CHO + OW~~,C~,

aldehydes dCObOk

Y

1.3.1 &$UQlfd(ll&$UQlfd(ll (vegetable oil)

Y

1.3.2 ~UWfl~Vli (animal oil)

-26--26-

oilsoulw T fatty acid oompodion(%)

6zOo-10~0 129 149 16~0 18~0 18:l 1 8 2 18:3

I

coconut 11.0-19.0 44.0-52.0 13.0-19.0 8.0-I 1 .o

co rn - 10.9

cotton 808d - 22.7

olh 11.0

pslm’ 6.0-13.0 40.0-52.0 14.0-18.0 7.0-9.0

psglut 9 .5

safflower b * - 6 .2

safnowar” 4 .8

- - 8 .9

i3oybQan - 10.3

8unfIower 5 .9

Wllblllh - 7 .0

’ dqnwn MlKKae llwnol:: [ 19 ]

bhigh linobic aoid

= highobiuaoid

LL

1.0-3.0 5.0-8.0 2 .5

1.8 58.0

2 .3 51.5

2 .2

1 .o-3.0

2 .2

2 .2

1 .3

4 .8

3 .8

4 .5

2 .0

24.2

17.0

72.5

11.019.0

44.8

11.7

75.3

39.3

22.8

19.5

22.2

7 .9

0.5-2.0

32.0

74.1

14.2

41.3

51.0

65.7

52.9

1

0 .7

0 .2

0 .6

-

0 .4

-

0 .3

6 .8

10.4

OilllomcQ fatty acid uxnpodion(%)

4zOo-12fl 149 16~0 16:l 18~0 18:l 18:2 OthQlX

lad 0 1 21 3 12 4 6 16 1

Paltry 0 1 2 7 9 7 4 5 1 1 1

beef 0 3 2 6 9 8 4 5 2 0

lamb 0 3 21 4 2 0 41 5 6

dkcOW 10 12 2 6 3 1 1 2 9 2 4

milk-goat 21 1 1 2 7 3 10 2 6 2 0

Qgg Yolk 0 0 2 9 4 0 4 3 1 1 4

-27-

Fatty acid Fatty acid composition ( 96 w/w

140 1.4

16:o 19.6

16:l 3 . 53 . 5

18:O 3 . 83 . 8

18: 1 1 3 . 81 3 . 8

18:2 0 . 70 . 7

18:3 0.1

18:4 0 . 40 . 4

2&l 3 . 03 . 0

20:4 2 . 52 . 5

20:s20:s 17.0

221221 1.0

22:s22:s 1.3

2233 29.8

Haddock

1.5

2 0 . 02 0 . 0

4 . 04 . 0

6 . 16 . 1

1 4 . 21 4 . 2

2 . 22 . 2

0 . 40 . 4

O SO S

2 . 62 . 6

3 . 33 . 3

1 2 . 01 2 . 0

0.1

2 . 42 . 4

246

Mackerel

8 . 68 . 6

1 7 . 61 7 . 6

10.0

2 . 22 . 2

1 4 . 81 4 . 8

1 . 01 . 0

0 . 80 . 8

2 . 02 . 0

8 . 68 . 6

0.9

9.4

1 0 . 21 0 . 2

1 . 21 . 2

8 . 78 . 7

Sprat8

6 . 06 . 0

2 1 . 52 1 . 5

5 . 35 . 3

2 . 42 . 4

1 6 . 51 6 . 5

1 . 61 . 6

1 . 31 . 3

3 . 33 . 3

7 . 07 . 0

0 . 70 . 7

8.1

1 2 . 01 2 . 0

1

Rainbow trout

3 . 53 . 5

1 3 . 31 3 . 3

4 . 84 . 8

3 . 83 . 8

1 8 . 71 8 . 7

5.5

5 .9

2.1

-

7 . 27 . 2

solsol

6 . 26 . 2

2 1 . 02 1 . 0

-2s

*1.3.3 Jhu'ufll~Q~Jhu'ufll~Q~ (microbial oil)

1.3.3.1 nlMtiltrnlMtiltr ( a l g a e ) ( a l g a e )

1.3.3.2 ALIEd& (bacteria)

-29--29-

dative % (w/w) of fat&y apyl Jpupll in lipids

Microorganism 140 MD 169 161 189 18:l 18:2 183 18~3 18t4 20x1

(a) (9

Artbrobnctor AK 19 3 43 5 - 15 24 0.3 - - - -

cMo#lln~ti 2 26 8 9 2 2 34 20 - - -

Dun~primabota 5 11 10 21 tr 6 6 10 2 7 14

Nowhkuin olwabdn 2 15 4 3 11 36 7 - - - 5

sowodwmunncufun tr 15 1 25 1 8 20 30 tr - -

Chdidarp.l07(NCYC 911) 3 44 5 - 8 31 9 1 - - -

ZMomycopkvwdis -15- - 2 5 7 2 4 1 - - -

HnnmrllIlnnniumun 1 24 3 - 4 30 25 12 - - -

tipomyoeetipafer h 37 4 - 7 48 3 - - - -

L.starkqi 32 2 - 7 53 5 - - - -

Rtlod~tio~ 1 24 1 - 13 46 13 3 1 - -

R&odotonliaghrtiaiz? 1 13 - - 7 53 20 5 - - -

R.gWifiS 21 - - 13 52 11 3 - - -

rtl-ichonparonoutnwum tr 30 - - 13 46 11 - - - -

MOldi

Atzpgibniclulnnn tf 20 3 - 12 39 13 - - - -

A.kJlWS 2 23 tr - tr 14 40 21 - - -

hf- lfhaoa 2346-24494--ic

-w= 1 20 2 - - 8 48 11 - 6 -

Spllcclolbeoa~n 8 23 1 - 3 37 1 - - - 7

-3o-

1.3.3.3 &IjhEIl (yeast and mold)

d*

w1.4.1 sY

-31-

-32-

-33-

Radwan iin: soliman [ 3 7 ] Ylcw.l@hl orlndlmn!9lolniiuii Mllfi

Aspetgiuus SP., ~~nic~um q., opium sp., PaedomJxx?s sp., Tri&o&ma sp.,

-34-

-35

ikhMJ,,,D,,l~t~u"~ 12.3%, "VtAA0~r/l~tU~u'" 38.5% AA~~"~~AA"lJJJldtUdw'" 0.9%

-36

1.4.2.1 m (substrate)

-37--37-

carbon source biomass lipid lipid yield fatly acid composition

(30(30 g/l)g/l) wbwb (w/w) (g11OOg. substrate) (%I

1 6 : 01 6 : 0 18:0 l&l l&2

glucose 10.2 3 3 . 23 3 . 2 1 1 . 91 1 . 9 3 3 . 03 3 . 0 1 2 . 01 2 . 0 4 2 . 94 2 . 9 7 . 37 . 3

sucrose 11.2 3 7 . 43 7 . 4 1 4 . 81 4 . 8 3 2 . 43 2 . 4 1 1 . 31 1 . 3 4 2 . 04 2 . 0 6 . 76 . 7

l a c t o s el a c t o s e 1 2 . 51 2 . 5 3 9 . 23 9 . 2 1 6 . 51 6 . 5 3 2 . 53 2 . 5 1 1 . 01 1 . 0 4 9 . 04 9 . 0 6 . 06 . 0

xylose 9 . 99 . 9 4 3 . 64 3 . 6 1 7 . 41 7 . 4 4 1 . 24 1 . 2 1 4 . 01 4 . 0 4 3 . 04 3 . 0 3 . 53 . 5

ethanol 8.5 30.1 10.0 2 6 . 52 6 . 5 1 2 . 51 2 . 5 4 9 . 04 9 . 0 8 . 98 . 9

Growth Substrate Fatty Acids ( relative 9b, w/w )

l&O 16:l 18:o l&l l&2 18:3 2o:o2o:o

corncorn o i lo i l F e dF e d 1 1 . 61 1 . 6 1 1.7 24.7 61.5 0.5 -

Recovered 11.3 5 .8 2.1 3 6 . 03 6 . 0 4 4 . 84 4 . 8 -- --

Linseed oil Fed 7,37,3 -- 3 . 83 . 8 2 0 . 02 0 . 0 1 5 . 01 5 . 0 5 3 . 95 3 . 9 --

Recovered 6 .7 2 .8 6 .7 3 6 . 03 6 . 0 1 8 . 41 8 . 4 2 9 . 42 9 . 4 --

Palm oil Fed 2 9 . 72 9 . 7 7 . 17 . 1 2 . 02 . 0 4 5 . 44 5 . 4 1 0 . 71 0 . 7 5 . 15 . 1 --

Recovered 26.3 7.3 7.6 46.5 1 0 . 31 0 . 3 1 . 91 . 9 --

Olive oil Fed 12.5 1.8 2.5 69.3 10.7 1.3 1.8

Recovered 11.7 2.5 2.5 6 9 . 76 9 . 7 1 2 . 51 2 . 5 1.1 -

-3a-

1 . 4 . 2 . 2 &+A71~17AQ3tj.&& ( g r o w t h r a t e )

-39-

Dillltioll

rate(hr5

0.02

0.04

0.06

0.08

0.10

Biomass Lipid Fatty acid composition

Cl40 Cl&O C18:O C18:l C18:2 C18:3

6.7 57.2 9.1 24.0 14.4 26.6 22.1 3.8

3.9 37.0 9.8 20.6 13.4 22.2 29.5 4.5

2.6 16.9 6.5 18.5 11.7 24.7 34.2 4.4

1.6 16.2 12.6 14.9 8.9 20.4 37.7 5.5

1.4 13.6 10.2 14.3 8.6 20.2 39.6 7.1

a&mole

0.822

0.947

1.063

1.123

1.207

aA / mole designates the degree of unsahration [ 51 ]

-4o--4o-

Growth Lxssolved-oxygen Fatty acid composition

kmp- ( Oc ) tensim@= Of Hg.) 160 16~1 17:l 1 8 : 0 18:l 18:2 l&3 &nolea

b3 0 7 5 17.5 5.5 1.4 tr 41.8 34.4 tr 1.18

2 0 14.7 3.4 tr tr 33.8 48.2 tr 1.31

5 15.1 3.4 2.4 2.9 29.0 45.0 tr 1.25

2 0 7 5 16.9 7.0 3.2 tr 27.4 36.8 8.6 1.37

2 0 17.2 6.8 3.0 tr 27.6 37.9 7.6 1.36

5 19.1 6.6 2.7 tr 27.7 37.0 6.7 1.29

1 5 7 5 13.9 7.1 1.0 tr 23.9 32.6 19.2 1.55

2 0 14.8 6.9 1.1 tr 24.2 31.6 21.3 1.60

5 15.6 7.5 tr tr 23.3 34.0 20.0 1.59

adegee of unsaturation [ 511

btrace amount detected less than 0.1 %

41-

-42-

2 N,H,, + 3 / 2 0, + oleic acid + 2 N, + 3 H,O + stearic acid

v-4 + 0, - 2 N, + 2 H,O

43-

-44--44-

1.4.3.3 fi?rllnzn?ofi?rllnzn?o

1.4.3.4 ihhUKffl¶fltlfld~ihhUKffl¶fltlfld~ (Catalyst and Oxidizing agent)

2 KOOCN=NCOOK + 4H,O +H,N - NH, + 4KHC0, + N,

47-

48-

49-

2.1.1 d

&u”l$ubfi (purified linseed oil) lJOW%l Daler-Rowney n)5EAVW&“snQ%J

d4AflU commercial grade, lilu’wll*l8~A%J commercial grade, 1BFl519W18AFi59l (hydrazine

hydrate, H4N2.H20) FI~lUAu’olu’u 24% kJ~%.Al~5 AAK!til58Fl~ (pyrrolidine) A8U

analytical grade lJOW%l Fluka, f’l59Wfl.h~AWl~~Uo”tl (heptadecanoic acid, C16H33COOH)

&law technical grade, &n^AnRkWI58fl6~VlQ%J (butylaled hydroxytoluene:BHT) llOW%l

Sigma, ~FIAWlt~?IAIlEli ( diethyl ether, (QH5 )20 ) &A&J analylical grade UOW%l J.T.

Baker Chemical, I~AA~nA3uUA~o;~5r~al?ud; ( potassium ferricyani&, &Fe(cN)G ) ‘IJO9

ll%?Vl May & Baker Ltd. Dagenham ~5tAW’&flr\IA, AWl!li~Uflfl8%56 (methylene chloride,

CH2C12 ), A8fIAW.A (hexane), AUYIIUOQ (methanol), AElVllUOlt (ethanol), fI5F1~6l~FWlEl~~~

(hydrochloric acid), fW&$?rl (sulfinic a c i d ) , Id66m763uulatwm%~4 (potassium

hydroxide), nWlEWi%nAv’u~U (glacial acetic acid), kl~%AkJ¶JFll~YOAUS+l (disodium

carbonate), np%la @(+)-glucose), ~~AA~~A~~ol~~~a~~5A~U~~~A~~ (potassium dihydrogen

phosphate), rAY~A~uUls~~5AoUr~~A~~ (disodium hydrogen phosphate), AAWl~A&l&lAv(~

(magnesium sulfate), AAOlJ%JAilWJ&AArl9l (ammonium sulfate), AAfWAA3tJllflAlDl5a' (calcium

chloride)

2.1.2 DlH?sA&&lPs

~tT~AEln~AAVl5fl (yeast extract) lJEl4 Difco Laboratories Detroit, sabouraud

dextrose 4% , sabouraud dextrose agar

-51-

2.1.3 ti

pH meter: Fisher Accumet Model 325 expanded scale research, analytical balance:

Sartorius model A2OOS, Centrifuge : Centronic uow% P.Selecta l&l S - 577, shakiug

incubator : Lab term WN Adoff Kuhrur A.G. ilJ CH-4052 ~5~65lfEf%T6%8~66tlU~, water

bath, heater: Sybrou thermolyue type 1000, n5~~l~nsQ~luR~laalU6~5n (celhrlose nitrate)

UUlFt~nWN 1.2 !lJnSOU, HnOWlWlFNlJUl~ 2.5 x 12 L’W~IlJ995, 6fl~W666dfl5lJldPln5l~6fl~W666dfl5lJldPln5l~

(gas chromatography, G-C.) model GC 14 A UENlJ%l shimadzu kJ~5Wkt66IJlJ flame

ionization detector (PID) dE6t%J6f&~~5XJ?tWn model C-R4A, 6n~fI~66f%~n5IJlkln51~

66tl~66lJ~~6l.kl%Ps6lJVl~ (gas chromatography-mass spectrometry, GC-MS) model GC 8086 MS

d53.lXlWd’XJ~d566nToJ Mass Lab ~&lE&J~66Yll66dtltd~&lE&J~66Yll66dtltd SP-2330 6fi#tW&J 80%

biscyauopropyl, 20% cyauopropylphenyl-polysiloxaue KllU~l1KllU~l1 30 6UPlT llUlF66#U~l~U&6tll~

0.25 0.25 u"tln"6WI5u"tln"6WI5 66fltflfIn'oJu'66fltflfIn'oJu' Gmegawax 320 (o-wax) 6fln”DIJkXJ polyethylene glycol fl1lUQ111fl1lUQ111

30 6I.6995 UUl9l6t&w’l~U&W 0.32 u”nii6Wls UEIW%l Supelco

44 4 22 4'4 'YY2 . 3 2.3 fI156Bl5tJll'PI'1SLfiOJLLAwfJlMl56PU~fI156Bl5tJll'PI'1SLfiOJLLAwfJlMl56PU~

-52-

2.3.2.1 Sabouraud dextrose agar (SDA) 66BE sabouraud dextrose 4% $4

sabouraud dextrose 30 fI%l n=a?u9Urf;“~Ulln=11~~~~~~~5~U*5~ 1 &I5 66#&.h6&1

2.3.2.2 Production media piaUlls=nDulla~~lU~l5~~~ 3.1

2.3.3.2 3.5% methanolic H2SO4 (~llf~~~~WWl5): h.h5F&l~~~ 1.9

&Id%5 &MllU8tI 80 &&%l5 166+-dYhl.hFl56fhJ 100 ih%h5#Jti6WllUtM

2.4.1.1 “?56&JUH”16& (inoculmn) 6%~6&hh=lWU SDA-slant lhd

QQ6M$hhN 24 l+‘Jh ol”~Ui~a~~d6o~~66~~rls~u?ol 10 Qti (loop) iWhll?F@IfU~

(erylenmayer flask) Wlfl 250 u”&ol5 &J55~tllH15 sabouraud dextrose 25 h%hl5 \ql!d

lhJ$! 30°91. QfWXfll56lJth66YY reciprocal 120 5OlJ~Ol.61~ UIU 24 &JhlJ 66hI!l?~%‘#6fh

-53--53-

-54-54

4 wd WCI2.4.4 ~156Pl5~~~~~U~dlws~1RsPl~~~51~ (G.C.)

-55-

-56-

-5s

PKNO TIME4 4.375 4.978 7.339 8.33

1 0 10.271 1 13.3

AREA HEIGHT CONC NAME-7099 1293 17.34 ME PALMITATE2 5 6 1 8 0.63 ME PALMITOLEATE

1660 1 6 6 4.06 ME STEARATE17776 1585 43.43 ME OLEATE6631 4 6 2 16.2 ME LINOLEATE1457 7 9 3.56 ME ARACHIDATE

-59-

PKNO TIME AREA6 4.33 62977 4.87 a 2 0

1 0 7.27 31381 1 8.27 426161 2 10.17 14501 3 13.17 9 5 31 4 14.1 7 3 4

HEIGHT CONC NAME1107 9.41 ME PALMITATE

48 1.22 ME PALMITOLEATE3 1 4 4.69 ME STEARATE

3928 63.66 ME OLEATE7 9 2.17 ME LINOLEATE

39 1.42 ME ARACHIDATE4 2 1 . 1 ME LINOLENATE(w31

-0

- 5

- 1 0

- 1 5

- 2 0

.

- 2 5

,J 1

rr-14 Cl8:3,n-3

PKNO T I M E AREA2 2.98 923 3. 33 144 3:97 73.5 4.88 37196 5.4 125‘7 6.49 55118 7.23 2179 8 . 8 2172

10 9.33 108741 1 10.38 40112 10.74 1184413 11’. 97 49714 13.19 4860315 14.66 1899

HE I GHT353

15 ’909

301029

36286

133346

129045

4229145

COW0.11 140.02 14.50.09 15.234.62 16.02

,o. 15 16.380 17.020.27 17.382.7 18.02

13.5 18.20. 5 18.54

14. -il 18.640.62 18.9-i

60.36 19.272. 36 19.59

CiRBOh; $

fll5El1HlTfll5El1HlT ( g / l ) ( g / l )

yeast extract

glucose

w2p04

Na2HPO4

MgS04.7H20

(NHq)2s04

CaC12.2H20

FeS04.7H20

CaC12.2H20

%lso,$7H’~o

CUSO4SH2O

CoCl2.6H2O

MIlSO4.H2O

(NHd2M0207

0 . 50 . 5

20.0

7.0

2.0

0.73

1.0

0.134

-

-

-

m?lJl~UOL%Okyl5il2

1.0

20.0

2.4

2.3

1.0

4.0

0.024

0.036

0.008

0.001

0.001

0.005

0.001

-62-

-63-

25 r

20t

tB medium 1

43 medium ltsoap

f medium 2

* medium 2tsoap

+ medium 9

* medium Stsoap

.0’ I I I I I I I

0 24 48 72 ee 120 144 166

Hours

4%

25c

20-

ClS-m

%$

ElO-

@ medium 1

+S medium l+soap

* medium 2

+ medium 2+soap

+ medium 3

* medium S+soap

-65-

-66-

01w1qmil 1 %I Lllo~~rU~nY~~uu’ulUlc~~U~~~~~

C16:O C16:l Cl&O C18:l C18:2 Cl&3 C22:O

1 36.7 1.8 24.7 28.3 4.3 4.0 0.4

lliihff$uaR 2 23.3 1.5 5.5 48.5 17.7 3.1 0.4

3 21.1 1.3 3.3 38.2 26.3 9.2 0.6

5 20.6 1.7 1.6 40.0 25.7 10.2 0.3

1 16.7 2.2 2.7 39.7 25.9 10.7 2.1

l~Ufl~~Ui)R 2 8.8 0.4 6.0 25.6 16.4 42.2 0.7

3 12.0 0.0 4.7 30.9 20.2 32.2 0.0

5 13.6 0.5 11.4 44.6 13.9 15.0 1.1

Dlwlq91’1il 1 Td 6llo~l~~~~luu’u~ul~nn;o~u”ao;

C16:O C16:l C18:O C18:l C18:2 C18:3 C22:O

1 20.1 0.9 3.0 41.4 27.0 7.5 0.0

lti13U~Yj~U~R 2 14.0 1.8 6.8 53.6 19.8 4.2 0.0

3 17.8 0.6 3.6 49.1 24.0 4.9 0.0

5 16.8 1.2 2.6 49.1 25.6 4.7 0.1

1 12.2 0.2 9.3 30.0 15.7 32.5 0.1

&Jfl$hJZJR 2 14.1 0.3 7.8 39.9 15.0 22.0 0.8

3 13.9 0.5 8.3 45.3 13.9 17.3 0.9

5 12.6 0.7 6.2 32.5 22.7 24.5 0.9

-67-

+=--l-! Cl8:O

f 15 Cl8:l,rv9- 1 0 f- - 1 i Cl8:2,n4

19 C18:3,n-3

- 1 5

PKNO6

A9

10

:s.1;141, ;16l/

- l-819302 1

-20

T I SlE4. ai4.514.993. 666.196.547.157. 3;- 71

*;:j9. 559.89

1-l-L 3 9-14.9413.6318.38

AREA2;

3383322

284581720

20124184167

44i351418

6235444

61512335

430- - -I/3

HE I GHT COW CXRBOh: ;12 0.02 12.63

699 2.42 13.951 0.23 14.89

4237 20.35 15.92216 1.23 16.56

2311 0 16.9322 0.13 1-i. 4930 0.12 1;. 6’;

451 3.3 1;. 914805 36.78 18.48

13 0.04 19.13000 25.35 19.37

31 0.34 19.97878 8.82 20.19

36 0.31 20.4530 0. 55 22.06

>

-69-

--00

lK(NO?

256

8899

10101 11 1

TIYE ARE-44. jr 883j. 73 i9966. 26 2906.61 48377.45 106i. 8 16108. 5; 320999.97 lOi90

12:05 2209

.

HE I GHT188

140843

-iQ919

20224201031

179

CONC1.92

17.390.6300.23? -

4;: ;623.474 . 8

CARBON q14. OS1616.64171;. 741818.5319.3120.23

-7o-

- 0

- -3

-10

- 1 5

- 2 0

-25

PKNO4567‘St9

101 1121314151617181920

T I ?ilEil.335.03- -

;: ;66. 397. 217.137.88.629.389.629.97

Il.5313.0714.9818.1233.85 -

-iREX1670369

387341474

2346928747

33077126787

287733&Ml

-- 38860869

48252341

2548339

HE I GHT CONC CiRBON 7358 0.59 14. 16

32 0.09 15.133945 13.64 16. 11

109 0. 52 16.653069 0 17.08

28 0-l li-. 6317 0.02 17.82

3409 8. 12 18.0712872 44.63 18.63

25 0. 1 19.0533 0. 08 19.17

3633 13.68 19.3560 0.31 20.04

3741 16.99 30.2521 0. 12 21.2

102 0.9 22.0611 0.12 23.w

-71-

-72-

-73-

e R~taDap+ 572

+ 5l2tmp

24 4 8 7 2 95

Hours

120 144 155

-74-

ti

2

3

4

5

2

3

4

5

C16:O C16:l C18:O C18:l C18:2 C18:3 C22:O I

38.4 2.1 7.0 50.1 1.8 0.4 0.4

32.6 1.7 4.7 51.4 8.4 1.2 0.0

39.5 2.9 4.3 51.9 0.6 0.5 0.3

31.1 3.7 2.8 56.0 4.9 0.4 1.1

8.3 0.3 6.4 29.5 18.4 36.1 0.9

22.3 0.4 12.6 28.0 15.4 19.2 2.2

30.4 0.0 11.5 27.2 14.2 16.7 0.0

30.2 0.6 10.4 30.3 15.2 12.4 0.9

2 30.9 0.8 8.8 58.9 0.4 0.3 0.0

3 24.9 0.3 6.4 66.0 2.5 0.0 0.0

4 15.8 0.3 3.3 50.0 24.5 6.2 0.0

5 13.6 0.9 1.9 51.4 25.9 5.9 0.3

2 24.4 0.5 17.7 56.4 0.7 0.4 0.0

3 38.9 0.0 20.3 34.8 4.3 1.8 0.0

4 33.7 0.4 15.8 42.2 5.7 2.2 0.0

5 32.0 0.0 14.2 29.9 11.3 11.1 1.4

-75--75-

-76-

-0

- 5

- 1 0- 1 0

- 1 5- 1 5

- 2 0- 2 0

- 2 5- 2 5

-- 3 0 3 0

PWO IlYEIlYEi JJ8 - 1 .- 1 . 56560 4.974.97

IO j . . 3j . . 3I I 3 . 7 23 . 7 2I:! 6 .6 . I I I II3 6 . 5 96 . 5 9II 7.077.071 51 5 7. 3 61 61 6 7 . 77I iI i R. 051 81 8 8 . 4 08 . 4 0I OI O 9 . 1 99 . 1 92 02 0 I O . I O . 552 12 1 1 11 12 22 2 1 2 . 0 61 2 . 0 62 32 3 I ? .I ? . 4 3 4 32 42 4 1 3 . 0 91 3 . 0 92 52 5 13.4713.472 62 6 1 3 . 8 31 3 . 8 32 i2 i I - 1 . 6 9I - 1 . 6 92 s2 s 16.2716.272 92 9 1 6 . 7 31 6 . 7 33 03 0 I i . 1 5I i . 1 53 13 1 1 8 . 1 91 8 . 1 93 '3 ' 1 9 . 6 21 9 . 6 23 33 3 1 9 . 9 91 9 . 9 93 43 4 29.2929.293 53 5 2 3 . 2 3 . SFSF16 2 1 . 7 42 1 . 7 4

1 dd Cl&OCl&Ozrzr Cl&l,n-0Cl&l,n-0

I-lo.f---- 4 14 1 tXM,n-3tXM,n-3

AREA H E I G H TH E I G H T3 1 03 1 0 9 59 5

3 8 1 93 8 1 9 9 ' ) '9 ' ) '715 I J OI J O318318 3 93 9

i 6i 6 I OI OIOi520IOi520 185381853816655'16655'

141;141;2790927909

1 8 01 8 04 1 34 1 3 4 84 8

2 0 1 62 0 1 6 2 0 02 0 048524852 i40i405 6 3 35 6 3 3 7187184 2 84 2 8 j lj l

3 7 0 3 3 ‘ I 0 9 1‘ I 0 9 11 8 7 5 5 6 I85i 1

3 2 6 13 2 6 1 36X36X12311231 2 82 83 5 53 5 5 I ?I ?6 1 36 1 3 S TS T

12211221 I I OI I O1 7 0 51 7 0 5 I70I70

5 . 1 65 . 1 6 3 33 3I304I304 9 59 55 1 65 1 6 3 83 8337337 2 82 8

2 5 9 72 5 9 7 2542547 37 3 ii

3 0 03 0 0 2 42 41 0 1 01 0 1 0 1 31 37 8 6 17 8 6 1 i l li l l

C O N C0 . 0 50 . 0 50 . 6 30 . 6 30.120.120 . 0 50 . 0 50 . 0 . 0 10 1

I i . 7 9I i . 7 92 9 . 62 9 . 60 . 2 30 . 2 30 . 0 70 . 0 70 . 3 30 . 3 3000 . 9 30 . 9 30 . 0 76 . 1 36 . 1 3

3 1 . 0 43 1 . 0 40 . 0 . 5 . 15 . 10 . 20 . 20 . 0 60 . 0 60 . 0 . II0 . 20 . 20 . 2 80 . 2 80 . 0 90 . 0 90 . 2 20 . 2 20 . 0 90 . 0 90 . 0 60 . 0 60 . 1 30 . 1 30 . 0 10 . 0 10 . 0 . OSOS0 . 0 . I iI i1 . 31 . 3

15.13IS.41IS.41Ii.72Ii.72l bl b16.2816.2816.5516.55

22Ii.03 ,I i . 2 3I i . 2 31 7 . 5 21 7 . 5 2I 8I 818.1;18.1;I S . I S . 4949I S . 6 1I S . 6 1I C 3 . i ;I C 3 . i ;18.8718.87I S 9 6I S 9 619.1719.1719.5219.521 9 . 6 21 9 . 6 21 9 . 71 9 . 71 9 . 8 91 9 . 8 92 0 . 1 52 0 . 1 52 0 . 2 12 0 . 2 12 0 . 2 62 0 . 2 62 0 . 3 42 0 . 3 42 0 . . 1 92 0 . . 1 9

-77-

- 0

-- 5 5

--1 0

-1 5

- 2 0

- 25

---- A I CZ6.n3- 30

Pi\0 T I NT I N AFiEA8 4 . 1 94 . 1 9 16759 5. ?I lil

IO 5.09 3042111 6.41 8 1 9 911 6.95 3 6 013 i. 26 132I II I i. 5 3 2 3

15 7 . 6 7 18421 6 i. 93 2 2 5 31; 5.3; 122-1IS 9.07 4 3 719 10.34 8656‘0 10.76 2 0 9 3 3‘1 11.31 2 1 412 11.93 1 8 6 82 3 12.71 135521 1 3 . 1 453IS 13.713.7 10562 6 14.56 I18427 16.15 6 3 02s 16,61 20X32 9 16.92 2 3 03 0 I i . 6i 3 3 831 18.04 3213 2 15.81 22s3 3 19.47 2 7 3 03 1 20.9 83;3 5 21.5 i S 9 7

3 6 22. T3 5 7 0;i 23. o-l 10973 8 2 6 . 3 6 1 23 9 2;. 21 2 4 5 240 28.46 2 3 6 411 29.4 3 3 8 6 2

I I E I C I I TI I E I C I I T321

2 544321092

3i1.l4 9

2 3 02 3 2II

.lO863Ii32Ii32

2 62 6IS1119

488 2

1 2 16 2

1 5 71 5 7

::3 32 0

264G O

7SO33i ii i2 9

?OiIi3

3231

-12 30 OR 2514 116

I--- I O C16:O

2 52 6

35 C205,n-3

co\c cm:o\ =I. 2 I-l.820.12 15.-I-i

31.T 1 65.8; 16. ?b0 . 26 16 . 560.09 16.730.23 16.530 16.931.61 IT.060 . 8.5 Ii. 250.31 17.516.21 IS

14.9s IS. 140.15 1s. 311 . 3 - i 1% 50.9i 18.i2 ,0.35 IS . 810.5; I S . 97O.SS 19. Ii0.45 19.521.49 19.610 . 16 19.670.21 19.820.23 19.ss0.16 20.03I. 9s 20.130.6 20.373. 63 20.4i0.41 20. GJ0. is 20.650.34 21.11I.75 21.221.69 21. 36

2-1 .23 2 1 . 4 7

1.8 21.51

-0

-3

-10

89 , ~ C16:Oll

li 15Cl6:l,n-7

Cl&O _- iF3 Clt%l,n4

PKSO T IYE6 3.19

9 1.971 0 6 . 11 1 6.5612 7. o.!

l-1 s. 03

1 6 8.7li IO. 3i1 8 10.961 9 13.032 0 12.4-L2 1 13.2122 !6.2623 16.762-I 17.1415 22.862 6 7- -/.31

1 9 9 53i96

15526521

1 4 2 6 7 83323 0 61 2 24 8 14 1 13573 8 4235

HEIGHT467

822 6 41 0 9

1080521315

IS10

q-.7>>I4 2 3

2 33250

146113 61 41 15 - l2923331 5

:o.\c0.330.090. 380.15

L5.536.13

0.0-l0.0200.760.047.19

38.70. 10.080.03

-0.130.110. 10 . 10. 06

C.?IRBO\ =1 3 . 5 914.0-L14.815. i71 616.2816.5316.521;. 0-Ili. 2-l1,. 3-l1 818.1618.4918.618.82

- 19.5219.6219.6920.6321.21

-79-

-8O--8O-

1010 Cl6:OCl6:O

-15

-20- 25 11 Cl@0Cl@01212 Cl6:lA9;n-9Cl6:lA9;n-9

13 Cl8:lAl2;n-6

- 301515 Cl6:2A9,12;n-6Cl6:2A9,12;n-6

1616 Cl6:2A9,15;n-3Cl6:2A9,15;n-3

1s Cl8:3A9,12,15;n3

iREi HE I GHT COSC CARBOX =610: 7 3 0 5.51 16

-71531 137S 20.5 1S2 2 6 0 6 1 x4 21.53 1s. 161 3 3 6 0 ;-‘2 12.72 1 s . 2 9

6717 306 6.42 1s. 5 213631 69s 1’. 9s 1s. 58

7829 40s 7.46 1s. 716 0 7 0 153 5.78 1 s . 97 1 3 5 310 6.79 15. !P

-81-

-82-

J

-84--84-

o~l~6anlnl5r~uo~"5a%uu"u~~ 2 n'urtcjulu"iln5du~u~~n'urtcjulu"iln5du~u~~ 1 1 w5ua:Fjw5ua:Fj hJ6hmlhJ6hml

dlhJnl5?f~n"E1dlhJnl5?f~n"E1 C18:ti9,12,C18:ti9,12, C18:2C18:2 A9,1S 66flf66flf C18:2A12,15C18:2A12,15 09lUdlkl09lUdlkl

20r

16

15 16 21 24 27

mole ratio* 4

+ 8

f 12

+ 16

+20

- 8 6 -- 8 6 -

oo 33 66 88 1 21 2 1 51 5 1 81 8 2 12 1 2 42 4 2 72 7 3030

Reactbnthne(hours)

mole ratio-- 4 4

++ 88

ff 12 12

* 16

++ 20 20

-87-

-88-

18-

18 -

zp14 -

. r

&2-

.-ilo-& _

h 8-

8 -

4 -

-m- 0.1 g.

+ 0 . 2 g .

t- 0 . 5 g .- l.Og.

+ 1.59.

0 8 8 0 12 15 18 21 24 27 80

-89--89-

l*r

88

Q 0.1 g.

-e 0 . 2 9 .0 . 2 9 .

f 0 . 5 g .

- 1 . 0 g .+ 1.6g.

i---ri---r 11 11 11II II II II iibb ~-m~-m

II 99 66 99 1 2 12 15 1 8 21 2 4 2 7 3 0

Reaction time ( hours )

-9O-

-91-

2 0 -

1 0 -1 0 -

1 6 1 6 --

14 -

ap

?2-?2--9

,o,o0

vv10 -

N-

i -

8 -

06-

r

8 73 %+ 4 6 %f 31 %+ 16 %+ 0 %

0 3 6 9 12 15 18 2 1 24 27 30

R-ume(hows)

-92-

IO-

Q -

. -

7-ap

I

2J a -

u

K 5-

9

e

! 4- -

3-

-a-a 7 3 %%44 4 6 %4 6 %

ff 3 1 %%++ 16 %%++ 0 %0 %

-93--93-

-94--94-

0 3 6 9 12 1 5 18 21 2 4 2 7 3 0

Reaction times(hours)

* pH 0.0

* pH 0.0

* pH 11.0

4 pH 12.0

+ pH 13.0

-95-95

10-

Q-

u-

7-ap-

u3 o-

8S-

g _

3'liil 4-

8a-

2-

l -

* pti 0.0

* pH 9 . 8f pH 11.0

* pH 12.0+ pH 19.0

0 3 Q 1 2 15 lb 2 1 24 27 30

Rsadion timens(houm)

-96-

Octadec-124noic add, 96

-9%

0 S 8 0 12 1S 18 21 24 2 72 7 soso

Fbwtion timcw, ( hours )

* 3o”c

+- 50°C* 7o”c* 90°C

-99--99-

~~olnnolmsvo~mltn"ali~~~~~6flij~~olnnolmsvo~mltn"ali~~~~~6flij [[ 8 5 ] 8 5 ] 66ftE66ftE [86][86] fJd#fJd#

-d[A] / d t = k [ A ]

i%Jn"6~~~~ll~l~~Xi%Jn"6~~~~ll~l~~X In Ml, / [Alo= -kt

6dO6dO [Al : concentration of substrate

(1)

(2)

WI,: concentration of substrate at any times

Ml0 : concentration of substrate at time zero

t : reaction times

k : rate constant

66tl~6lhUD~~U~d66tl~6lhUD~~U~d exponential hW~l~6?hJhW~l~6?hJ

- k tCAlt/[Al, = e (3)(3)

dD- k t

C A It = [Q-p (4)(4)

ki logarithms 1#6?hJ h[AJ - ln[A]* = -kt (5)

H% lxl[A]tlxl[A]t = = -kt + ln[A], (6)

~~6~uum'ln"ynu"lr6~u~1J~~6~uum'ln"ynu"lr6~u~1J , , y=mx+by=mx+b

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9O”V. +h6lZldlQdi 3.28 - 3.31 o~:r~~11lU~Ul~Ut~U~~~6~1~~ -k 66R=ihJd$%Jl~U~~

db%ildb%il k 1Tilfh1Tilfh 3.077~10-~,3.077~10-~, 8.025~10-~,8.025~10-~, 15.55oX1O-315.55oX1O-3 66Qt66Qt 20.740~16~20.740~16~ 9WdlkJ9WdlkJ $0$0

6ihJUflW'htiVf~l~6ihJUflW'htiVf~l~ InkInk fhfh l/rl/r (OK.(OK. ) ) f1lfWlYl4~f1lfWlYl4~ 3 . 6 3 . 6 66~~nlP1?lu~uuo~6~unslrl~~~~66~~nlP1?lu~uuo~6~unslrl~~~~

I&I& 3.32 3.32

k(mi.t?)

-33 . 0 7 73 . 0 7 7 xx 10

-38 . 0 2 58 . 0 2 5 x 10

lnk

-- 5 . 7 8 45 . 7 8 4

-- 4 . 8 2 54 . 8 2 5

T ( OK. ) l,T(IP)

-33 0 33 0 3 3.30x3.30x 10

-33 2 33 2 3 3 . 1 0 3 . 1 0 x 10

-3 -31 5 . 5 5 51 5 . 5 5 5 x 10 - 4.163 3 4 33 4 3 2 . 9 2 2 . 9 2 x 10

-3 -32 0 . 7 4 02 0 . 7 4 0 x 10 -- 3 . 8 7 63 . 8 7 6 3 6 33 6 3 2 . 7 5 2 . 7 5 x 10

-lOO-

-2 , I I I

-3 -3.077x10 -3 mill-1

slope =

lIl[C18:3] A

-5

~~

ln[C18:3]ln[C18:3] 44 _ __ _

-8 - -

-lOl-

- 3-3

-1. dOp= -15.550x10 min

lIl[C18:3] 4

4

-5

1~

4

-2 I I I I

-3 - --1

lIl[C18:3] 4 - -

-5 - -

00 30 60 90

time (minute)

120 150

-102-

-3

-4

I n kI n k

l/Tl/T xl&K-'1xl&K-'1

-103--103-

-Ea/RTk = A e

A : frequency factor

R : gas constant , 8.314 j/mol.‘K

T : Kelvin temperature

( 7 )( 7 )

Ea : activation energy

Iti logarithm t%wwlnun1s~Xt%wwlnun1s~X

- Ea/RTInk = h eh e +lnA (8)

N%l Ink = -Ea/R( l/r)+lnA ( 9 )( 9 )

PllU?arRn~~Uuo~~~~"57dQl"~11~PllU?arRn~~Uuo~~~~"57dQl"~11~ 3 . 3 2 3 . 3 2 1414 mm = - = - 4.258~10~4.258~10~ K lnlWi1lnlWi1

*??u~~lwnunl~~(s)ua=*??u~~lwnunl~~(s)ua= mm = = -- Ea/ R !#

-Es/R = - Ea / 8.314 J/mol. K

= - 4.258~10’ K

-105-105

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5 .5 .

6 .6 .

7 .7 .

8 .8 .

9 .9 .

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lhdlhd :hlicgw',:hlicgw', I%lfII%lfI il&il& UtEn^ql~-iJjUtEn^ql~-iJj Wlihhd,253O,+Jlfd,Wlihhd,253O,+Jlfd, IJMl?WtJl&JIJMl?WtJl&J

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1 6 .1 6 .

1 7 .1 7 .

18.

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2 0 .2 0 .

21.

2 2 .2 2 .

2 3 .2 3 .

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