Composition and origin of complex organic matter in recent marine sediments;: Significance of bacterial biomarkers

C O M P O S I T I O N A N D O R I G I N O F

C O M P L E X O R G A N I C M A T T E R

I N R E C E N T M A R I N E S E D I M E N T S

Significance of bacterial biomarkers

COMPOSITION A N D ORIGIN OF

C O M P L E X ORGANIC MATTER

IN RECENT MARINE SEDIMENTS

Significance of bacterial biomarkers

f o co o vO GO I n l l l M H 1« MU It 1:1 I t i l ' 1 III ill :« B I B L I O T H E E K T U D e l f t P 2 0 9 2 5 0 2 0 C 15310 8 6 5 1 8 9

COMPOSITION A N D ORIGIN OF

C O M P L E X ORGANIC M A T T E R

IN R E C E N T M A R I N E SEDIMENTS

Significance of bacterial biomarkers

PROEFSCHRIFT ter verkrijging van de graad van doctor in de technische wetenschappen

aan de Technische Hogeschool Delft, op gezag van de Rector Magnificus,

prof.ir. B.P.Th. Veltman, in het openbaar te verdedigen

ten overstaan van het College van Dekanen op donderdag 24 mei 1984 te 14.00 uur

door

JACOB K L O K doctorandus in de chemie

geboren te Brielle

prof.drs. P . A . Schenck

00

Dr. H . C . Cox en dr. J.W. de Leeuw hebben in hoge mate bijgedragen aan de

begeleiding bij het tot standkomen van het proefschrift en zijn als zodanig aangewezen door het College van Dekanen.

Aan mijn ouders

Aan Monique

CONTENTS

1. INTRODUCTION 1

References 5

2. QUALITATIVE AND QUANTITATIVE CHARACTERIZATION OF THE TOTAL ORGANIC

MATTER IN A RECENT MARINE SEDIMENT I I

Abstract 7 Introduction 8 Experimental 8 Results 12 Discussion 14 Conclusion 24 Acknowledgement 24 References 25

3. CAPILLARY GAS CHROMATOGRAPHIC SEPARATION OF MONOSACCHARIDES AS THEIR ALDITOL ACETATES

Abstract 29 Experimental 30 Results and discussion 30

References 32

4. ANALYSIS OF SYNTHETIC MIXTURES OF PARTIALLY METHYLATED ALDITOL ACETATES BY CAPILLARY GAS CHROMATOGRAPHY , GAS

CHROMATOGRAPHYELECTRON IMPACT MASS SPECTROMETRY AND GAS CHROMATOGRAPHY -CHEMICAL IONIZATION MASS SPECTROMETRY

Summary 33 Introduction 34 Experimental 34 Results and discussion 38

Conclusions 42 Acknowledgements 43 References 43

5. CARBOHYDRATES IN RECENT MARINE SEDIMENTS I

ORIGIN AND SIGNIFICANCE OF DEOXY- AND 0-METHYL-MONOSACCHARIDES

Abstract 45 Introduction ' 46 Sample description 47 Experimental 49 Results 51 Discussion 54 A cknowledgements 61 References 61

6. CARBOHYDRATES IN RECENT MARINE SEDIMENTS I I

OCCURRENCE AND FATE OF CARBOHYDRATES IN A RECENT STROMATOLITIC DEPOSIT : SOLAR LAKE , SINAI

Abstract 67 Introduction 68 Experimental 68

Discussion 74 Conelusions 80 References 81

7. THE MODE OF OCCURRENCE OF L I P I I S I N A NAMIBIAN SHELF DIATOMACEOUS OOZE WITH EMPHASIS ON THE 6-HYDROXY FATTY ACIDS

Abstract 83 Introduction 84 Experimental 84 Results 87 Discussion 90 References 92

8. LOLIOLIDES AND DIHYDROACTINIDIOLIDE I N A RECENT MARINE SEDIMENT PROBABLY INDICATE A MAJOR TRANSFORMATION PATHWAY OF CAROTENOIDS

Abstract 95 Experimental 96 Results and discussion 97

Acknowledgements 100 References 100

SUMMARY AND CONCLUSIONS 101

SAMENVATTING EN CONCLUSIES 105

CURRICULUM VITAE 109

1

C H A P T E R 1

I N T R O D U C T I O N

The o c c u r r e n c e o f o r g a n i c m a t t e r on e a r t h i s n o t l i m i t e d t o t h e c o n s p i c u o u s forms l i k e biomass and t h e e x p l o r a b l e f o s s i l r e s o u r c e s c o a l , p e t r o l e u m and n a -t u r a l g a s . I n f a c -t -t h e s e forms c o n s -t i -t u -t e o n l y a v e r y s m a l l p a r -t o f -t h e -t o -t a l o r g a n i c c a r b o n p r e s e n t t h r o u g h o u t t h e e a r t h ' s c r u s t . The m a j o r p a r t i s p r e s e n t i n s e d i m e n t a r y r o c k s i n a d i s s e m i n a t e d and i n s o l u b l e form w h i c h i s g e n e r a l l y known as k e r o g e n . I t has been e s t i m a t e d t h a t about 90% o f a l l t h e o r g a n i c c a r -bon on e a r t h i s p r e s e n t as k e r o g e n (Hunt, 1979).

The word k e r o g e n d e r i v e d f r o m t h e Greek "keros" (= wax) was p r o p o s e d t o d e s c r i b e t h e o r g a n i c c o n t e n t o f o i l s h a l e w h i c h p r o d u c e s , by d e s t i n a t i o n , o i l w i t h a waxy ( p a r a f f i n i c ) c o n s i s t e n c y ( S t e u a r t , 1912). However t h e term k e r o g e n c o v e r e d and s t i l l c o v e r s a v a r i e t y o f c o n c e p t s w h i c h h i s t o r i c a l l y o r i g i n a t e from t h e e x p l o i t a t i o n o f o i l s h a l e s and w h i c h a r e c l o s e l y l i n k e d t o r e s e a r c h d e a l i n g w i t h t h e o r i g i n o f p e t r o l e u m , e s p e c i a l l y w i t h t h e o r i e s on i t s o r g a n i c o r i g i n . I n t h e d e f i n i t i o n s t a t e d by Durand (1980) and used h e r e , k e r o g e n i s d e f i n e d as t h e f r a c t i o n o f s e d i m e n t a r y o r g a n i c m a t t e r w h i c h i s i n s o l u b l e i n the u s u a l o r g a n i c s o l v e n t s . I n p r a c t i s e t h e t e r m k e r o g e n d e s i g n a t e s a p o l y c o n d e n -sed o r p o l y m e r i z e d s t a t e o f o r g a n i c m a t t e r n o t b e l o n g i n g t o the l i v i n g r e a l m . I n t h e c u r r e n t c o n c e p t o f k e r o g e n f o r m a t i o n (Hunt, 1979; T i s s o t and W e l t e , 1979; Durand, 1980) b i o p o l y m e r s a r e a t t h e v e r y s t a r t o f t h e d i a g e n e s i s o f o r -g a n i c m a t t e r . A f t e r t h e d e a t h o f o r -g a n i s m s , d u r i n -g s e d i m e n t a t i o n and a f t e r w a r d s t h e a c t i v i t y o f b u r r o w i n g o r g a n i s m s and m i c r o b e s , governed by e n v i r o n m e n t a l c o n d i t i o n s , r e s u l t s i n t h e p a r t i a l d e c o m p o s i t i o n o f b i o g e n i c p o l y m e r s . D u r i n g t h i s p r o c e s s b i o p o l y m e r s a r e p a r t l y m i n e r a l i z e d and p a r t l y r e s y n t h e s i z e d i n ( m i c r o b i a l ) c e l l m a t e r i a l , thus u l t i m a t e l y l e a v i n g an o r g a n i c r e s i d u e u n a s s i m i l a b l e by m i c r o o r g a n i s m s under t h e s e c o n d i t i o n s . T h i s r e s i d u e i s now i n c o r p o -r a t e d i n t o a new, humic p o l y c o n d e n s a t e p -r e c u -r s i n g k e -r o g e n ( T i s s o t and W e l t e , 1979). I n a s p i n - o f f f r o m t h e b i o g e o c h e m i c a l c y c l e p a r t o f t h e m i c r o b i a l l y

r e l e a s e d biomonomers a r e t h o u g h t to condense and to be i n c o r p o r a t e d i n the p o l y c o n d e n s a t e as w e l l (Hunt, 1979). A p a r t f r o m the i n s o l u b l e f r a c t i o n

( k e r o g e n ) s e d i m e n t a r y o r g a n i c m a t t e r c o m p r i s e s a m i n o r amount o f f r e e h y d r o -carbons and r e l a t e d compounds a t t h e end o f the d i a g e n e t i c p e r i o d . These h y d r o c a r b o n s o r i g i n a t e f r o m compounds s y n t h e s i z e d by l i v i n g o r g a n i s m s and a r e i n c o r p o r a t e d i n the sediment w i t h no o r minor changes and w h i c h can be c o n s i -dered as g e o c h e m i c a l f o s s i l s r e f l e c t i n g the e n v i r o n m e n t o f d e p o s i t i o n .

Because o f i t s q u a n t i t a t i v e i m p o r t a n c e and because i t i s c o n s i d e r e d to c o n s t i t u t e the s o u r c e m a t e r i a l f o r o i l and n a t u r a l gas i t i s w o r t h w h i l e to u n d e r s t a n d the c o m p o s i t i o n and o r i g i n o f k e r o g e n . Due t o i t s i n s o l u b i l i t y k e r o g e n i s l e s s amenable t o s t r u c t u r a l i n v e s t i g a t i o n f r o m an a n a l y t i c a l p o i n t o f v i e w . Moreover when s t u d y i n g i t s o r i g i n t h e absence o f a m o r p h o l o g i c a l r e l a t i o n s h i p w i t h b i o l o g i c a l s t r u c t u r e s i s a g r e a t d i s a d v a n t a g e . However, the c o o c c u r r e n c e o f g e o c h e m i c a l f o s s i l s i n t h e s o l u b l e f r a c t i o n o f s e d i m e n t a r y o r g a n i c m a t t e r c o u l d y i e l d some i n f o r m a t i o n about t h e o r i g i n . I n the case o f c o a l s t u d i e s the morphology o f the n a t u r a l p r e c u r s o r i s p a r t l y r e t a i n e d upon c o a l i f i c a t i o n , w h i c h s i m p l i f i e s b o t h the c h e m i c a l c h a r a c t e r i z a t i o n and the i n t e r p r e t a t i o n o f t h e p r o c e s s e s t h a t l e d t o the p r e s e n t c o n d i t i o n o f t h i s type o f o r g a n i c m a t t e r . I n o r d e r t o make k e r o g e n a c c e s s i b l e t o s t r u c t u r a l e l u c i d a t i o n c h e m i c a l d e g r a d a t i o n t e c h n i q u e s must be employed. D e g r a d a t i o n p r o c e s s e s s h o u l d be as s p e c i f i c as p o s s i b l e i n o r d e r t o o b t a i n s m a l l e r i d e n t i f i a b l e compounds w h i c h s t i l l r e t a i n a s t r u c t u r a l r e l a t i o n s h i p w i t h the k e r o g e n . V a r i o u s t y p e s o f c h e m i c a l d e g r a d a t i o n and f u n c t i o n a l group a n a l y s e s have been c a r r i e d o u t , many of w h i c h have been r e v i e w e d by V i t o r o v i c ( 1 9 8 0 ) . F u r t h e r m o r e p y r o l y s i s methods have shown t o be v a l u a b l e t o o l s i n s t r u c t u r a l c h a r a c t e r i z a t i o n o f k e r o g e n s

( e . g . L a r t e r and D o u g l a s , 1980; S o l l i et at., 1980; van de Meent et at., 1980). However the above methods a r e o f l i m i t e d v a l u e f r o m a q u a n t i t a t i v e p o i n t o f v i e w . Moreover i t i s o f t e n i m p o s s i b l e to r e l a t e the s t r u c t u r e of the p r o d u c t s r e l e a s e d to t h a t o f the o r i g i n a l complex o r g a n i c m a t r i x .

S i n c e i n the c u r r e n t c o n c e p t the g e n e s i s o f k e r o g e n i s c o n s i d e r e d t o be the r e s u l t of a sequence o f b i o c h e m i c a l and g e o c h e m i c a l t r a n s f o r m a t i o n s o f o r g a n i c m a t t e r p r i m a r i l y d e r i v e d f r o m b i o m a s s , one c o u l d c o n s i d e r the o r g a n i c m a t t e r p r e s e n t i n a r e c e n t sediment t o r e p r e s e n t an i n t e r m e d i a t e s t a g e between biomass and k e r o g e n . F o r t h i s r e a s o n s t u d i e s o f t h e o r g a n i c m a t t e r f r o m r e c e n t s e d i m e n t s c o u l d y i e l d v a l u a b l e i n f o r m a t i o n about the s t a r t i n g s t r u c t u r e s o f f u t u r e k e r o g e n . When t h e o r g a n i c m a t t e r i n a r e c e n t m a r i n e sediment i s r e g a r d e d as an a c c u -m u l a t i o n o f b i o p o l y -m e r s i n v a r i o u s s t a g e s o f d e c o -m p o s i t i o n the s t r u c t u r a l c h a r a c t e r i z a t i o n i s d i r e c t e d i n t h e f i r s t p l a c e by a n a l y t i c a l methods a i m i n g a t

3

the r e l e a s e and q u a n t i t a t i o n o f b i o p o l y m e r s o r b u i l d i n g b l o c k s of b i o p o l y m e r s . I n the second p l a c e o r g a n i c compounds n o t r e c o g n i z a b l e as s u c h , b u s t most p r o b a b l e d e r i v a t i v e s t h e r e o f , must be i n v e s t i g a t e d .

V a r i o u s a t t e m p t s have been u n d e r t a k e n t o q u a n t i f y the c o n t r i b u t i o n o f the m a j o r compound c l a s s e s i n the m a r i n e e n v i r o n m e n t v i z . c a r b o h y d r a t e s , p r o t e i n s ,

l i p i d s and n u c l e i c a c i d s and o f "humic s u b s t a n c e s " to s e d i m e n t a r y o r g a n i c c a r -bon. C a r b o h y d r a t e s d e t e r m i n e d i n m a r i n e and l a k e sediments ( e . g . Swain and R o g e r s , 1966; Swain et at., 1967; Swain et at., 1970; S w a i n , 1971; M o d z e l e s k i

et at., 1971; F l e i s c h e r , 1972; Handa a n d M i z u n o , 1973; Degens and Mopper, 1976;

Mopper, 1977; H a t c h e r et at., 1977; Bohm et at., 1980; F e r g u s o n and I b e , 1981; U z a k i and I s h i w a t a r i , 1983) have been shown t o r e p r e s e n t f r o m s e v e r a l ppm i n a n c i e n t s e d i m e n t a r y r o c k s to about 45% o f the t o t a l o r g a n i c c a r b o n i n a r e c e n t mangrove l a k e sediment. S i m i l a r l y p r o t e i n s ( e . g . Brown e t at., 1972; C a s a g r a n -de, 1974; Whelan, 1975; M o r r i s , 1975; H a t c h e r et at., 1977; Degens and Mopper, 1976; H e n r i c h s and F a r r i n g t o n , 1979) have been e s t i m a t e d t o c o m p r i s e up to 33% o f the t o t a l s e d i m e n t a r y o r g a n i c c a r b o n . L i p i d s c o n s t i t u t e i n g e n e r a l l e s s t h a n 10% o f t h e o r g a n i c c a r b o n (Boon, 1978; H u n t , 1979 and r e f e r e n c e s c i t e d t h e r e i n ) . The c o n t r i b u t i o n of n u c l e o t i d e s i s g e n e r a l l y an o r d e r of magnitude l e s s t h a n t h o s e o f c a r b o h y d r a t e s , p r o t e i n s or l i p i d s (van d e r V e l d e n , 1976 and r e f e r e n c e s c i t e d t h e r e i n ) . O r g a n i c m a t t e r r e l e a s a b l e f r o m sediment samples by e x t r a c t i o n w i t h b a s e and s t r u c t u r a l l y l e s s d e f i n e d ( o f t e n denoted as humic s u b s t a n c e s ) c o n s t i t u t e s up to 40% o f the t o t a l s e d i m e n t a r y o r g a n i c c a r b o n (De-b y s e r et at., 1977; P e l e t and D e (De-b y s e r , 1977; H u n t , 1979; J o c t e u r - M o n r o z i e r and J e a n s o n , 1981).

Because o f the p o o r s p e c i f i c i t y o f the common m o n o s a c c h a r i d e s and amino a c i d s the above a u t h o r s i n t e r p r e t t h e i r d a t a w i t h some c a u t i o n when t h e o r i g i n of the b u l k c o n s t i t u e n t s l i k e c a r b o h y d r a t e s and p r o t e i n s i s c o n c e r n e d . However d u r i n g c a r b o h y d r a t e component a n a l y s e s i n t h e d i s t r i b u t i o n o f m o n o s a c c h a r i d e s i n g e n e r a l the c h a r a c t e r i s t i c s o f s t r u c t u r a l c a r b o h y d r a t e s o f the c o n t r i b u t i n g o r g a n i s m s ( a l g a e ) a r e r e c o g n i z e d . I n the case o f amino a c i d a n a l y s e s some m i n o r amino a c i d s w i t h a marker v a l u e f o r the p r e s e n c e o f b a c t e r i a i n d i c a t e a b a c t e r i a l c o n t r i b u t i o n t o the s e d i m e n t a r y o r g a n i c m a t t e r i n a d d i t i o n t o t h a t o f the p r i m a r y a l g a l p r o d u c e r . Among the l i p i d s r e l e a s a b l e from m a r i n e s e d i -ments i n g e n e r a l b o t h t h e c o n t r i b u t i o n s o f the p r i m a r y a l g a l p r o d u c e r and o f b a c t e r i a a r e d i s t i n g u i s h a b l e .

I n t h e p r e s e n t s t u d y samples f r o m r e c e n t m a r i n e s e d i m e n t s a r e i n v e s t i g a t e d i n o r d e r t o e l u c i d a t e the c o m p o s i t i o n and o r i g i n o f the o r g a n i c m a t t e r . An a t -tempt i s made t o s e t up a c o m p l e t e i n v e n t o r y o f r e c o g n i z a b l e , w e l l d e f i n e d o r g a n i c compounds v i z . p r o t e i n s , c a r b o h y d r a t e s and l i p i d s . T h i s i s p a r t l y

r e a l i z e d by n o n - d e g r a d a t i v e e x t r a c t i o n o f b i o p o l y m e r s u s i n g w a t e r o r w a t e r based e x t r a c t a n t s . B i o p o l y m e r s n o t r e l e a s a b l e i n t h i s way a r e d e p o l y m e r i z e d t o y i e l d t h e biomonomers e.g. m o n o s a c c h a r i d e s , amino a c i d s and l i p i d compo-n e compo-n t s . By meacompo-ns o f the a p p l i c a t i o compo-n o f a c i d h y d r o l y s i s o r base s a p o compo-n i f i c a t i o compo-n i t i s p o s s i b l e t o b r e a k down the g r e a t e r p a r t o f the c o v a l e n t l i n k a g e s between monomer b u i l d i n g b l o c k s o f b i o m a c r o m o l e c u l e s v i z . e s t e r - , g l y c o s i d i c - and amide bonds. The n a t u r e and s t r e n g t h o f the c h e m i c a l t r e a t m e n t and t h e c h o i c e o f the e x t r a c t a n t o f f e r the p o s s i b i l i t y t o d i s t i n g u i s h between t h e v a r i o u s c l a s s e s o f monomeric b u i l d i n g b l o c k s r e l e a s e d .

The v a r i o u s c l a s s e s thus o b t a i n e d were a n a l y s e d q u a l i t a t i v e l y and q u a n t i -t a -t i v e l y . F o r -t h a -t p u r p o s e i -t was n e c e s s a r y -t o adap-t a number o f a n a l y -t i c a l methods i n o r d e r t o make them a p p l i c a b l e t o the a n a l y s i s o f sediment samples.

I n C h a p t e r 2 t h e i n v e s t i g a t i o n o f o r g a n i c m a t t e r p r e s e n t i n a r e c e n t marine diatomaceous ooze sample f r o m the N a m i b i a n S h e l f (S.W. A f r i c a ) i s p r e s e n t e d . The s e d i m e n t was a n a l y s e d f o r c a r b o h y d r a t e s , p r o t e i n s and l i p i d s r e l e a s a b l e upon e x t r a c t i o n w i t h w a t e r and a c i d o f i n c r e a s i n g c o n c e n t r a t i o n o r w i t h o r g a n i c s o l v e n t s u s i n g a n a l y t i c a l p r o c e d u r e s w h i c h a r e i n t r o d u c e d h e r e a f -t e r . The o r g a n i c componen-ts r e l e a s e d b u -t n o -t i d e n -t i f i e d by -t h e s e p r o c e d u r e s and the u l t i m a t e i n s o l u b l e o r g a n i c m a t t e r a f t e r e x t r a c t i o n were a n a l y s e d f u r t h e r by p y r o l y s i s mass s p e c t r o m e t r y and p y r o l y s i s gas chromatography -mass s p e c t r o m e t r y i n o r d e r to make a complete i n v e n t o r y o f the o r g a n i c m a t t e r .

A method was d e v e l o p e d f o r the a n a l y s i s o f c a r b o h y d r a t e s i n samples f r o m r e c e n t m a r i n e s e d i m e n t s . Upon a c i d h y d r o l y s i s complex m i x t u r e s o f monosaccha-r i d e s wemonosaccha-re o b t a i n e d , w h i c h wemonosaccha-re a n a l y s e d as t h e i monosaccha-r a l d i t o l a c e t a t e s by gas chromatography ( C h a p t e r 3 ) . Gas c h r o m a t o g r a p h i c - mass s p e c t r o m e t r i c a n a l y s i s r e v e a l e d the p r e s e n c e o f a l a r g e number o f p a r t i a l l y m e t h y l a t e d and deoxy a l d i t o l a c e t a t e s . The i d e n t i f i c a t i o n o f t h e s e components r e q u i r e d t h e s y n t h e -s i -s o f a p p r o p r i a t e -s t a n d a r d -s . I n C h a p t e r 4 the a n a l y -s i -s o f -s y n t h e t i c m i x t u r e -s o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s i s d e s c r i b e d . The r e s u l t s o f the u l t i m a t e i d e n t i f i c a t i o n o f m i x t u r e s o f a l d i t o l a c e t a t e s o b t a i n e d f r o m sediment h y d r o l y z a t e s a r e summarized i n C h a p t e r 5. I n t h i s c o m p a r a t i v e s t u d y t h e geo-c h e m i geo-c a l s i g n i f i geo-c a n geo-c e o f p a r t i a l l y m e t h y l a t e d and deoxy m o n o s a geo-c geo-c h a r i d e s i s d i s c u s s e d .

The a l d i t o l a c e t a t e method i s a l s o a p p l i e d t o the a n a l y s i s o f c a r b o h y d r a t e s i n a r e c e n t s t r o m a t o l y t i c d e p o s i t i n o r d e r t o s t u d y t h e d i a g e n e s i s o f c a r b o h y -d r a t e s upon b u r i a l . The c a r b o h y -d r a t e c o m p o s i t i o n an-d the c o n t r i b u t i o n o f c a r b o h y d r a t e c a r b o n r e l a t i v e t o the t o t a l o r g a n i c c a r b o n are d i s c u s s e d i n terms o f the p o t e n t i a l t o become p a r t o f the o r g a n i c m a t t e r t h a t s u r v i v e s g e o l o g i c a l p e r i o d s o f t i m e ( C h a p t e r 6 ) .

5

I n C h a p t e r 7 a s p e c i a l and p a r t l y new method i s p r e s e n t e d f o r the a n a l y s i s o f t o t a l l i p i d s i n s e d i m e n t s . L i p i d components o b t a i n e d a f t e r e x t r a c t i o n w i t h w a t e r o r o r g a n i c s o l v e n t s and r e l e a s e d a f t e r a c i d h y d r o l y s i s and/or s a p o n i f i

-c a t i o n w i t h base were a n a l y s e d w i t h o u t any p r e s e p a r a t i o n by gas -chromatography and gas chromatography - mass s p e c t r o m e t r y a f t e r a p p r o p r i a t e d e r i v a t i z a t i o n . I n t h i s way f r e e , e s t e r i f i e d and amide bound l i p i d s were d i s t i n g u i s h e d . Two c l a s s e s o f l i p i d s were d e t e c t e d i n s i g n i f i c a n t amounts. On t h e one hand t h e abundance o f B-hydroxy f a t t y a c i d s , amide bound t o the o r g a n i c m a t r i x , p o i n t t o a b a c t e r i a l c o n t r i b u t i o n t o t h e o r g a n i c m a t t e r p r e s e n t i n t h e s e d i m e n t . On the o t h e r hand the l o l i o l i d e - t y p e l a c t o n e s p o i n t t o an a l g a l c o n t r i b u t i o n t o the s e d i m e n t a r y o r g a n i c m a t t e r as w e l l . Moreover t h e s e l a c t o n e s a r e t h o u g h t t o be i n d i c a t o r s f o r e a r l y d i a g e n e t i c t r a n s f o r m a t i o n s o f c a r o t e n o i d s f r o m an a l g a l s o u r c e ( C h a p t e r 8 ) .

REFERENCES

Bohm L., Dawson R., L i e b e z e i t G. and Wefer G. (1980) S u i t a b i l i t y o f monosaccha-r i d e s as mamonosaccha-rkemonosaccha-rs f o monosaccha-r p a monosaccha-r t i c l e i d e n t i f i c a t i o n i n c a monosaccha-r b o n a t e s e d i m e n t s .

Sedimentology Z7_, 167-177.

Boon J . J . (1978) M o l e c u l a r b i o g e o c h e m i s t r y o f l i p i d s i n f o u r n a t u r a l e n v i r o n -ments. T h e s i s , D e l f t U n i v e r s i t y o f T e c h n o l o g y , The N e t h e r l a n d s .

Brown F.S., B a e d e c k e r M.J., Nissenbaum A. and K a p l a n I.R. (1972) E a r l y d i a g e n e s i s i n a r e d u c i n g f j o r d , S a a n i c h I n l e t , B r i t i s h C o l u m b i a - I I I . Changes i n o r g a n i c c o n s t i t u e n t s o f s e d i m e n t . Geochim. Cosmoahim. Acta 36, 1185-1203.

Casagrande D.J. and G i v e n P. (1974) G e o c h e m i s t r y o f amino a c i d s i n some F l o r i d a p e a t a c c u m u l a t i o n s - I . A n a l y t i c a l a p p r o a c h and t o t a l amino a c i d c o n c e n t r a t i o n s . Geochim. Cosmoahim. Acta 3_8, 419-434.

Degens E. and Mopper K. (1976) F a c t o r s c o n t r o l l i n g the d i s t r i b u t i o n and e a r l y d i a g e n e s i s o f o r g a n i c m a t e r i a l i n m a r i n e s e d i m e n t s . Chem. Ooeanogr. 3_, 59-113.

Debyser Y., P e l e t R. and D a s t i l l u n g M. (1977) Geochemie o r g a n i q u e de s e d i m e n t s m a r i n e r e c e n t s : Mer N o i r e , B a l t i q u e , A t l a n t i q u e ( M a u r i t a n i e ) . I n Advances

in Organic Geochemistry 1975 (eds R. Campos and J . G o n i ) , pp. 289-320.

Enadimsa. M a d r i d .

Durand B. (1980) K e r o g e n . E d i t i o n s T e c h n i p . P a r i s .

F e r g u s o n J . and Ibe A.C. (1981) O r i g i n o f l i g h t h y d r o c a r b o n s i n c a r b o n a t e o o l i t e s . J. Petrol. Geol. 4_, 103-107.

F l e i s c h e r S. (1972) Sugars i n t h e s e d i m e n t s o f Lake Trummen and r e f e r e n c e l a k e s . Arch. Hydrobiol. 70, 392-412.

Handa N. and Mizuno K. (1973) C a r b o h y d r a t e s f r o m l a k e s e d i m e n t s . Geoahem. J. 7, 215-230.

H a t c h e r P.G., S i m o n e i t B.R., Gerchakov S.M. (1977) The o r g a n i c g e o c h e m i s t r y o f a r e c e n t s a p r o p e l i c e n v i r o n m e n t : Mangrove Lake , Bermuda. I n Advances in

Organic Geochemistry 1975 (eds R. Campos and J . G o n i ) , pp. 469-484.

H e n r i c h s S.M. and F a r r i n g t o n J.W. (1980) Amino a c i d s i n i n t e r s t i t i a l w a t e r s of m a r i n e s e d i m e n t s : A c o m p a r i s o n o f r e s u l t s from v a r i e d s e d i m e n t a r y e n v i r o n m e n t s . I n Advances in Organic Geochemistry 1979 (eds A.G. Douglas and J.R. M a x w e l l ) , pp. 435-443. Pergamon.

Hunt J.M. (1979) P e t r o l e u m g e o c h e m i s t r y and g e o l o g y . Freeman W.H. and Company. San F r a n c i s c o .

J o c t e u r - M o n r o z i e r L. and J e a n s o n P. (1981) Caractères généraux e t évolution de l ' a z o t e o r g a n i q u e dans l e s sédiments s u p e r f i c i e l s . I n Géochemie Organique

des Sédiments Marins Profonds. Orgon I V . E d i t i o n s CNRS. P a r i s .

L a r t e r S.R. and Douglas A.G. (1980) A p y r o l y s i s - g a s c h r o m a t o g r a p h i c method f o r k e r o g e n t y p i n g . I n Advances in Organic Geochemistry 1979 (eds A.G. Douglas and J.R. M a x w e l l ) , pp. 579-583. Pergamon.

v a n de Meent D., Brown S.C., P h i l p R.P. and S i m o n e i t B.R.T. (1980) P y r o l y s i s -h i g -h r e s o l u t i o n gas c-hromatograp-hy-mass s p e c t r o m e t r y o f k e r o g e n s and k e r o g e n p r e c u r s o r s . Geochim. Cosmochim. Acta 44_, 999-1013.

M o d z e l e s k i J . E . , L a u r i e W.A. and Nagy B. (1971) C a r b o h y d r a t e s f r o m S a n t a B a r b a r a B a s i n s e d i m e n t s : Gas c h r o m a t o g r a p h i c - m a s s s p e c t r o m e t r i c a n a l y s i s of t r i m e t h y l s i l y l d e r i v a t i v e s . Geochim. Cosmochim. Acta 35_, 825-838. Mopper K. (1977) Sugars and u r o n i c a c i d s i n s e d i m e n t and w a t e r from t h e B l a c k

Sea and N o r t h Sea w i t h emphasis on a n a l y t i c a l t e c h n i q u e s . Mar. Chem. 5_, 585-603.

M o r r i s R . J . (1975) The amino a c i d c o m p o s i t i o n o f deep-water m a r i n e s e d i m e n t s from t h e u p w e l l i n g r e g i o n n o r t h w e s t o f A f r i c a . Geochim. Cosmochim. Acta 39, 381-388.

P e l e t R. and Debyser Y. (1977) O r g a n i c g e o c h e m i s t r y o f B l a c k Sea c o r e s .

Geochim. Cosmochim. Acta 4J_, 1575-1586.

S o l l i H., L a r t e r S.R. and Douglas A.G. (1980) A n a l y s i s o f k e r o g e n s by p y r o l y s i s gas chromatography-mass s p e c t r o m e t r y u s i n g s e l e c t i v e i o n m o n i t o r i n g . I n

Advances in Organic Geochemistry 1979 (eds A.G. Douglas and J.R. M a x w e l l )

pp. 591-597. Pergamon.

S t e u a r t D.R. (1912) I n The oil shales of the Lothians-Part III : The Chemistry of

the o i l shales , 2nd ed. , Memoirs o f the G e o l o g i c a l S u r v e y , S c o t l a n d , pp. 143.

Swain F.M. and Rogers M.A. (1966) S t r a t i g r a p h i e d i s t r i b u t i o n o f c a r b o h y d r a t e r e s i d u e s i n M i d d l e D e v o n i a n Onondaga beds o f P e n n s y l v a n i a and W e s t e r n New Y o r k . Geochim. Cosmochim. Acta 30, 497-509.

Swain F.M., Rogers M.A., Evans R.D. and W o l f e R.W. (1967) D i s t r i b u t i o n o f c a r b o h y d r a t e r e s i d u e s i n some f o s s i l specimens and a s s o c i a t e d s e d i m e n t a r y m a t r i x and o t h e r g e o l o g i c a l s a m p l e s . J. Sediment. Petrol. 37_, 12-24. Swain F.M., B r a t t J.M. and K i r k w o o d S. (1970) C a r b o h y d r a t e s f r o m P r e c a m b r i a n

and Cambrian r o c k s and f o s s i l s . Geol. Soc. Am. Bull. 81_, 499-504. Swain F.M. (1971) B i o g e o c h e m i s t r y o f s e d i m e n t samples f r o m B r o a d k i l l M a r s h ,

D e l a w a r e . J. Sediment. Petrol. 4J_, 549-556.

T i s s o t B.P. and W e l t e D.H. (1979) P e t r o l e u m f o r m a t i o n and o c c u r r e n c e . A new a p p r o a c h t o o i l and gas e x p l o r a t i o n . S p r i n g e r V e r l a g .

Vitorovié D. (1980) S t r u c t u r e e l u c i d a t i o n o f k e r o g e n by c h e m i c a l methods. I n Kerogen (ed B. Durand) pp. 320-338. E d i t i o n s T e c h n i p . P a r i s .

Whelan J.K. (1975) Gas c h r o m a t o g r a p h i c c o n f i r m a t i o n o f amino a c i d s t r u c t u r e v i a d i a s t e r e o m e r p r e p a r a t i o n . J. Chromatogr. I l l , 337-346.

U z a k i M. and I s h i w a t a r i R. (1983) D e t e r m i n a t i o n o f c e l l u l o s e and n o n - c e l l u l o s e c a r b o h y d r a t e s i n r e c e n t s e d i m e n t s by gas chromatography. J. Chromatogr. 260, 487-492.

7

C H A P T E R 2

Q U A L I T A T I V E AND Q U A N T I T A T I V E C H A R A C T E R I Z A T I O N OF THE T O T A L O R G A N I C M A T T E R I N A RECENT M A R I N E S E D I M E N T I I . *

J . K l o k , M. B a a s , H.C. Cox, J.W. de Leeuw, W.I.C. R i j p s t r a and P.A. Schenck

D e l f t U n i v e r s i t y o f T e c h n o l o g y Department o f C h e m i s t r y and C h e m i c a l E n g i n e e r i n g O r g a n i c G e o c h e m i s t r y U n i t De V r i e s v a n H e y s t p l a n t s o e n 2 2628 RZ D e l f t , The N e t h e r l a n d s ABSTRACT

The t o t a l o r g a n i c m a t t e r o f a r e c e n t m a r i n e diatomaceous ooze i s s t u d i e d by a n a l y s i s o f i t s w a t e r and a c i d e x t r a c t s and r e s i d u e s . The e x t r a c t i o n d a t a r e l a t i v e t o t h e t o t a l o r g a n i c c a r b o n r e v e a l m a j o r c o n t r i b u t i o n s o f c a r b o h y -d r a t e s ( 2 2 % ) , " v o l a t i l e s " (12%) an-d p r o t e i n s (11%) an-d a s m a l l e r c o n t r i b u t i o n o f l i p i d s (4%) and v o l a t i l e f a t t y a c i d s ( 1 % ) . The r e s i d u a l o r g a n i c c a r b o n (50%) m a i n l y shows a l i p h a t i c c h a r a c t e r i s t i c s . Among t h e compounds i d e n t i f i e d i n t h e e x t r a c t s some w i t h an a l g a l and o t h e r s w i t h a b a c t e r i a l o r i g i n a r e d i s t i n -g u i s h a b l e .

INTRODUCTION

O r g a n i c m a t t e r i n b o t h r e c e n t and a n c i e n t s e d i m e n t s i n s o l u b l e i n o r g a n i c s o l v e n t s , has been s u b j e c t o f many s t r u c t u r a l i n v e s t i g a t i o n s because o f i t s q u a n t i t a t i v e i m p o r t a n c e when compared t o t h e s o l u b l e f r a c t i o n . The s t u d y o f the o r g a n i c m a t t e r i n r e c e n t s e d i m e n t s , b e i n g a t t h e s t a r t o f a sequence d i a g e n e t i c p r o c e s s e s , may o f f e r t h e p o s s i b i l i t y t o o b t a i n i n f o r m a t i o n on t h e s t a r t i n g s t r u c t u r e s o f t h e i n s o l u b l e o r g a n i c m a t t e r i n a n c i e n t s e d i m e n t s , the s o - c a l l e d k e r o g e n . I n c o n n e c t i o n w i t h a f o r m e r s t u d y ( K l o k et al. , 1983) an a t t e m p t i s made h e r e t o c h a r a c t e r i z e t h e t o t a l o r g a n i c m a t t e r i n a r e c e n t m a r i n e s e d i m e n t . F o r t h i s p u r p o s e we have i n v e s t i g a t e d a N a m i b i a n S h e l f diatomaceous ooze sample. T h i s s e d i m e n t i s c h a r a c t e r i z e d by u n i q u e i n p u t p a r a m e t e r s and t h e absence o f

t e r r e s t r i a l m a t e r i a l (Boon, 1978). Because o f t h e r e l a t i v e l y young age and t h e s h a l l o w depth o f t h e sediment i t may be e x p e c t e d t h a t a t l e a s t p a r t o f t h e b i o p o l y m e r s i n t h e remains o f c o n t r i b u t i n g o r g a n i s m s a r e s t i l l p r e s e n t . By means o f a sequence o f e x t r a c t i o n s w i t h w a t e r and a c i d o f i n c r e a s i n g

concen-t r a concen-t i o n , concen-t h e i n concen-t e n concen-t i o n was concen-t o r e l e a s e concen-t h e maximum amounconcen-t o f concen-t h e o r g a n i c c a r b o n p r e s e n t , w i t h m i n i m a l c h e m i c a l d e g r a d a t i o n . The o r g a n i c c a r b o n thus r e l e a s e d i s f u r t h e r a n a l y s e d f o r t h e c o n s t i t u e n t monomeric b u i l d i n g b l o c k s o f b i o p o l y -mers e.g. amino a c i d s , m o n o s a c c h a r i d e s , v o l a t i l e f a t t y a c i d s and l i p i d compo-n e compo-n t s . The r e s i d u a l , i compo-n s o l u b l e o r g a compo-n i c m a t t e r i s f u r t h e r i compo-n v e s t i g a t e d a p p l y i compo-n g p y r o l y s i s t e c h n i q u e s .

I n t h i s way some i n s i g h t i s o b t a i n e d , n o t o n l y i n t o t h e amount and mode o f o c c u r r e n c e o f t h e r e l e a s e d m o i e t i e s , b u t a l s o i n t o t h e p r i m a r y d i a g e n e t i c changes o c c u r r i n g i n t h e s e d i m e n t and/or i n t h e p r e d e p o s i t i o n a l s t a g e o f t h e s e d i m e n t a r y o r g a n i c m a t t e r .

EXPERIMENTAL

Ooze samples f r o m t h e N a m i b i a n S h e l f were c o l l e c t e d on a c r u i s e d u r i n g December 1968 - J a n u a r y 1969 ( E i s m a , 1969). The KD6 c o r e (22°30'S 14°05.7'E), sampled u n d e r n e a t h a w a t e r column o f 106 m, was s e c t i o n e d and s t o r e d a t -20°C u n t i l u s e . The 40-75 cm s e c t i o n used f o r t h i s s t u d y was homogenized and l y o p h i l i z e d p r i o r t o e x t r a c t i o n as o u t l i n e d i n F i g . 1.

A 50.0 gram a l i q u o t o f t h e d r y sample ( R l ) was t r e a t e d u l t r a s o n i c a l l y w i t h 400 ml c o l d w a t e r d u r i n g 3*5 m i n u t e s and t h e m i x t u r e was s u b s e q u e n t l y a l l o w e d t o h e a t under r e f l u x f o r 69 h o u r s . A f t e r c o o l i n g t h e s u s p e n s i o n was c e n t r i f u g e d f o r

10 m i n u t e s (2700 g) and t h e s u p e r n a t a n t (and s e v e r a l w a s h i n g s ) were f i l t e r e d o v e r a G4 f i l t e r . The r e s i d u e (R2) was l y o p h i l i z e d . An a l i q u o t o f t h e combined

9 NAM1BIAN SHELF diatom aceous ooze sample K D 6 lyophilization R 1 H20 , reflux , 69 hr R 2 E2 concentration GPC PP L M M M H M 2N HCl , reflux . 22 hr R 3 "8Utra.liza,iion ,

LB

Fig. 1. Extraction scheme. For description of the fractions (PP, LM, etc.) see Experimental.

e x t r a c t s and w a s h i n g s (E2) was d r i e d i n an oven ( a t 90 - 100°C) and subsequent-l y in vacuo o v e r KOH f o r a d r y w e i g h t d e t e r m i n a t i o n .

The l y o p h i l i z e d r e s i d u e a f t e r w a t e r e x t r a c t i o n (R2, 25.0 gram) was suspen-ded i n 400 m l 2N H C l and h e a t e d under r e f l u x f o r 22 h o u r s . S i m i l a r l y , 10.0 gram o f t h e d r y s t a r t i n g m a t e r i a l ( R l ) and r e s i d u e R3 were each suspended i n

100 ml 6N H C l and h e a t e d under r e f l u x f o r 24 h o u r s . About 50 mg o f c e l l u l o s e powder (flN 300 HR, Macherey, N a g e l & C o . ) was h y d r o l y s e d i n a s i m i l a r way. The v a r i o u s r e s u l t i n g h y d r o l y z a t e s were t r e a t e d as d e s c r i b e d above. A l i q u o t s o f t h e e x t r a c t s were d r i e d f o r d r y w e i g h t and c h l o r i d e ( V o l h a r d t i t r a t i o n ) d e t e r m i n a t i o n s . The pH o f o t h e r a l i q u o t s was r a i s e d t o 3 w i t h s o -dium h y d r o x i d e b e f o r e t o t a l l i p i d e x t r a c t i o n . The r e m a i n i n g p o r t i o n s o f t h e e x t r a c t s were s t o r e d a t -20°C u n t i l use.

Gel permeation chromatography (GPC)

A l i q u o t s o f the e x t r a c t s were l y o p h i l i z e d ( i f n e c e s s a r y a f t e r n e u t r a l i z a -t i o n w i -t h ammonia -t o pH 7) and s -t o r e d in vacuo o v e r KOH. The d r y e x -t r a c -t was suspended i n w a t e r . The i n s o l u b l e f r a c t i o n ( d e f i n e d p r e c i p i t a t e = PP) was r e -moved by c e n t r i f u g a t i o n , washed w i t h w a t e r , d r i e d in vacuo o v e r KOH and w e i g h e d . The s o l u b l e p a r t o f the e x t r a c t i n c l u d i n g the p r e c i p i t a t e w a s h i n g s was chromatographed o v e r a B i o - g e l P2 column ( B i o r a d , 200-400 mesh, 17x2.4 cm) w i t h w a t e r as e l u e n t ( f l o w r a t e : 1 m l / m i n ) . The r e f r a c t i v e i n d e x o f the e l u a t e was r e c o r d e d u s i n g a Waters-R401 R l - d e t e c t o r . Three f r a c t i o n s were c o l l e c t e d r e p r e s e n t i n g h i g h m o l e c u l a r w e i g h t m a t e r i a l (HM, mol.w. > 2000 D) , medium m o l e c u l a r w e i g h t m a t e r i a l (MM, ^400 <mol.w. < ^2000 D) and low m o l e c u l a r w e i g h t m a t e r i a l (LM, mol.w. <^400 D). Each f r a c t i o n was a n a l y s e d f o r o r g a n i c c a r b o n and i t s dry w e i g h t was d e t e r m i n e d .

Carbon measurements

T o t a l c a r b o n i n r e s i d u e s and p r e c i p i t a t e s was d e t e r m i n e d u s i n g an a u t o m a t i c P e r k i n E l m e r 240 C H N a n a l y s e r . C a r b o n a t e c a r b o n was d e t e r m i n e d t i t r i -m e t r i c a l l y . C a r b o n a t e c a r b o n and t o t a l c a r b o n p r e s e n t i n aqueous s o l u t i o n s were d e t e r m i n e d u s i n g a wet c o m b u s t i o n a p p a r a t u s o p e r a t e d a t 150°C and 900°C r e s p e c t i v e l y . The amount o f o r g a n i c c a r b o n i s c a l c u l a t e d by s u b t r a c t i o n .

Amino acids

Amino a c i d s i n l y o p h i l i z e d r e s i d u e s and i n e x t r a c t s were r e l e a s e d upon h y d r o l y s i s w i t h 6N HC1 d u r i n g 24 h o u r s a t 105°C under n i t r o g e n i n s e a l e d ampoules. N o r - l e u c i n e was added b e f o r e h y d r o l y s i s as an i n t e r n a l s t a n d a r d . The amino a c i d s p r e s e n t i n t h e h y d r o l y z a t e s were a n a l y s e d u s i n g a K o n t r o n L i q u i m a t I I I e q u i p p e d w i t h a Durrum R e s i n DC-4A and P i c o b u f f e r s y s t e m 2 as e l u e n t . Q u a n t i t a t i o n was p e r f o r m e d a f t e r n i n h y d r i n d e r i v a t i z a t i o n and d e t e c -t i o n a -t 570 ran.

Carbohydrates

N e u t r a l m o n o s a c c h a r i d e s were d e t e r m i n e d i n the s t a r t i n g m a t e r i a l ( R l ) , r e s i d u e R2 and e x t r a c t E2. About 500 mg o f each l y o p h i l i z e d r e s i d u e and a l y o p h i l i z e d a l i q u o t o f about 50 ml o f e x t r a c t E2 were h y d r o l y s e d i n 15 ml 0.5N H2SO4 d u r i n g 18 h o u r s a t 100°C i n s e a l e d ampoules under n i t r o g e n . D e r i -v a t i z a t i o n i n t o the c o r r e s p o n d i n g a l d i t o l a c e t a t e s was p e r f o r m e d as d e s c r i b e d e a r l i e r ( K l o k et al. , i n p r e s s ) . M y o i n o s i t o l was added as an i n t e r n a l s t a n -d a r -d . I -d e n t i f i c a t i o n was b a s e -d on GC r e t e n t i o n -d a t a an-d on c o m p a r i s o n of the mass s p e c t r a o b t a i n e d by c a p i l l a r y GC-MS w i t h t h o s e p u b l i s h e d by J a n s s o n et al

11

Volatile fatty acids

V o l a t i l e f a t t y a c i d s r e l e a s e d f r o m the s t a r t i n g m a t e r i a l (R1) a f t e r sapon i f i c a t i o sapon w i t h 8N NaOH were a sapon a l y s e d as t h e i r b e sapon z y l e s t e r s as w i l l be d e s -c r i b e d e l s e w h e r e ( K l o k et at. , i n p r e p a r a t i o n ) .

Total lipids

The p r o c e d u r e f o r the d e t e r m i n a t i o n o f t o t a l l i p i d s i n r e s i d u e s i s sum-m a r i z e d b r i e f l y below. D e t a i l s on the a p p l i e d p r o c e d u r e a r e g i v e n e l s e w h e r e

(de Leeuw et al. , i n p r e s s ; K l o k et al. , s u b m i t t e d ) . The l i p i d s o b t a i n e d a f t e r s a p o n i f i c a t i o n were e s t e r i f i e d w i t h diazomethane and s i l y l a t e d w i t h T r i s i l - Z ( P i e r c e ) . The r e s u l t i n g d e r i v a t i v e s were s u b j e c t e d to column chromatography on L i p i d e x - 5 0 0 0 ( P a c k a r d ) f o r r e m o v a l o f v e r y p o l a r compounds. The e l u e n t was c o n c e n t r a t e d and a n a l y s e d by c a p i l l a r y GC and GC-MS.

T o t a l l i p i d s i n e x t r a c t s were e x t r a c t e d w i t h d i c h l o r o m e t h a n e and d e r i v a -t i z e d and a n a l y s e d i n a s i m i l a r way.

Gas chromatography o f t h e l i p i d f r a c t i o n s was p e r f o r m e d u s i n g a C a r l o E r b a 4160 gas chromatograph e q u i p p e d w i t h an FID and an on-column i n j e c t i o n system (Grob and G r o b , 1978). A g l a s s c a p i l l a r y column c o a t e d w i t h SE52 (20 m, I.D. 0.32 mm) was used w i t h h e l i u m as t h e c a r r i e r gas. Samples were i n j e c t e d a t

125°C and the t e m p e r a t u r e was programmed a t a r a t e o f 4°C p e r m i n u t e t o 310°C. Gas chromatography-mass s p e c t r o m e t r y was c a r r i e d out on a V a r i a n 3700 gas chromatograph c o n n e c t e d t o a V a r i a n Mat 44 q u a d r u p o l e mass s p e c t r o m e t e r . E l e c t r o n impact mass s p e c t r a were o b t a i n e d a t 80 eV.

Pyrolysis-mass spectrometry and pyrolysis-gas chromatography-mass spectrometry (Py-MS and Py-GC-MS)

The automated C u r i e - p o i n t Py-MS s y s t e m used has been d e s c r i b e d e l s e w h e r e ( W i n d i g et al. , 1980). The i n s t r u m e n t was o p e r a t e d u s i n g the f o l l o w i n g e x p e r i -m e n t a l c o n d i t i o n s : sa-mple s i z e , 5-25 yg ( i n 0.1M phosphate b u f f e r , pH 7 ) ; w i r e e q u i l i b r i u m t e m p e r a t u r e , 610°C; t e m p e r a t u r e r i s e t i m e , 0.1 s; t o t a l h e a t i n g t i m e , 0.9 s; MS i n l e t t e m p e r a t u r e , 150°C; s c a n r a t e 0.1 s / s c a n (m/z 15-180); t o t a l s c a n n i n g t i m e , 10 s. Each sample was a n a l y s e d i n q u a d r u p l i c a t e .

Py-GC-MS was c a r r i e d out u s i n g a p y r o l y s i s r e a c t o r as d e s c r i b e d by Meuze-l a a r et aMeuze-l. ( 1 9 7 5 ) , m o d i f i e d f o r use a t h i g h t e m p e r a t u r e s (van de Meent et aMeuze-l. 1980a). Gas c h r o m a t o g r a p h i c s e p a r a t i o n was p e r f o r m e d u s i n g t h e f o l l o w i n g c o n -d i t i o n s : g l a s s c a p i l l a r y column ( C P s i l 5 , 25 m, 0.3 mm I . D . ) ; c a r r i e r g a s , h e l i u m ; t e m p e r a t u r e program, 0°C (5 min) 3°C/min 300°C (20 m i n ) ; w i r e e q u i

-l i b r i u m t e m p e r a t u r e , 610°C; p y r o -l y s i s t i m e , 10 s; mass s p e c t r o m e t e r , V a r i a n Mat 44 o p e r a t e d a t 80 eV; c y c l e t i m e , 1 s; m/z 25-500 up to s c a n 250 and m/z 50-500 a f t e r s c a n 250; m/z 28,32,40,44 were o m i t t e d i n t h e r e c o n s t r u c t e d i o n c u r r e n t (RIC) .

RESULTS T a b l e 1 summarizes t h e d a t a on d r y w e i g h t and o r g a n i c c a r b o n o f r e s i d u e s , t o t a l w a t e r and a c i d e x t r a c t s and GPC f r a c t i o n s o f e x t r a c t s as m e n t i o n e d i n F i g . 1. The d r y w e i g h t d e t e r m i n a t i o n s a r e based on 50.0 g ( = 100%) l y o p h i l i z e d s t a r t i n g m a t e r i a l . S i m i l a r l y t h e o r g a n i c c a r b o n measurements a r e b a s e d on 2320 mg ( = 100%) o r g a n i c c a r b o n p r e s e n t i n t h i s amount o f s t a r t i n g m a t e r i a l .

Table 1. Results of mass balance and organic carbon balance determinations r e l a t i v e to Rl1 Mass b a l a n c e ( 5 0 . 0 g = 100.0%) sample %W _ %WD_ %WTM %WMM %WUM %W 2 v t o t PP LM MM HM r e c R l 100.0 -E2 1 7 . 33 1.0 15.0 0.2 0.1 16.3 R2 81.8 E3 16.6k 4.8 10.2h 0.9 0.3 16.2 R3 66.4 -E4 1.6 0. 3 5 1 . 0 6 0 . 1 7 0 . 0 6 1.6 R4 6 6. 2 -E5 3 3 . 2k 3 . 4 2 8 . 2k 2 . 0 0 . 2 3 3 . 8 R5 6 7 . 2 -O r g a n i c C b a l a n c e (2320 mg i n R l i 100%) %C ie_„ % CT„ %CV_, % C ™ %c 2 t o t PP LM MM HM r e c R l 100.0 -E 2 4.7 0.5 1.8 0.8 1.6 4 . 7 R2 9 1 . 8 -E3 24.1 6 . 9 6.5 5.8 2. 4 21.6 R3 60.8 -E4 4.5 0. 9 0 . 9 1.1 0.2 3.1 R4 54.4 -E5 32.5 6.6 10.3 10.8 0.8 28.5 R5 60.0 -1 R l : o r g a n i c c a r b o n , 4 . 6 4 % ; c a r b o n a t e c a r b o n , 1.12% 2 %W = %Wt)t) + %WT + %WMM + % W „M ( s i m i l a r l y %C ) r e c PP LM MM HM J r e c 3 i n d i g e n o u s c h l o r i d e c o n t e n t , 7.6% ( b a s e d on d r y w e i g h t R l ) 4 p e r c e n t a g e s a d j u s t e d f o r c o n t r i b u t i o n o f c h l o r i d e due t o HC1 t r e a t m e n t

The d r y w e i g h t - and o r g a n i c c a r b o n b a l a n c e s a r e seen i n F i g . 2 .

The m o l a r d i s t r i b u t i o n and t h e c o n t r i b u t i o n t o t h e o r g a n i c c a r b o n o f t h e i n d i v i d u a l amino a c i d s a r e summarized i n T a b l e 2. F i g . 3 r e p r e s e n t s t h e s e d a t a o b t a i n e d a f t e r 6N HC1 t r e a t m e n t o f t h e s t a r t i n g m a t e r i a l (E5) . F i g . 4 shows the gas chromatogram o f t h e n e u t r a l c a r b o h y d r a t e s p r e s e n t i n the w a t e r e x t r a c t ( E 2 ) , a n a l y s e d as a l d i t o l a c e t a t e s . I d e n t i f i c a t i o n s o f m a j o r peaks a r e g i v e n i n t h e f i g u r e c a p t i o n . T a b l e 3 summarizes the q u a n t i t a t i v e r e s u l t s o f t h e ana-l y s i s o f n e u t r a ana-l m o n o s a c c h a r i d e s i n t h e s t a r t i n g m a t e r i a ana-l ( R ana-l ) , t h e r e s i d u e a f t e r w a t e r e x t r a c t i o n (R2) a n d i n t h e w a t e r e x t r a c t ( E 2 ) . F I D r e s p o n s f a c t o r s of t h e i n d i v i d u a l a l d i t o l a c e t a t e d e r i v a t i v e s have b e e n assumed e q u a l on a

1 3

w e i g h t b a s i s , compared t o m y o - i n o s i t o l h e x a a c e t a t e ( i n t e r n a l s t a n d a r d )

DRY WEIGHT BALANCE

100 8 2 17 6 6 3 4 6 6 3 5 6 7 3 3 H20 2N HCl 6N HCl directly

ORGANIC CARBON BALANCE

100 9 2 ₁₅ 61 2 9 54 3 3 H20 2N HCl 6N HCl 6 0 Fig. 3 3 6N HCl directly 2. Dry weight and organic carbon balances. All percentages refer to the

total amount of dry s t a r t i n g material (50.0 g Rl = 100%) and total amount of organic carbon (2320 mg = 100%) present therein (Table 1).

Among t h e i n d i v i d u a l chromatograms o f the d e r i v a t i z e d t o t a l l i p i d s e x t r a c -t e d b o -t h f r o m w a -t e r - and a c i d e x -t r a c -t s and f r o m -the r e s i d u e s m e n -t i o n e d i n F i g . 1, t h r e e types c o u l d be d i s t i n g u i s h e d based on the d i s t r i b u t i o n o f the o b s e r -v e d compounds. From each t y p e a t y p i c a l example i s shown i n F i g . 5 : t o t a l l i p i d s p r e s e n t i n t h e e x t r a c t s (E3 shown), i n t h e s t a r t i n g m a t e r i a l and non-a c i d t r e non-a t e d r e s i d u e s ( R l shown) non-and i n t h e non-a c i d t r e non-a t e d r e s i d u e s (R5 shown) r e s p e c t i v e l y . Q u a n t i t a t i o n o f t h e l i p i d components o b s e r v e d i n the chromato-grams i s b a s e d on peak a r e a i n t e g r a t i o n . F o r c o n v e n i e n c e t h e F I D r e s p o n s e s o f t h e i n d i v i d u a l components a r e assumed e q u a l o i i a w e i g h t b a s i s . A b s o l u t e c a l i -b r a t i o n o f t h e GC a p p a r a t u s was a c h i e v e d w i t h p a l m i t i c a c i d m e t h y l e s t e r . The t o t a l amount o f e x t r a c t a b l e l i p i d c a r b o n was c a l c u l a t e d t o be 4.2% of t h e t o t a l o r g a n i c c a r b o n p r e s e n t i n t h e s t a r t i n g m a t e r i a l ( R l ) . S h o r t c h a i n f a t t y a c i d s (CjCij) a n a l y s e d as t h e i r b e n z y l e s t e r s c o m p r i s e 0.7% o f t h e t o t a l o r g a -n i c c a r b o -n ( K l o k et al. , i -n p r e p a r a t i o -n ) .

The Py-MS d a t a o b t a i n e d f r o m r e s i d u e s and f r a c t i o n s o f e x t r a c t s a r e g i v e n i n T a b l e 4. Some t y p i c a l p y r o l y s i s mass s p e c t r a a r e shown i n F i g . 6. The r e -c o n s t r u -c t e d t o t a l i o n -c u r r e n t (RIC) o f the Py-GC-MS a n a l y s i s o f r e s i d u e R5 i s shown i n F i g . 7. I d e n t i f i c a t i o n s a r e l i s t e d i n T a b l e 5 ( s c a n numbers c o r r e s -pond t o t h e numbers i n F i g . 7 ) .

DISCUSSION

The v a r i o u s t r e a t m e n t s of a l y o p h i l i z e d sample o f t h e N a m i b i a n S h e l f s e d i m e n t , as d e p i c t e d i n F i g . 1, r e s u l t e d i n t h e s o l u b i l i z a t i o n o f one t h i r d o f the s t a r t i n g m a t e r i a l ( T a b l e 1, F i g . 2 ) . About 15% o f the d r y s t a r t i n g m a t e r i a l i s p r e s e n t as s a l t s ( m a i n l y c h l o r i d e s ) and i s r e c o v e r e d i n t h e low m o l e c u l a r w e i g h t f r a c t i o n of the w a t e r e x t r a c t (E2, LM). D u r i n g the s u c c e s s i v e e x t r a c t i o n s about 46% o f the o r g a n i c c a r b o n ( i n c l u d i n g v o l a t i l e compounds) o r i g i n a l l y p r e s e n t i n the s t a r t i n g m a t e r i a l ( R l ) was r e l e a s e d ( T a b l e 1, F i g . 2 ) . S i m i l a r l y t h e d i r e c t a c i d t r e a t m e n t w i t h 6N HC1 r e s u l t e d i n a r e l e a s e of about 40% ( i n c l u d i n g v o l a t i l e compounds). The d i s c r e p a n c y between the amount o f o r g a n i c c a r b o n r e t a i n e d i n the r e s i d u e s R4 and R5 m i g h t be a s c r i b e d t o t h e o c -c u r r e n -c e o f -c o n d e n s a t i o n r e a -c t i o n s , w h i -c h a r e more s e v e r e d u r i n g the d i r e -c t a c i d t r e a t m e n t .

About 12% and 8% o f t h e o r g a n i c c a r b o n was not r e c o v e r e d a f t e r the s u c c e s -s i v e e x t r a c t i o n -s and a f t e r t h e d i r e c t a c i d t r e a t m e n t r e -s p e c t i v e l y . The-se l o s s e s a r e e x p l a i n e d by t h e f a c t t h a t v e r y v o l a t i l e compounds, such as s h o r t c h a i n a l i p h a t i c s and organo s u l p h u r compounds, a r e not r e c o v e r e d d u r i n g the v a r i o u s p r o c e d u r e s . I n a s e p a r a t e s t u d y ( K l o k et al., i n p r e p a r a t i o n ) i t i s shown t h a t the f r e e and bound v o l a t i l e a c i d s ( C 1 - C 4 ) p r e s e n t i n t h i s sediment s a m p l e , a c c o u n t f o r 0.7% o f t h e o r g a n i c c a r b o n p r e s e n t i n the s t a r t i n g mate-r i a l . The amount o f o mate-r g a n i c c a mate-r b o n not mate-r e c o v e mate-r e d w i l l be f u mate-r t h e mate-r denoted as " v o l a t i l e " .

QlJ§2£i£§£!Y£ §!}d aHilitative d a t a on i n d i v i d u a l component c l a s s e s

Amino acids

From T a b l e 2 i t i s c l e a r t h a t a s u b s t a n t i a l p a r t of t h e o r g a n i c c a r b o n t h a t s o l u b i l i z e s d u r i n g the s u c c e s s i v e e x t r a c t i o n s and s i m i l a r l y d u r i n g the d i r e c t a c i d h y d r o l y s i s c o n s i s t s o f p r o t e i n a c e o u s c a r b o n . I n b o t h ways about 11% o f t h e t o t a l amount o f o r g a n i c c a r b o n p r e s e n t i n t h e s t a r t i n g m a t e r i a l was i d e n t i f i e d as amino a c i d c a r b o n .

F i g . 3 i l l u s t r a t e s b o t h t h e c o n t r i b u t i o n s of the i n d i v i d u a l amino a c i d s t o t h e o r g a n i c c a r b o n and t h e r e l a t i v e c o m p o s i t i o n o f amino a c i d u n i t s a n a l y s e d i n the d i r e c t 6N HC1 h y d r o l y z a t e of the s t a r t i n g m a t e r i a l ( e x t r a c t E 5 ) . The c o n t r i b u t i o n t o the o r g a n i c c a r b o n of the amino a c i d s l e u c i n e and p h e n y l -a l -a n i n e e x c e e d s 1%, w h i c h m-akes them the most -abund-ant b u i l d i n g b l o c k s t h -a t c o u l d be r e l e a s e d from the o r g a n i c m a t t e r p r e s e n t i n t h i s sample.

A l l amino a c i d s e n c o u n t e r e d i n t h i s s t u d y , e x c e p t o r n i t h i n e , a r e r e g a r d e d as p r o t e i n a c e o u s amino a c i d s . O r n i t h i n e i s t h o u g h t t o o r i g i n a t e f r o m o r n i t h i n e

15

Table 2. Amino acids quantitated in the extracts after hydrolysis in 6N HCl

m o l a r d i s t r i b u t i o n [mole %) % amino a c i d C o f o r g a n i c C amino a c i d E2 E3 E4 E5 E2 E3 E4 E5 cys 1.7 0 6 0. 6 0 .5 0.001 0.03 0.01 0.03 asp 12.6 12 4 5 6 10 2 0.009 0.85 0.13 0.93 t h r 5.2 6 0 4 8 5 8 0.004 0.41 0.11 0.53 s e r 6.5 5 9 4 7 5 .8 0.004 0.31 0.08 0.40 g l u 9.0 8. 4 5 9 7 .3 0.008 0.72 0.17 0.84 p r o 4.2 4 6 3. 9 4 1 0.004 0.40 0.11 0.46 g i y 19.7 16 8 10 2 13 .6 0.007 0.58 0.12 0.62 a l a 11.2 10 7 10. 3 10 2 0.006 0.55 0.08 0.70 v a l 5.6 5 8 8. 7 6 5 0.005 0.50 0.25 0.75 met 0.5 0 6 1 3 1 .6 0.000 0.06 0.04 0.18 i l e 5.9 6. 5 8. 9 7 I 0.006 0.67 0.30 0.98 l e u 7.2 7 5 13. 6 9 0 0.008 0.77 0.46 1.24 t y r 2.5 1 6 3. 1 2 .8 0.004 0.25 0.16 0.59 phe 2.8 4. 3 9. 2 5 3 0.005 0.66 0.17 1.09 o r n 0.5 0. 7 0. 8 0 8 0.001 0.06 0.02 0.10 l y s 1.9 3. 3 4 9 4 9 0.002 0.34 0.17 0.67 h i s 0.7 1 2 1 . 2 1 4 0.001 0.13 0.04 0.20 a r g 1 .1 3 1 2. 4 3 . 1 0.001 0.32 0.08 0.42 t o t a l (%) 98.8 100. 0 100. 1 100 0 0.08 7.6 2.9 10.7 t o t a l umole 35 3331 1090 4417

Fig. 3. Amino acids analysed in extract E5 expressed as percentage of the total organic carbon present in the starting material and of the molar distribution of the individual components (Table 2).

l i p i d s o c c u r r i n g i n b a c t e r i a ( L e c h e v a l i e r , 1977). These l i p i d s a r e a l s o c h a r a c t e r i z e d by t h e p r e s e n c e o f amide l i n k e d 8hydroxy f a t t y a c i d s . The s i g n i -f i c a n t p r e s e n c e o -f t h e B-hydroxy -f a t t y a c i d s among t h e l i p i d s a n a l y s e d i n t h e r e s i d u e s a f t e r a c i d t r e a t m e n t ( s e e below) may i n d i c a t e t h a t o r n i t h i n e c o n t a i -n i -n g l i p i d s a r e t h e s o u r c e o f t h e o b s e r v e d o r -n i t h i -n e . The e x t e -n t i -n w h i c h t h e h i g h c o n c e n t r a t i o n o f o t h e r amino a c i d s ( e . g . g l y c i n e and a l a n i n e ) m i g h t be i n t e r p r e t e d as c o n t r i b u t i o n s from a n o n - p r o t e i n a c e o u s s o u r c e i s n o t c l e a r .

Carbohydrates

F i g . 4 shows t h e gas chromatogram o f t h e a l d i t o l a c e t a t e s o b t a i n e d f r o m the w a t e r e x t r a c t (E2) a f t e r h y d r o l y s i s and d e r i v a t i z a t i o n . A p a r t f r o m t h e w e l l known, major components a l a r g e number o f l e s s common, m i n o r components a r e o b s e r v e d . giy 6-deoxy-hexitols rha rib hexitols fuc _pentitols i 1 xyl man gal ara U glu inod.s.)

u

Gas ohromatogram of the a l d i t o l acetates obtained from extract E2. Identifications: gly = glycerol, rha - rhamnitol, fuc = fucitol, rib = ribitol, ara = arabitol, xyl = xylitol, man - mannitol, gal galactitol, glu = g l u c i t o l and ino - myo-inositol (internal stan-dard) . The connected v e r t i c a l lines in the figure indicate the ob-served partially methylated alditols.

Table 3. Relative d i s t r i b u t i o n of neutral monosaccharides in starting mate-r i a l (Rl), mate-residue R2 and extmate-ract E2 mate-released upon hydmate-rolysis with 0.5N n2soh. m o n o s a c c h a r i d e1 R l R2 E2 rhamnose 12.7 12.5 12.8 f u c o s e 7.3 7.4 8.1 r i b o s e 3.9 4.6 2.4 a r a b i n o s e 5.1 5.9 5.0 x y l o s e 9.0 8.2 7.8 mannose 14.1 13.0 17.2 g a l a c t o s e 16.6 15.9 14.9 g l u c o s e 13.0 12.0 10.6 o t h e r s 18.3 20.5 21.3 t o t a l m o n o s a c c h a r i d e s (mg) 236 176 24 % m o n o s a c c h a r i d e C o f o r g a n i c C 4.1 3.1 0.4 1 a n a l y s e d as a l d i t o l a c e t a t e s ( c o n d i t i o n s see E x p e r i m e n t a l ) S i n c e t h e r e s u l t s o f t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f m o n o s a c c h a r i d e

17

b u i l d i n g b l o c k s i n complex c a r b o h y d r a t e s a r e i n f l u e n c e d by b o t h t h e e f f i c i e n c y o f t h e h y d r o l y s i s and t h e d e r i v a t i z a t i o n p r o c e d u r e ( D u t t o n , 1973; Mopper,1977; A l b e r s h e i m et at., 1967; T o r e l l o et at., 1980) t h e amount o f c a r b o h y d r a t e c a r -bon d e t e r m i n e d ( T a b l e 3 ) , c o m p r i s i n g more t h a n 4% o f t h e t o t a l o r g a n i c c a r b o n p r e s e n t i n t h e s t a r t i n g m a t e r i a l ( R l ) , i s i n t e r p r e t e d as a minimum v a l u e . T a b l e 3 a l s o r e v e a l s t h e s i m i l a r i t y i n t h e r e l a t i v e c o m p o s i t i o n o f t h e h y d r o l y z a b l e m o n o s a c c h a r i d e s o b t a i n e d f r o m t h e s t a r t i n g m a t e r i a l ( R l ) , t h e r e s i d u e a f t e r w a t e r e x t r a c t i o n (R2) and f r o m t h e w a t e r e x t r a c t ( E 2 ) . O b v i o u s l y a r e p r e s e n t a t i v e p a r t (about 10%, T a b l e 3) o f t h e h y d r o l y z a b l e c a r b o h y d r a t e s was s o l u b i l i z e d by t h e w a t e r e x t r a c t i o n p r o c e d u r e . The h y d r o l y z a t e o f t h e h i g h m o l e c u l a r w e i g h t f r a c t i o n o f t h i s w a t e r e x t r a c t ( E 2 , HM) o b t a i n e d by GPC r e v e a l e d t h e p r e s e n c e o f n e u t r a l m o n o s a c c h a r i d e s w i t h a d i s t r i b u t i o n p a t t e r n s i m i l a r t o t h a t o b t a i n e d from a h y d r o l y z a t e o f t h e t o t a l e x t r a c t (E2) . The c a r b o h y d r a t e n a t u r e o f t h e h i g h m o l e c u l a r w e i g h t f r a c t i o n was a l s o c o n f i r m e d by Py-MS d a t a as w i l l be d i s c u s s e d below.

The m a j o r m o n o s a c c h a r i d e s e n c o u n t e r e d d u r i n g t h i s s t u d y a r e o m n i p r e s e n t c o n s t i t u e n t s o f n a t u r a l c a r b o h y d r a t e s , w h i l e t h e l e s s common, p r e d o m i n a n t l y p a r t i a l l y m e t h y l a t e d - and deoxy m o n o s a c c h a r i d e s and h e p t o s e s ( K l o k et at. , i n p r e s s ) a r e m a i n l y r e p o r t e d t o o c c u r i n c a r b o h y d r a t e s a s s o c i a t e d w i t h b a c t e r i a l

c e l l w a l l s , such as l i p o p o l y s a c c h a r i d e s (Weckesser et at. , 1979). These l i p o -p o l y s a c c h a r i d e s a r e a l s o c h a r a c t e r i z e d by the -p r e s e n c e o f amide l i n k e d @-hy-dro xy f a t t y a c i d s (Weckesser et at., 1979). The abundance o f g-hy@-hy-droxy f a t t y a c i d s among t h e l i p i d s e x t r a c t a b l e f r o m t h e r e s i d u e s a f t e r a c i d t r e a t m e n t , as w i l l be d i s c u s s e d b e l o w , s u p p o r t s a common o r i g i n o f B-hydroxy f a t t y a c i d s and the m i n o r m o n o s a c c h a r i d e s from l i p o p o l y s a c c h a r i d e s . We a t t e m p t e d , t h e r e f o r e , to i s o l a t e f r o m t h e s e d i m e n t sample l i p o p o l y s a c c h a r i d e s p o s s i b l y p r e s e n t ( K l o k

et at. , s u b m i t t e d ) . A h i g h m o l e c u l a r w e i g h t s u b s t a n c e was i n d e e d o b t a i n e d ,

w h i c h upon h y d r o l y s i s p r o d u c e d m o n o s a c c h a r i d e s and a l s o f a t t y - and B-hydroxy f a t t y a c i d s t y p i c a l o f l i p o p o l y s a c c h a r i d e s . T a k i n g t h i s i n t o a c c o u n t t h e obs e r v e d m e t h y l a t e d m o n o obs a c c h a r i d e obs p r o b a b l y a r e good i n d i c a t o r obs f o r t h e p r e obs e n -ce o f s t r u c t u r e s a s s o c i a t e d w i t h b a c t e r i a l c e l l w a l l s i n t h e sediment sample.

Lipids

The gas chromatograms o f t h e t o t a l l i p i d m i x t u r e s o b t a i n e d f r o m b o t h t h e w a t e r - and a c i d e x t r a c t s and t h e r e s i d u e s m e n t i o n e d i n F i g . 1, c a n be d e v i d e d i n t o t h r e e t y p e s b a s e d upon t h e d i s t r i b u t i o n o f t h e o b s e r v e d compounds. F i g . 5 shows t h e gas chromatograms o f t y p i c a l r e p r e s e n t a t i v e s o f each t y p e : t h e e x t r a c t s E 2 - E 5 (E3 shown), t h e s t a r t i n g m a t e r i a l ( R l ) and r e s i d u e R2 ( R l shown) and t h e r e s i d u e s a f t e r a c i d t r e a t m e n t R3 R5 (R5 shown). The c o n t r i b u -t i o n o f -t h e e x -t r a c -t a b l e l i p i d c a r b o n -t o -t h e -t o -t a l o r g a n i c c a r b o n p r e s e n -t i n

- ° d i h y d r o - a c t i n i d i o l i d e B : iso - L o l i o l i d e C' H O ^ ^-0 l C l'0 l": l e A

B

mate-r i a l and C) the ultimate mate-residue R5. Fomate-r conditions: see Expemate-rimental.

t h e s t a r t i n g m a t e r i a l i s e s t i m a t e d t o be 4%.

A b r i e f r e v i e w o f t h e l i p i d compounds e n c o u n t e r e d i n t h e v a r i o u s chromato-grams i s g i v e n below. D e t a i l e d d a t a o n t h e s e l i p i d s w i l l be g i v e n e l s e w h e r e

( K l o k et al. , s u b m i t t e d ) .

The m i d d l e t r a c e shown i n F i g . 5 shows t h e d i s t r i b u t i o n p a t t e r n o f l i p i d c l a s s e s and i n d i v i d u a l l i p i d s e x t r a c t e d f r o m t h e s t a r t i n g m a t e r i a l . As i n o t h e r r e c e n t m a r i n e s e d i m e n t s a c i d s , a l c o h o l s ( e s p e c i a l l y p h y t o l ) and s t e r o l s a r e t h e m a j o r l i p i d c l a s s e s o b t a i n e d . The l o w e r chromatogram o f l i p i d s o b t a i -ned from t h e r e s i d u e a f t e r d i r e c t 6 N HC1 h y d r o l y s i s i s c h a r a c t e r i z e d by t h e

19

abundance o f f a t t y a c i d s and g-hydroxy f a t t y a c i d s ( t h e l a t t e r marked w i t h a r r o w s ) . The ghydroxy f a t t y a c i d s a r e r e l e a s e d from the s a m p l e , w i t h o u t s o l u -b i l i z a t i o n , d u r i n g the a c i d t r e a t m e n t and can -be e x p l a i n e d -by the p r e s e n c e of amide l i n k e d h y d r o x y f a t t y a c i d s . They a r e known t o o c c u r as such i n b a c t e r i a l l i p o p o l y s a c c h a r i d e s and o r n i t h i n e c o n t a i n i n g l i p i d s as a l r e a d y m e n t i o n e d b e f o r e . T h e i r abundance p o i n t s to a c o n s i d e r a b l e b a c t e r i a l c o n t r i b u t i o n t o the e x t r a c t a b l e l i p i d c a r b o n p r e s e n t i n t h i s sample.

The upper t r a c e i s dominated by the p r e s e n c e of t h r e e compounds w i t h a d i s t i n c t s t r u c t u r a l r e l a t i o n s h i p ( m o l e c u l a r s t r u c t u r e s d e p i c t e d i n the chroma-t o g r a m ) . Componenchroma-t A was i d e n chroma-t i f i e d as d i h y d r o a c chroma-t i n i d i o l i d e . The o chroma-t h e r and more abundant compounds were i d e n t i f i e d as i s o l o l i o l i d e and l o l i o l i d e (pounds B and C r e s p e c t i v e l y ) . The n a t u r a l o c c u r r e n c e o f t h e s e t e r p e n o i d com-pounds i s m a i n l y r e p o r t e d i n p l a n t m a t e r i a l (Marx and Sondheimer, 1966; P a i l e r and H a s c h k e - H o f m e i s t e r , 1969; Holub et al., 1975; Kodama et al., 1982) b u t a l s o as a sex pheromone o f the r e d f o x Vulpes vulpes ( A l b o n e , 1975), i n m o l l u s c s

( P e t i t t et al., 1980) and brown a l g a e ( R a v i et al. , 1982). These compounds are a l s o r e p o r t e d t o be g e n e r a t e d d u r i n g p h o t o - o x i d a t i o n o f c a r o t e n o i d s ( I s o e et

al. , 1969; I s o e et al., 1971; I s o e et al., 1972). S i n c e f u c o x a n t h i n i s t h e

m a j o r c a r o t e n o i d t o be e x p e c t e d i n the N a m i b i a n S h e l f u p w e l l i n g a r e a , because i t i s the p r e d o m i n a n t c a r o t e n o i d i n Baaillariophyceae and Phaeophyceae ( J o -h a n s e n et al. , 1974; Goodwin, 1980; R e p e t a and G a g o s i a n , 1983) i t i s s u g g e s t e d t h a t t h e l o l i o l i d e s a r e p r o d u c t s of the p h o t o - o x i d a t i o n o f t h i s c a r o t e n o i d

( K l o k et al. , i n p r e p a r a t i o n ) .

it. -he i f f i i d e n j j i f i e d £Slk2D

U s i n g the a n a l y t i c a l p r o c e d u r e s d e s c r i b e d above o n l y a p a r t (^20%) o f the t o t a l o r g a n i c c a r b o n p r e s e n t i n t h e s t a r t i n g m a t e r i a l ( R l ) c o u l d be i d e n t i f i e d as amino a c i d , m o n o s a c c h a r i d e , v o l a t i l e f a t t y a c i d o r l i p i d c a r b o n . We t h e r e

-f o r e a p p l i e d p y r o l y s i s m a s s - s p e c t r o m e t r y (Py-MS) and p y r o l y s i s - g a s chromato-graphy-mass s p e c t r o m e t r y (Py-GC-MS) t o i n v e s t i g a t e the s t r u c t u r e s of the as y e t u n i d e n t i f i e d o r g a n i c c o n s t i t u e n t s p r e s e n t i n r e s i d u e s and e x t r a c t s . F o r t h i s p u r p o s e the r e s i d u e s ( R ) , the p r e c i p i t a t e s ( P P ) , the medium (MM) and t h e h i g h m o l e c u l a r (HM) w e i g h t f r a c t i o n s were a n a l y s e d by Py-MS ( T a b l e 4, F i g . 6 ) . The s t a r t i n g m a t e r i a l ( R l ) and r e s i d u e R5 were a l s o s t u d i e d by Py-GC-MS ( F i g . 7, T a b l e 5 ) .

Py-MS d a t a of the r e s i d u e s as summarized i n T a b l e 4 and seen i n F i g . 6 r e v e a l t h e abundance of h y d r o c h l o r i c a c i d (m/z 36, 38) as a r e s u l t of i n d i g e -nous s e a s a l t and the h y d r o c h l o r i c a c i d u s e d . S i m i l a r l y , s p e c i f i c i o n s of s u l p h u r compounds a r e r e l a t i v e l y abundant (m/z: 34, 48, 64, 66, 7 6 ) . E l e m e n t a r y s u l p h u r (m/z 64, 96, 128, 160) p r e s e n t i n t h e s t a r t i n g m a t e r i a l ( R l ) was p a r t l y

Fig. 6. Pyrolysis mass spectra of some of the residues and fractions of extracts obtained after extraction of a lyophilized Namibian Shelf sediment sample and of the MM f r a c t i o n of cellulose treated with 6N HCl. For conditions: see Experimental.

Table 4. Py-MS data obtained from residues and fractions of extracts as mentioned in Fig. 1. For conditions: see Experimental.

R e s i d u e s P r e c i p i t a t e s MM f r a c t i o n s HM f r a c t i o n s c e l l u l o s e c h a r a c t e r i s t i c s 1 Rl R2 R3 R4 R5 E2 E3 E4 E5 E2 E3 E4 E5 E2 E3 E4 E5 f r a c t i o n s c h l o r i d e s ( H C l ) +++ +++• ++ + +++ ++•+ ++ + + + + s u l p h u r compounds ++ +-•- ++ +•+• +++ + + ++ + + ++ + + + + ++ p r o t e i n s f + c a r b o h y d r a t e s + + ++ +++ amino c a r b o h y d r . " l e v u l i n i c a c i d " + + ++ ++ ++• ++ ++ ++ ++ +++ ¿ilkar.es ++ ++ ++ ++ + + + + •f + + a l k e n e s + + + + + + + + f a t t y a c i d s + + + + + + + + + + + + 1 c h a r a c t e r i s t i c m/z v a l u e s : H C l : 36,38 ; s u l p h u r compounds : 34,48,64,66,76 ; p r o t e i n s 67,69,83, 92,94,108,117,131 ; c a r b o h y d r a t e s : 43,68,82,96,98,110,112,114,126,128 ; amino c a r b o h y d r a t e s : 59, 109,125,137,151 ; " l e v u l i n i c a c i d " : 43,56,98,101,1 16 ; a l k a n e s : 57,71,85,99,... ; a l k e n e s : 55,69, 83,97,... ; f a t t y a c i d s : 60,73,87,101,115,129,... ( a f t e r M e u z e l a a r et at., 1982) + a r b i t r a r y u n i t s ( + • p r e s e n t , ++ • a b u n d a n t , +++ = p r e d o m i n a n t )