UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The distribution of lipid sulfur in soils of British Columbia Chae, Yeh Moon 1979

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1979_A1 C43.pdf [ 6.5MB ]
Metadata
JSON: 831-1.0094753.json
JSON-LD: 831-1.0094753-ld.json
RDF/XML (Pretty): 831-1.0094753-rdf.xml
RDF/JSON: 831-1.0094753-rdf.json
Turtle: 831-1.0094753-turtle.txt
N-Triples: 831-1.0094753-rdf-ntriples.txt
Original Record: 831-1.0094753-source.json
Full Text
831-1.0094753-fulltext.txt
Citation
831-1.0094753.ris

Full Text

THE DISTRIBUTION OF L I P I D SULFUR IN SOILS OF BRITISH COLUMBIA BY YEH MOON|CHAE B. Sc., S e o u l N a t i o n a l U n i v e r s i t y , S e o u l , K o r e a , 1962 M. Sc., Washington S t a t e U n i v e r s i t y , P u l l m a n , Wa., U.S.A., 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY xn THE FACULTY OF GRADUATE STUDIES (Department of S o i l S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA November 1979 0 Yeh Moon Chae, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l ' f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i i -ABSTRACT The s i g n i f i c a n c e o f s o i l l i p i d s t o man's environment i s no t f u l l y y e t u n d e r s t o o d , b u t t h e r e a r e some i n d i c a t i o n s t h a t s u g g e s t p o s s i b l e r o l e s i n i n f l u e n c i n g s o i l s t r u c t u r e , h y d r o p h o b i c i t y and i n f i l t r a t i o n c h a r a c t e r i s t i c s , p h y t o t o x i c i t y , and n u t r i e n t c y c l i n g p a r t i c u l a r g y o f P and S. The l i p i d s may a l s o a c t as a s i n k f o r p o l y n u c l e a r h y d r o c a r b o n s and some p e s t i c i d e s . The p r e s e n t s t u d y c o n s t i t u t e s t h e f i r s t r e p o r t e d i n v e s t i g a t i o n o f s u l p h o l i p i d s i n s o i l . The d i s t r i b u t i o n o f s o i l l i p i d s u l f u r was s t u d i e d p a r t i c u l a r l y i n r e l a t i o n t o t h e f a c t o r s w h i c h i n f l u e n c e t h e c o n t e n t of t h e l i p i d s u l f u r under d i f f e r e n t s o i l e n v i r o n m e n t s . F u r t h e r s t u d i e s were c a r r i e d out t o f r a c t i o n a t e t o t a l l i p i d s and l i p i d s u l f u r by means of column chromatography and t o c h a r a c t e r i z e s o i l l i p i d s u l f u r , u s i n g t h i n - l a y e r and g a s - l i q u i d chromatography. The i n v e s t i g a t i o n o f t h e d i s t r i b u t i o n o f l i p i d s u l f u r i n s o i l s showed t h a t l i p i d s u l f u r was found i n a l l s o i l s examined b u t the amount was v e r y v a r i a b l e . The l i p i d s u l f u r c o n t e n t s were h i g h e r i n o r g a n i c h o r i z o n s t h a n i n m i n e r a l h o r i z o n s , and p o o r l y d r a i n e d s o i l s had h i g h e r l i p i d s u l f u r t h a n f r e e l y d r a i n e d s o i l s . The h i g h e s t l i p i d s u l f u r c o n t e n t was o b s e r v e d i n p o o r l y d r a i n e d o r g a n i c s o i l s . The l i p i d s u l f u r a c c o u n t e d f o r a s m a l l p e r c e n t a g e o f t o t a l s u l f u r and o f t o t a l l i p i d s . The l i p i d s u l f u r c o n t e n t s were on ave r a g e n e a r l y t h r e e t i m e s h i g h e r t h a n t h e l i p i d phosphorus c o n t e n t s . The l i p i d s u l f u r c o n t e n t was s i g n i f i c a n t l y c o r r e l a t e d o n l y w i t h t o t a l and H l - r e d u c i b l e s u l f u r and o r g a n i c c a r b o n c o n t e n t , among v a r i o u s s o i l f a c t o r s examined. - i i i -The d i s t r i b u t i o n of l i p i d s u l f u r i n s o i l can be best explained by the two s o i l f a c t o r s , i . e . , t o t a l l i p i d content and t o t a l s o i l s u l f u r content, when the l i p i d s u l f u r content was expressed as part per m i l l i o n of s o i l . Therefore, s o i l f a c t o rs can be chosen accordingly when a sui t a b l e expression f o r the l i p i d s u l f u r content was used f o r the d i s t r i b u t i o n of the l i p i d s u l f u r i n s o i l . The f r a c t i o n a t i o n of s o i l t o t a l l i p i d s , f o r selected s o i l s , i nto three general classes, i . e . , neutral l i p i d s , g l y c o l i p i d s and polar l i p i d s , on a s i l i c i c acid column has shown that the d i s t r i b u t i o n pattern of the three classes of l i p i d s was s i m i l a r f o r a l l eight s o i l s studied regardless of s o i l type. The uniformity of the d i s t r i b u t i o n patterns suggests that the l i p i d s i n these s o i l s were si m i l a r i n type and o r i g i n , or that t h e i r d i s t r i b u t i o n s were affected by, and made more uniform through, i n t e r a c t i o n of microorganisms with other s o i l environmental fa c t o r s . The s o i l l i p i d s u l f u r , however, had no such a consistent s i m i l a r i t y i n the d i s t r i b u t i o n pattern, d i f f e r i n g from s o i l to s o i l . The most s i g n i f i c a n t f i n d i n g of the study was that s i g n i f i c a n t amounts of s u l f u r were, i n a l l cases, recovered i n each general c l a s s , i n contrast to the fi n d i n g that most of the t o t a l l i p i d s were recovered i n both neutral l i p i d and g l y c o l i p i d classes, e x c l u s i v e l y . This f i n d i n g c l e a r l y suggests that s o i l l i p i d s u l f u r i s present i n a v a r i e t y of forms. Observation of the thi n - l a y e r and ga s - l i q u i d chromatographic behavior of general l i p i d classes, fractionated from two s o i l samples, was conducted to characterize s o i l l i p i d s u l f u r . Thin-layer chromato-- i v -graphic behavior of the corresponding l i p i d classes of the two s o i l s were not s i m i l a r to each other, although column chromatographic behavior of the l i p i d classes of the two s o i l s were s i m i l a r to each other. These d i s s i m i l a r i t i e s i n d i c a t e that the i n d i v i d u a l component of one general l i p i d class fractionated from one s o i l d i f f e r s from that of the other s o i l . Observation on the g a s - l i q u i d chromatographic behavior of general l i p i d classes suggested that GLC could be used i n monitoring the sulfur-containing compounds i n l i p i d extracts of s o i l by choosing a s u i t a b l e column, and organic solvents of high p u r i t y . Although the attempt to separate and characterize i n d i v i d u a l s u l f u r -containing l i p i d components by chromatographic methods i n t h i s l a s t phase of i n v e s t i g a t i o n f a i l e d to s i g n i f i c a n t l y advance our knowledge of i n d i v i d u a l l i p i d constituents, p a r t l y because of t e c h n i c a l problems and p a r t l y because of lack of time, i t served to re-emphasize the complexity both of s o i l l i p i d s i n general, and of t h e i r s u l f u r -containing constituents i n p a r t i c u l a r . It also indicated some of the more promising l i n e s of i n v e s t i g a t i o n for future studies. - v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i CHAPTER 1. INTRODUCTION 1 CHAPTER 2. REVIEW OF LITERATURE 4 1. SOIL L I P I D S 4 1.1 I n t r o d u c t i o n 4 1.2 Methods o f E x t r a c t i o n 5 1.3 C o n t e n t o f S o i l L i p i d s 8 1.4 S i g n i f i c a n c e o f L i p i d s i n S o i l s . . .11 1.5 P e r s i s t e n c e o f L i p i d s i n S o i l s . . . 13' 1.6 C h e m i s t r y o f S o i l L i p i d s 15 1.6.1 Waxes 15 1.6.2 A c i d s 17 1.6.3 Hy d r o c a r b o n s 21 1.6.4 F a t s 25 1.6.5 P h o s p h o l i p i d s 25 1.6.6 S t e r o i d s and T r i t e r p e n o i d s . .27 1.6.7 C a r o t e n o i d s and C h l o r o p h y l l s . 2 8 1.6.8 Ket o n e s 29 1.6.9 M i s c e l l a n e o u s 29 - v i -2. NATURALLY OCCURRING SULFOLIPIDS 30 2.1 I n t r o d u c t i o n 30 2.2 Mammalian S u l f o l i p i d s 32 2.3 P l a n t S u l f o l i p i d s 35 2.4 M i c r o b i a l S u l f o l i p i d s . 37 2.5 A n a l y t i c a l Methods o f S u l f o l i p i d s . . 41 2.6 I s o l a t i o n o f S u l f o l i p i d s 44 2.7 S o i l S u l f o l i p i d s .47 REFERENCES 48 CHAPTER 3. THE DISTRIBUTION OF SULFUR IN L I P I D EXTRACTS OF SOILS , . 60 INTRODUCTION 60 METHODS AND MATERIALS 61 S o i l s .61 E x t r a c t i o n 63 A n a l y t i c a l Methods 64 RESULTS AND DISCUSSION . 65 O r g a n i c Carbon and T o t a l Phosphorus 65 S u l f u r 69 L i p i d and L i p i d Phosphorus 72 The D i s t r i b u t i o n o f t h e L i p i d S u l f u r i n S o i l s ^ CONCLUSION . 86 REFERENCES 89 CHAPTER 4. THE COLUMN CHROMATOGRAPHIC FRACTIONATION OF TOTAL LIPIDS AND L I P I D SULFUR IN SOME SELECTED BRITISH COLUMBIAN SOILS 92 INTRODUCTION . . 92 METHODS AND MATERIALS. 93 - v i i -RESULTS AND DISCUSSION 95 CONCLUSION 102 REFERENCES 104 CHAPTER 5. OBSERVATIONS ON THE GAS-LIQUID AND THIN-LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS IN TWO SELECTED SOILS 105 INTRODUCTION. 105 METHODS AND MATERIALS 106 S o i l s 106 A n a l y t i c a l Methods J06 Column Chromatography 108 T h i n - L a y e r Chromatography 109 G a s - L i q u i d Chromatography H O RESULTS AND DISCUSSION H I T h i n - L a y e r Chromatography 112 G a s - L i q u i d Chromatography. 121 CONCLUSION. 137 REFERENCES 140 GENERAL SUMMARY AND CONCLUSIONS 141 - v i i i -T a b l e CHAPTER 3. 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 LIST OF TABLES Page THE DISTRIBUTION OF SULFUR IN L I P I D EXTRACTS OF SOILS O r i g i n and c h e m i c a l c h a r a c t e r i s t i c s o f s o i l samples ( a n a l y s i s e x p r e s s e d on oven d r y b a s i s ) 62 R e l a t i o n s h i p between o r g a n i c c a r b o n and t o t a l phosphorus c o n t e n t s and pH v a l u e s among s o i l groups 67 C o r r e l a t i o n m a t r i x o f some c h e m i c a l c h a r a c t e r i s t i c s o f s o i l samples 68 R e l a t i o n s h i p s o f t o t a l s u l f u r w i t h t h e H i - r e d u c i b l e s u l f u r (HI-S) and c a r b o n -bonded s u l f u r (C-S) f o r s o i l groups 70 The d i s t r i b u t i o n o f l i p i d and l i p i d p hosphorus i n s o i l s 74 C o r r e l a t i o n c o e f f i c i e n t s between l i p i d c o n t e n t and o t h e r s o i l p r o p e r t i e s 76 The d i s t r i b u t i o n o f l i p i d s u l f u r i n s o i l s 80 C o r r e l a t i o n c o e f f i c i e n t s f o r l i p i d s u l f u r v e r s u s some s o i l p r o p e r t i e s 82 C o r r e l a t i o n c o e f f i c i e n t s f o r L i p i d s S/ T o t a l S and L i p i d S / L i p i d v e r s u s some s o i l p r o p e r t i e s 83 R e g r e s s i o n e q u a t i o n s and c o e f f i c i e n t o f d e t e r m i n a t i o n (R^) f o r r e l a t i o n s h i p s between l i p i d s u l f u r as ppm o f s o i l , as % of t o t a l s u l f u r , and as ppm o f l i p i d and s o i l f a c t o r s o f samples o f some B r i t i s h C olumbian s o i l s 85 - i x -T a b l e Page CHAPTER 4. THE COLUMN CHROMATOGRAPHIC FRACTIONATION OF TOTAL LIPIDS AND L I P I D SULFUR IN SOME SELECTED BRITISH COLUMBIAN SOILS 4.1 4.2 Some c h e m i c a l a n a l y s e s o f s o i l samples . . . 94 L i p i d s u l f u r and l i p i d d i s t r i b u t i o n s i n f r a c t i o n s o f S i l i c i c a c i d column chromatography 98 4.3 A s s o c i a t i o n w i t h l i p i d c l a s s e s o f s o i l l i p i d s u l f u r and t o t a l r e c o v e r y o f l i p i d and l i p i d s u l f u r .101 CHAPTER 5. OBSERVATIONS ON THE GAS-LIQUID AND THIN-LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS IN TWO SELECTED SOILS 5.1 Some c h e m i c a l a n a l y s e s o f s o i l samples 107 5.2 L i p i d and l i p i d s u l f u r d i s t r i b u t i o n s i n f r a c t i o n s f r o m S i l i c i c a c i d column 113 - x -LIST OF FIGURES F i g u r e Page CHAPTER 5. OBSERVATIONS ON THE GAS-LIQUID AND THIN-LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS IN TWO SELECTED SOILS 5.1 T w o - d i m e n s i o n a l mapping TLC o f F r a c t i o n s 1, 2, 3 and 4 e l u t e d f r o m S i l i c i c a c i d column o f t o t a l l i p i d e x t r a c t o f s o i l 1 . . . 114 5.2 T w o - d i m e n s i o n a l mapping TLC of F r a c t i o n s 1, 2, 3 and 4 e l u t e d from S i l i c i c a c i d column o f t o t a l l i p i d e x t r a c t o f s o i l 2 115 5.3 M u l t i p l e development TLC of F r a c t i o n s 1, 2, 3 and 4 e l u t e d f r o m S i l i c i c a c i d column o f t o t a l l i p i d e x t r a c t o f s o i l 1 ' 117 5.4 M u l t i p l e development TLC o f F r a c t i o n s 1, 2, 3 and 4 e l u t e d f r o m S i l i c i c a c i d column o f t o t a l l i p i d e x t r a c t o f s o i l 2 118 5.5 M u l t i p l e development TLC of F r a c t i o n s A, B, C, D, E and F e l u t e d f r o m a second S i l i c i c a c i d column o f F r a c t i o n 1 from t h e f i r s t S i l i c i c a c i d column o f s o i l 1 • • 119 5.6 O n e - d i m e n s i o n a l m i n i - T L C o f t h e F r a c t i o n s A - F e l u t e d f r o m S i l i c i c a c i d columns o f s o i l 1 120 5.7 GLC o f c o n c e n t r a t e d r e a g e n t grade c h l o r o f o r m 122 5.8 GLC o f t o t a l l i p i d e x t r a c t e d from s o i l 1 123 5.9 GLC o f F r a c t i o n 1 o f l i p i d e x t r a c t e d from s o i l 1 124 5.10 GLC o f c o n c e n t r a t e d e x t r a c t i o n b l a n k 126 - x i -5.11 GLC o f t o t a l l i p i d e x t r a c t e d s o i l 2 127 5.12 GLC o f F r a c t i o n 1 o f t o t a l l i p i d e x t r a c t e d f r o m s o i l 2 128 5.13 GLC o f F r a c t i o n 1 o f l i p i d e x t r a c t e d f rom s o i l 1 130 5.14 GLC o f F r a c t i o n A f r o m s o i l 1 131 5.15 GLC of F r a c t i o n B f r o m s o i l 1 132 5.16 GLC o f F r a c t i o n C from s o i l 1 133 5.17 GLC o f F r a c t i o n D f r o m s o i l 1 134 5.18 GLC o f F r a c t i o n E from s o i l 1 135 5.19 GLC of F r a c t i o n F f r o m s o i l 1 136 - x i i -ACKNOWLEDGEMENT S My sincere appreciation i s extended to Dr. L.E. Lowe, professor, Department of S o i l Science, f o r his encouragement and valuable guidance at a l l stages of the study and preparation of t h i s t h e s i s . Suggestions by Dr. CA. Rowles, Dr. A.A. Bomke, Dr. Mary Barnes and Dr. J.F. Richards are also g r a t e f u l l y acknowled A s p e c i a l thanks to my wife f o r her constant patience and encouragement throughout the period of my study. CHAPTER 1 INTRODUCTION P a s t r e s e a r c h on s o i l o r g a n i c m a t t e r has been c o n c e r n e d l a r g e l y w i t h t h e n a t u r e and o r i g i n of t h e humic s u b s t a n c e s , a l t h o u g h , i n r e c e n t y e a r s , i n c r e a s i n g a t t e n t i o n has been g i v e n t o r e l a t i v e l y s i m p l e r o r g a n i c compounds such as t h e amino a c i d and c a r b o h y d r a t e f r a c t i o n s . I n f o r m a t i o n on s o i l l i p i d f r a c t i o n s i s b o t h meager and f r a g m e n t a r y . They have r e c e i v e d v i r t u a l l y no s y s t e m a t i c a t t e n t i o n from s o i l c h e m i s t s , no doubt p a r t l y because of t h e a l m o s t i n s u p e r a b l e d i f f i c u l t y o f t h e s e p a r a t i o n and i d e n t i f i c a t i o n of components u n t i l r e c e n t l y , b u t a l s o because t h e s e l i p i d s seemed of l i t t l e s i g n i f i c a n c e f rom t h e p o i n t o f v i e w o f a g r o n o m i s t s . However, t h e p e r f e c t i o n of new c h r o m a t o g r a p h i c methods f o r h i g h - r e s o l u t i o n s e p a r a t i o n and a n a l y s i s of l i p i d s has been an i m p o r t a n t f a c t o r i n ope n i n g t h i s f i e l d t o more p e n e t r a t i n g i n v e s t i g a t i o n . F u r t h e r m o r e , t h e r e a r e i n d i c a t i o n s s u g g e s t i n g p o s s i b l e r o l e s i n f l u e n c i n g s o i l s t r u c t u r e , h y d r o p h o b i c i t y and i n f i l t r a t i o n c h a r a c t e r i s t i c s , p h y t o t o x i c i t y and n u t r i e n t c y c l i n g p a r t i c u l a r l y of phosphorus and s u l f u r . The l i p i d f r a c t i o n may a l s o a c t as a s i n k f o r p o l y n u c l e a r h y d r o c a r b o n s and some p e s t i c i d e s . Of t h e v a r i o u s l i p i d components, t h e s u l f o l i p i d i n s o i l has been c o m p l e t e l y i g n o r e d . T h e r e a r e some s t u d i e s f o c u s s e d on t h e p h o s p h o l i p i d s o f a g r i c u l t u r a l s o i l s . P h o s p h o l i p i d s have been f o u n d i n s o i l s and i n a l l o r g a n i s m s i n w h i c h t h e y have been sought t o d a t e . W h i l e s u l f o l i p i d s were o r i g i n a l l y c o n s i d e r e d l e s s common t h a n - 2 -p h o s p h o l i p i d s , i t now appears t h e y a r e u b i q u i t o u s . The r e t a r d e d development of s u l f o l i p i d r e s e a r c h has been due t o t h e p o o r e r a n a l y t i c a l methods f o r s u l f a t e , whereas phosphate i s c o n v e n i e n t l y a s s a y e d by t h e molybdenum b l u e method. The p r i m a r y f o c u s o f t h i s i n v e s t i g a t i o n , t h e n , was t o c o n s i d e r some a s p e c t s on t h e d i s t r i b u t i o n o f l i p i d s u l f u r i n s o i l s . More s p e c i f i c a l l y some of t h e f a c t o r s were c o n s i d e r e d w h i c h i n f l u e n c e t h e c o n t e n t o f t h e l i p i d s u l f u r under d i f f e r e n t s o i l e n v i r o n m e n t s . F u r t h e r s t u d y was c a r r i e d o ut t o d e t e r m i n e i f g a s - l i q u i d chromatography i s u s e f u l i n m o n i t o r i n g t h e l i p i d s u l f u r i n e a c h . f r a c t i o n e l u t e d f r o m a s i l i c i c a c i d column. A s i g n i f i c a n t p r o p o r t i o n o f t h e work was de v o t e d t o a s t u d y of t h e c h a r a c t e r i z a t i o n o f l i p i d s u l f u r u s i n g g a s -l i q u i d , t h i n - l a y e r and s i l i c i c ' a c i d column c h r o m a t o g r a p h i e s . Because a l l c o m m e r c i a l s o l v e n t s e x h i b i t e d s i g n i f i c a n t s u l f u r i m p u r i t i e s , t h e development o f a " s u l f u r b ase l i n e " f o r t h e s o l v e n t b l a n k s p r o v e d t o be a major p r o b l e m i n c h a r a c t e r i z i n g s o i l s u l f u r l i p i d s u s i n g a gas chromatograph e q u i p p e d w i t h a s u l f u r d e t e c t o r . The second c h a p t e r has r e v i e w e d a r t i c l e s on s o i l l i p i d s and n a t u r a l l y o c c u r r i n g s u l f o l i p i d s . The subsequent c h a p t e r s a r e i n t h e for m o f a s e r i e s o f c h a p t e r s t h a t would be s u i t a b l e f o r p u b l i c a t i o n i n s c i e n t i f i c j o u r n a l s , each c h a p t e r b e i n g i m p o r t a n t i n t h e c o n s i d e r a t i o n o f t h e t o p i c o u t l i n e d . The t h i r d c h a p t e r examines t h e d i s t r i b u t i o n o f l i p i d s u l f u r i n s o i l s . The c o n t e n t of l i p i d s u l f u r was c l o s e l y examined i n r e l a t i o n t o o t h e r s o i l f a c t o r s such as pH, o r g a n i c c a r b o n c o n t e n t , t o t a l and H I - r e d u c i b l e s u l f u r , and t o t a l and l i p i d p hosphorus c o n t e n t s . R e l a t i o n s h i p s and c o n c l u s i o n s were a t t e m p t e d - 3 -from the analyses that were done, pr i m a r i l y to gain information on c h a r a c t e r i s t i c s which would be most u s e f u l f o r further studies i n r e l a t i o n to the i s o l a t i o n and structure-determination of the l i p i d s u l f u r i n s o i l s . The f r a c t i o n a t i o n of l i p i d s u lfur on a s i l i c i c a c i d column of eight selected s o i l samples i s described i n the fourth chapter. In t h i s i n v e s t i g a t i o n a s i l i c i c acid column chromatography for the f r a c t i o n a t i o n of the l i p i d s u l f u r which had not previously been used i n s o i l studies, was adopted. The d i s t r i b u t i o n of t o t a l l i p i d s and l i p i d s u l f u r i n s o i l s were compared i n r e l a t i o n to the s o i l environments. The f i f t h chapter describes a study that examines methods of i s o l a t i n g and characterizing i n d i v i d u a l l i p i d s u l f u r components i n two selected s o i l samples. The r e s u l t s of t h i s study have demonstrated the effectiveness of a combination of g a s - l i q u i d , thin-layer and column chromatographies i n i s o l a t i n g one p a r t i c u l a r sulfur-containing l i p i d component. In the l a s t chapter, a l l topics outlined i n chapters threes four and f i v e are summarized and an attempt i s made to draw conclusions on the basis of the experimental r e s u l t s obtained from the relevant sections. - 4 -CHAPTER 2 REVIEW OF LITERATURE 1. SOIL LIPIDS 1.1. Introduction The importance of s o i l organic matter i n modern s o i l science has long been recognized. Past research has been concerned l a r g e l y with .humic substances and has paid l e s s attention to other components of s o i l organic f r a c t i o n s . These components of s o i l organic matter are v a r i o u s l y known i n s o i l science as waxes and r e s i n s , bitumens, or simply as organic substances extracted with an alcohol-benzene mixture. According to separation conditions from s o i l and to many properties, t h i s f r a c t i o n corresponds to substances defined, as l i p i d s i n bi o -chemistry. The alcohol-benzene f r a c t i o n may contain some s p e c i f i c humic substances, e.g., hymatomelanic acid, and some r e s i n o i d substances and free amino acids. S o i l s undoubtedly contain a wide v a r i e t y of l i p i d s ranging from the rather stable p a r a f f i n i c hydrocarbons to ephemeral c h l o r o p h y l l degradation products. L i p i d s are d i s t r i b u t e d extensively throughout the s o i l s of the world, ranging from the highly weathered l a t e r i t e s of the humid t r o p i c s to the weekly developed tundras of the a r c t i c zone. In normal aerobic s o i l s , the l i p i d s probably exist l a r g e l y as remnants of m i c r o b i a l ti s s u e ; low and v a r i a b l e quantities of these constituents - 5 -may be a s s o c i a t e d w i t h undecomposed p l a n t r e s i d u e s and t h e b o d i e s of l i v i n g and dead m i c r o f l o r a l o r o t h e r o r g a n i s m s . I n t h e e a r l y y e a r s o f t h e c e n t u r y S c h r e i n e r and Shorey (1907-1911), i n t h e i r now c l a s s i c works on t h e i s o l a t i o n of o r g a n i c s u b s t a n c e s from s o i l s , o b t a i n e d s e v e r a l l i p i d p r e p a r a t i o n s w h i c h t h e y were a b l e a t l e a s t p a r t l y t o c h a r a c t e r i z e . S i n c e t h e n t h e most e x t e n s i v e s t u d i e s of t h e l i p i d s o f m i n e r a l s o i l s have been t h o s e r e p o r t e d by M e i n s h e i n and Kenny (1957) and Wang e t a l . (1969 and 1971). The l i p i d s , o r , more s p e c i f i c a l l y , t h e waxes of p e a t have been r e v i e w e d by Howard and Hamer (1960) and W o l l r a b and S t r e b l ( 1 9 6 9 ) . E x t e n s i v e r e v i e w s of t h e s u b j e c t o f s o i l l i p i d s were p u b l i s h e d by S t e v e n s o n ( 1 9 6 6 ) , M o r r i s o n ( 1 9 6 9 ) , B r a i d s and M i l l e r ( 1 9 7 5 ) , and F r i d l a n d ( 1 9 7 6 ) . 1.2 Methods o f E x t r a c t i o n The e x t r a c t i o n o f l i p i d s f rom s o i l i s c o m p l i c a t e d by a number of f a c t o r s . Much of t h e l i p i d s may be l i n k e d i n some fo r m of c o m b i n a t i o n w i t h p r o t e i n o r c a r b o h y d r a t e , and t h e s e complexes a r e g e n e r a l l y 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 . Wagner and Muzorewa ( 1 9 7 7 ) , i n t h e i r s t u d y on l i p i d s o f m i c r o b i a l o r i g i n , p o i n t e d out t h a t m i c r o -b i a l l y s y n t h e s i z e d p r o d u c t s of a l i p i d n a t u r e i n s o i l may become i n c o r p o r a t e d i n t o s o i l humus w i t h o u t u n d e r g o i n g major d e g r a d a t i v e m o d i f i c a t i o n and t h a t t h i s presumed c o n d e n s a t i o n i m p a r t s s i g n i f i c a n t r e s i s t a n c e t o e x t r a c t i o n by o r g a n i c s o l v e n t s t o t h e l i p i d m a t e r i a l s . - 6 -I t i s o f t e n p o s s i b l e t o l i b e r a t e p r o t e i n - b o u n d l i p i d by use of e t h a n o l . However, f o r most l i p i d s , e t h a n o l i s a poor s o l v e n t . The a d d i t i o n o f e t h e r o r benzene improves t h e e f f e c t i v e n e s s of e t h a n o l , and s u c h s o l v e n t m i x t u r e s a r e f r e q u e n t l y used f o r e x t r a c t i o n of a n i m a l and p l a n t t i s s u e s ( B l o o r , 1915; Rewald, 1944) and of s o i l s as w e l l ( M o r r i s o n and B i c k , 1967; F u s t e c ^ M a t h o n e t a l . , 1977; Wagner and Muzorewa, 1977). The s o l u b i l i t y o f s o i l l i p i d s i s l i k e l y t o be f u r t h e r a f f e c t e d by t h e p r e s e n c e o f l a r g e amounts of i n o r g a n i c m a t e r i a l s u c h as c l a y m i n e r a l s and c a t i o n s of. aluminum o r i r o n . P a r t of t h e l i p i d s , f a t t y a c i d s f o r example, may o c c u r i n s o i l i n t h e form of aluminum o r i r o n s a l t s and t h e y cannot be i s o l a t e d by s i m p l e . e x t r a c t i o n because t h e s a l t s a r e p o o r l y s o l u b l e . i n o r g a n i c s o l v e n t s . An a c i d p r e t r e a t m e n t i s r e q u i r e d t o e x t r a c t them ( F r i d l a n d , 1976). P r e t r e a t m e n t w i t h a m i x t u r e of h y d r o f l u o r i c - h y d r o c h l o r i c a c i d s as shown by Hance and A n d e r s o n (1963) g r e a t l y i n c r e a s e d t h e y i e l d of l i p i d p hosphate d u r i n g e x t r a c t i o n . Wang et a l . (1969) a l s o showed t h a t c l a y - b o u n d u n s a t u r a t e d t r i g l y c e r i d e was e x t r a c t e d t h r o u g h h y d r o f l u o r i c - h y d r o c h l o r i c a c i d p r e t r e a t m e n t . The e x t r a c t a b i l i t y of s o i l l i p i d s may be a f f e c t e d by t h e d r y i n g of t h e s o i l , s i n c e i t i s g e n e r a l l y c o n s i d e r e d t h a t ' d r y i n g r e d u c e s l i p i d s o l u b i l i t y by p r o d u c i n g changes i n t h e f a t t y a c i d c o n s t i t u e n t s . These changes i n t h e c o n s t i t u e n t s p r o b a b l y enhance t h e a d s o r p t i o n of t h e f a t t y a c i d on c l a y m i n e r a l s and l e s s e n t h e l i a b i l i t y of t h e b o n d i n g between f a t t y a c i d and c l a y m i n e r a l t o be b r o k e n . Hance and A n d e r s o n (1963) p o i n t e d o u t t h a t t h e s o l u b i l i t y of s o i l l i p i d s was g r e a t e r i n - 7 -fresh than i n dried s o i l s , but i f the l a t t e r were pretreated with a mixture of hydrofluoric and hydrochloric acids the di f f e r e n c e was eliminated. The n o n - l i p i d contaminants i n l i p i d extracts may also be a factor complicating the extraction of l i p i d s from s o i l s , because the n o n - l i p i d contaminants may not a l l be water soluble and many l i p i d s are not soluble i n a l l f a t solvents. Some l i p i d solvents, p a r t i c u l a r l y acetone and ethanol, can extract inorganic or no n - l i p i d organic substances, and t h i s p o s s i b i l i t y should be considered i n investigations of s o i l l i p i d s . The water-washing procedure also i s not an e n t i r e l y s a t i s f a c t o r y way to remove n o n - l i p i d contaminants because t h i s procedure may r e s u l t i n some loss of l i p i d and retention of n o n - l i p i d substances i n the l i p i d extract (Nazir and Rouser, 1966). Both the y i e l d and chemical nature of the material extracted by organic solvents from s o i l s are influenced by the nature of the solvent and the conditions of extraction. More material i s extracted by strongly polar than by weakly polar solvents and the greatest amount by a mixture of the two. The extracted material consists of a complex mixture of compounds of f a i r l y high molecular weight. Morrison and Bick (1967) a r b i t r a r i l y separated, these into asphalt, r e s i n and wax fr a c t i o n s by means of the s e l e c t i v e a c t i o n of polar and non-polar solvents. Very recently S c i a c o v e l i et al_. (1977) extracted d i f f e r e n t l i p i d f r a c t i o n s from a brown s o i l of I t a l y using weak organic solvents with d i f f e r e n t p o l a r i t i e s . The extraction sequence was ethyl ether, benzene, acetone, dioxane, tetrahydrofuran, ethyl alcohol, - 8 -dimethylformamide, pyridine, dimethylsulfoxide and formamide. By inf r a - r e d and para-magnetic resonance spectrometries they found f r a c t i o n s extracted with ethyl ether, benzene and acetone were characterized by a preponderance of a l i p h a t i c structures; the fr a c t i o n s with dioxane, tetrahydrofuran and ethyl alcohol were characterized by a high content of oxygenated compounds and by the presence of hydroquinone units or hydroquinone polymeric chains; and the f r a c t i o n s with dimethylformamide and dimethylsulfoxide were characterized by the presence of p a r t i c u l a r skeletons, probably based upon hydroxylated naphtho- or anthraquinone u n i t s . There are c l e a r l y many possible combinations of solvents, and t h e i r s e l e c t i o n w i l l depend on the type of material and the nature of the predominant l i p i d s . However, the most e f f e c t i v e solvent for s o i l s cannot be predicted because the nature of s o i l l i p i d s i s obscure. The use of a v a r i e t y of solvents and combinations of them seems e s s e n t i a l . 1.3 Content of S o i l L i p i d s The l i p i d content of s o i l organic matter i s only a small and va r i a b l e part of the organic matter. No precise value can be estimated since no standard or generally accepted method of determination i s av a i l a b l e . Much information i n the l i t e r a t u r e on the amount of l i p i d extracted from s o i l s by organic solvents cannot be coordinated because of the use of d i f f e r e n t solvents and conditions. However, the method of proximate analysis proposed by Waksman and Stevens (1930), - 9 -although.the method i s neither complete nor s e l e c t i v e for l i p i d s , provides the best comparative values a v a i l a b l e f or a f a i r l y wide range of s o i l s . The range for the l i p i d content of humus found by Waksman and his coworkers (1930 and 1935), with few exceptions, f e l l within the range of 1 to 6% of the t o t a l organic matter. Similar r e s u l t s were obtained by Shewan (1938) for some s o i l s from Scotland. This range for the l i p i d content i s probably c h a r a c t e r i s t i c of most a g r i c u l t u r a l l y important s o i l s of the world. For the mineral s o i l s the highest values, up to 16% of the humus, occurred i n the podzolic s o i l s . In the case of the organic s o i l s the highmoor peats contained larger amounts than the lowmoor and sedimentary peats (Stevenson, 1966). The highest value reported for the l i p i d content of s o i l humus appears to be that of P i e t t r e (reference i n Stevenson, 1966) who found that nearly one half of the organic matter i n some coffee plantation s o i l s i n B r a z i l was i n the form of f a t t y or waxy material. Nearly one-third' of the organic matter i n some pollen peats examined by Minsen (reference i n Stevenson, 1966) was recovered as l i p i d . Stevenson (1966), on the basis of r e s u l t s obtained by such methods, indicated that the v a r i a t i o n noted i n the l i p i d content of humus can be explained by di f f e r e n c e s i n vegetation, pH or a combination of these two f a c t o r s . The high l i p i d contents are t y p i c a l l y associated with low pH, and t h i s i n turn suggests a c o r r e l a t i o n with m i c r o b i a l a c t i v i t y . The f a c t that the amount of l i p i d recovered from peats derived from Phragmites and Sphagnum was lower - 10 -than from peats derived from cottongrass, heather and Scirpus explains the v a r i a t i o n i n the l i p i d contents due to di f f e r e n c e s i n vegetation. Fustec-Mathon e t . a l . (1975 and 1977) i n t h e i r study on the l e v e l of bitumens i n two s o i l types under very s i m i l a r vegetation and from the same sandy region, observed that podzols contained c l e a r l y higher l e v e l s of bitumens than those of hydromorphic s o i l s . With respect to the podzols, they observed slower transformation and decomposition of plant debris and a more s i g n i f i c a n t m i c r o b i a l production of new chemical compounds. They concluded that the high l e v e l s of bitumens i n these sandy podzols arose from the low microbial a c t i v i t i e s due to the increase i n the a c i d i t y of the environment. They also concluded that the a c i d i t y of the environment had a more s i g n i f i c a n t r o l e i n the production of the bitumens than hydromorphy. Fridlarid (1976), i n h i s review a r t i c l e , pointed out that there were s i m i l a r r e l a t i o n s h i p s between the l i p i d extracted with alcohol-benzene mixtures and pH or m i c r o b i a l a c t i v i t y . The maximum l i p i d content of s o i l organic matter i s confined to s o i l s with low b i o l o g i c a l a c t i v i t y , i . e . , to hydromorphic and very a c i d i c s o i l s , and the minimum to steppe s o i l s with higher b i o l o g i c a l a c t i v i t y and to fo r e s t s o i l s on calcareous parent material, i . e . , s o i l s with a neutral pH. He also noted that the content of l i p i d s increases, as a r u l e , with an increase i n the t o t a l carbon content, except i n some s o i l s , and that the d i s t r i b u t i o n of l i p i d s i n s o i l horizons was the same i n most s o i l groups. The l i p i d content as percentage of s o i l organic matter (the r e l a t i v e l i p i d content) increases s l i g h t l y with depth while the - 11 -l i p i d content as percentage of s o i l weight (the absolute l i p i d content) decreases sharply because of the general decrease i n carbon content. The s l i g h t increase i n the r e l a t i v e l i p i d content with depth i s probably associated with the decrease i n microbial a c t i o n on them, r e s u l t i n g i n t h e i r preservation. However, sometimes the r e l a t i v e l i p i d content also decreases down the p r o f i l e , apparently because the preservation of the l i p i d s cannot be compensated by the decrease i n t h e i r supply to deeper horizons. I t i s also uncertain whether l i p i d s as they are, can migrate down the p r o f i l e because they are insoluble i n water per d e f i n i t i o n , although l i p i d s of low water-s o l u b i l i t y are expected to migrate with s o i l humic and f u l v i c acids as a bound l i p i d f r a c t i o n (Schnitzer, 1975). Fridland (1976) also found that the curve of the r e l a t i v e d i s t r i b u t i o n of the l i p i d content i n the horizon of the zonal s o i l s e r i e s i s a mirror image of the same curve of the Ch:Cf r a t i o s , i . e . , s o i l s with the highest Ch:Cf r a t i o have a minimal l i p i d content. This shows that l i p i d s experience the e f f e c t of the same factors as humic and f u l v i c acids. 1.4 S i g n i f i c a n c e of L i p i d s i n S o i l s Accumulation of l i p i d s i n s o i l has been considered as one of the possible causes of s o i l 'fatigue'. Waxy substances of l i p i d nature may waterproof s o i l p a r t i c l e s to prevent movement of nutrients from mineral surfaces into s o l u t i o n for plant uptake. They may s i m i l a r l y retard m i n e r a l i z a t i o n of organic nitrogen (Wagner and Muzorewa, 1977). Low molecular weight components may behave as organic - 12 -n u t r i e n t s , and a r e a l s o l i k e l y t o p r o d u c e h a r m f u l e f f e c t s on p l a n t g r o w th. The a c i d f r a c t i o n and t h e h y d r o c a r b o n s a r e t h e s t r o n g e s t i n h i b i t i n g a g e n t s f o r s o i l m i e r o f l o r a l p o p u l a t i o n s . F u s t e c - M a t h i o n e t a l . (1975) p o i n t e d out t h a t l i p i d s as a whole a r e i n h i b i t o r s f o r m i c r o o r g a n i s m s o f s o i l . The i n t e n s i t y o f i n h i b i t i o n depends on t h e c o n c e n t r a t i o n o f e t h a n o l - b e n z e n e e x t r a c t s , on pH of s o i l and on t h e o r i g i n o f m i c r o f l o r a . The d e p r e s s i v e a c t i o n a l s o depends t o some e x t e n t on t h e l e n g t h o f t h e c h a i n s and t h e n a t u r e o f f u n c t i o n a l groups of l i p i d s ( F u s t e c - M a t h o n e t a l . ; , 1977) . T h e r e i s , however, e v i d e n c e t h a t t h e p r e s e n c e i n s o i l s of l o n g - c h a i n compounds c a n r e s u l t i n i n c r e a s e d m i c r o b i a l p r o d u c t i o n o f s u b s t a n c e s , p o s s i b l y p o l y s a c c h a r i d e gum, w h i c h i n c r e a s e t h e s t a b i l i t y o f s o i l crumbs ( F e h l and Lange, 1965; M a r t i n et a l . , 1959). Numerous l i p i d components c o u l d e x e r t s t a b i l i z i n g f o r c e s upon s o i l a g g r e g a t e s by ce m e n t i n g m i n e r a l p a r t i c l e s t o g e t h e r , , a n d . i m p a r t i n g w a t e r r e p e l l e n c y t o l o o s e l y h e l d a g g r e g a t e s (Wagner and Muzorewa, 1977). An i n d i r e c t e f f e c t on s o i l f e r t i l i t y i s t h u s p o s s i b l e . A t t e m p t s t h a t had been made t o r e l a t e t h e p r o d u c t i v i t y of s o i l s t o the p r e s e n c e o f l i p i d s o r t h e i r t r a n s f o r m a t i o n p r o d u c t s , r e v e a l e d t h a t an i n c r e a s e i n t h e amount of l i p i d i n s o i l r e d u c e s s o i l p r o d u c t i v i t y . D e t a i l e d i n f o r m a t i o n c o n c e r n i n g t h e r o l e o f l i p i d s i n s o i l has been g i v e n i n a r e v i e w by S t e v e n s o n ( 1 9 6 6 ) . - 13 -1.5 Perslstance of L i p i d s i n S o i l s The primary source of s o i l humus i s the remains of b i o l o g i c a l systems i n and on the s o i l . In the decomposition of these residues i n s o i l , carbohydrates and proteins undergo hydrolysis to y i e l d water soluble products of low molecular weight, that are ult i m a t e l y destroyed i n biochemical and chemical processes. In contrast, many l i p i d s which are also subject to some changes following elimination from b i o l o g i c a l systems, are either preserved i n t a c t or converted into transformation products that are stable and tend to be preserved. Only l i g n i n , among the b i o l o g i c a l products, behaves more or le s s as do the l i p i d s with respect to preservation (Stevenson, 1966; Breger, 1966). Therefore, one might expect to f i n d l i p i d s i n sea water de s p i t e ' t h e i r low water s o l u b i l i t y ( J e f f e r y , 1966). I t i s generally recognized that l i p i d s decompose slowly i n s o i l s and t h e i r decomposition i s retarded mainly by the anaerobic nature of gleyed or saturated s o i l s and a c i d i c s o i l s . Very l i t t l e i s known of the rate at which i n d i v i d u a l l i p i d s decompose i n s o i l s . I t seems, however, that those compounds most r e s i s t a n t to microbial decomposition, such as higher alcohols and alkanes, should p e r s i s t f o r longer periods of time than those that are attacked r e a d i l y by microorganisms, such as the glycerides, phospholipids and unsaturated f a t t y acids. Waxes of higher plants are p a r t i c u l a r l y r e s i s t a n t to decomposition and survive e s s e n t i a l l y unchanged over long g e o l o g i c a l periods (Stevenson, 1966). - 14 -A study by Fustec-Mathion et a l . (1977) of the.acid and neutral f r a c t i o n s of the bitumens extracted from surface vegetation and various horizons of s o i l s , showed that the acid f r a c t i o n s and the hydrocarbons underwent les s transformation and less degradation i n A l horizons of the podzols than i n the corresponding hydromorphic s o i l horizons. The neutral f r a c t i o n s are for the most part only s l i g h t l y transformed, but aremore r a p i d l y degraded, i n s o i l s . Jones (1970) also has shown-that the plant derived alkanes i n s o i l appeared to be r e s i s t a n t to mi c r o b i a l a l t e r a t i o n under the experimental conditions. From the r e s u l t s of a laboratory incubation study T u r f i t t (1943) concluded that s t e r o l s were decomposed r a p i d l y i n s o i l , although i n one experiment i n which c h o l e s t e r o l was added to a mull s o i l only 60% was decomposed within a year; He also pointed out that lack of aeration and high water content were f a c t o r s which i n h i b i t e d decomposition. Gorham and Sanger (1967) reported that lake muds were very much r i c h e r than woodland.soils i n pigments of ch l o r o p h y l l d e r i v a t i v e s and carotenoids. They a t t r i b u t e d the r i c h e r pigments i n lake muds to the anaerobic condition provided. Except for the paper by P i e t t r e (reference i n Stevenson, 1966), no report has been published showing a s i g n i f i c a n t accumulation of d i f f i c u l t l y decomposable l i p i d s i n productive a g r i c u l t u r a l s o i l , even when large annual increments of residues and manures have been applied. Most c u l t i v a t e d s o i l s apparently contain s u f f i c i e n t numbers of the proper kinds of micro-organisms to destroy the l i p i d material contained i n plant and animal residues. - 15 -1.6 Chemistry of S o i l L i p i d s S o i l l i p i d s were found to contain waxes, f a t t y acids, hydrocarbons, glycerides, phospholipids, steroids, terpenoids, carotenoids, chlorophylls and others. Early workers lacked e f f i c i e n t methods to separate and i d e n t i f y c l o s e l y r e l a t e d components i n complex mixtures. In recent years, use-has teen made of modern a n a l y t i c a l methods for characterizing l i p i d constituents i n s o i l . In the following survey of l i p i d s i d e n t i f i e d i n s o i l s , only a few examples can be c i t e d where the newer methods have been applied to the study of s o i l l i p i d s because our knowledge of t h i s material i s highly fragmentary. 1.6.1 Waxes Ava i l a b l e evidence indicates that mixtures of waxes comprise a large part of the l i p i d s of s o i l s . I n dividual components are, however, d i f f i c u l t to separate, and evidence of composition i s usually based on i d e n t i f i c a t i o n of degradation products (see review by Stevenson, 1966). In the study conducted by Meinschein and Kenny (1957) four f r a c t i o n s were recovered by successive e l u t i o n with n-heptane, carbon t e t r a c h l o r i d e , benzene, and. methanol from s i l i c a g e l chromatography of benzene-methanol s o i l extracts. The material recovered with benzene consisted l a r g e l y of waxes. The types of acids and alcohols i n the wax esters were determined by converting the esters to saturated - 16 -hydrocarbons by high pressure hydrogenation; the hydrocarbons were further fractionated by chromatography and analyzed by mass spectrometry. Even carbon numbered (C-even) waxes were present i n considerably higher amounts than th e i r odd carbon numbered (C-odd) homologs. The wax ranged from to C^^ and the C-even waxes were formed from C-even normal a l i p h a t i c acids and normal primary a l i p h a t i c alcohols. The waxes i n urea non:-adduct- samples were c h i e f l y esters of c y c l i c alcohols and normal a l i p h a t i c acids. The wax f r a c t i o n examined accounted for about 5% of the benzene-methanol extracts. Butler et_ a l . (1964) obtained from an A u s t r a l i a n green s o i l a wax which was a mixture of hydrocarbons and esters of normal f a t t y acids with normal primary alcohols. By s a p o n i f i c a t i o n and column chromatography i t was separated into hydrocarbon, acid and alcohol f r a c t i o n s . The acids ranged from G-^ t o ^30' w i t n the even numbers predominating. The acids present i n greatest amounts were (13%), C„. (22%) and C„, (21%). The alcohols gave a range of compounds 2 H lb s i m i l a r to that of the acids. Himes and Bloomfield (1967) i s o l a t e d a mixture of waxes from an organic mat i n an orchard which had accumulated i n a s o i l as the r e s u l t of the copper induced i n h i b i t i o n of b i o l o g i c a l a c t i v i t y . From an examination of the wax by IR, NMR and mass spectrometry they concluded that the general structure was CH 3(CH 2) xCOO(CH 2)^GH 3. Values obtained for x and y indicated that the acid components were n-C^, n-C^g and n-C^g, and the primary alcohols were n-C^, n~^28 a n c * n -^30" Alcohols and acids of these chain length-have been reported as constituents of plant waxes. However, the carbon numbers of the acids - 17 -seem s u r p r i s i n g l y low and may not be t y p i c a l of s o i l waxes i n general. Recently Morrison and Bick (1967) extracted the wax f r a c t i o n of a garden s o i l and a peat by extracting them successively with a mixture of benzene-ethanol, .lig h t petroleum, and a mixture of isopropanol-ethanol. By s a p o n i f i c a t i o n and column chromatography of the methyl esters, they obtained a f r a c t i o n consisting of long-chain saturated f a t t y acids. Gas-liquid chromatography showed that the acids n - C ^ g t o n_<-'34 w e r e present and that about 80% were C-even; about 60% of these acids were n-C„., n-C„.,, n-C 0 0 and n-C o r i. The unsaponifiable f r a c t i o n contained normal primary alcohols from n-C. „ to.n-C,„. Similar r e s u l t s were obtained with wax from peat. They also obtained, from IR spectra, indications of the presence of hydroxy acids i n the saponifiable f r a c t i o n of the neutral l i p i d s from the same sources as lactones. The same authors also found i n the e a r l i e r study (1966) that the r e s i n and asphalt-free wax accounted for 0.6% of the organic matter i n the garden s o i l s and 1.59% i n the peat. 1.6.2 Acids Crude s o i l l i p i d extracts contain much a c i d i c material, the precise nature of which i s s t i l l obscure. Organic acids are of various types; more than a dozen free f a t t y acids both unsaturated and hydroxylated, and low molecular weight organic acids, including formic, a c e t i c , o x a l i c and but y r i c , have been i s o l a t e d from l i p i d extracts of both mineral and peaty s o i l s (see review by Stevenson, 1966). .- 18 -I t was also reported that organic acids of various types occur i n small quantities i n the rhizosphere of plant roots (Rovira, 1962) and i n raw podzol humus (Sallans, et a l . , 1937). Parker and Leo (1965) observed that there were f a t t y acids containing 12 to 20 carbon atoms i n a l g a l mat communities; underlying layers of black mud became progressively depleted i n the unsaturated acids. Schreiner and Shorey (1910) i s o l a t e d two organic acids, p a r a f f i n i c acid (C^g) from a s i l t loam and l i g n o c e r i c acid (C24) ^ r o m a peat. In the l i g h t of present knowledge i t seems.probable that both of these preparations were mixtures, mainly of unsaturated long-chain f a t t y acids. Another hydroxy f a t t y acid which they c a l l e d agrocerie acid (&2±) was.also i d e n t i f i e d from a North Dakota chernozemic type s o i l . A couple of years e a r l i e r , the same authors (1908) also i d e n t i f i e d 9,10-dihydroxystearic acid (C-^g) i n 27 d i f f e r e n t types of s o i l s and a mono-hydroxystearic acid probably o£-hydroxystearic acid (G-^ g) of which the p o s i t i o n of the hydroxy group was not rigorously established. The presence of both o l e i c (O^g) and e l a i d i e (C^g) acids i n s o i l organic matter and an apparently unsaturated acid which i s c a l l e d humoceric acid (C n) i n peat were demonstrated by K h a i n s k i i ( r e f . i n Morrison, iy 1969) and Aschan (ref. i n Morrison, 1969), r e s p e c t i v e l y . Morrison and Bick (1966), i n a study of the decomposition of wax from a garden s o i l and from a peat, obtained long-chain f a t t y acids and hydroxy acids. The same authors (1967), l a t e r obtained a p u r i f i e d l i p i d preparation from a garden s o i l which contained about 20% of f r e e acids, 55% of which consisted of a mixture of f a t t y acids n-C^rj t o n-C Q /, with 80% of even carbon number. The even-numbered acids, - 19 -n-C^^ to n - C ^ j together made up 75% of the f r a c t i o n . They also obtained evidence from IR spectra of the presence of a s i g n i f i c a n t f r ee hydroxy f a t t y acid f r a c t i o n i n s o i l l i p i d s , but no i n d i v i d u a l components have been i d e n t i f i e d . More recently Wang•et a l . (1969) reported that the main free higher f a t t y acids i n the s o i l s they studied were palmitic (C^.) , pa l m i t o l e i c (C..,) and o l e i c (C, 0) acids. Later Wang et a l . (1971) i o l o investigated by means of thin-layer and gas l i q u i d chromatography, the l i p i d content of s o i l s under various crops i n d e t a i l . The predominant free f a t t y acids they found were m y r i s t i c (0-^)> palmitic (C. ,) , p a l m i t o l e i c (C,,), s t e a r i c ( C 1 Q ) , o l e i c (C. „) and arachidic l b ±b lo l o ( C ^ ) acids, a l l even-carbon acids. They also found d i o c t y l p h t h a l i c acid of which IR spectra was i d e n t i c a l to that of dioctylphthalate found i n the documentation of molecular spectroscopy. Very recently Fustec-Mathon et a l . (1977) found that f a t t y acids (mono- and di-) obtained from acid sandy s o i l s had. n-C o n to n-C 0 0 range with the zu zo majority being C „ O J C 0 / , C„, and C 0 0 with preponderance of C-even. ZU z4 zo Zo Povoledo _et al. (1972) reported that analysis of l i p i d s extracted from,the humic acid preparations of some Canadian lake sediments revealed the^presence of odd, even, saturated, unsaturated and branched-chain f a t t y acids as major constituents. The p u r i f i e d e s t e r i f i c a t i o n products of the l i p i d s gave r i s e to some 40 gas l i q u i d chromatographic peaks, 22 of which were i d e n t i f i e d as methyl esters of a wide v a r i e t y of f a t t y acids ranging i n chain length between a n c ^ C^g. They also indicated that these findings are i n keeping with a preponderance of l i p i d s derived from microorganisms rather than from - 20 -t e r r e s t r i a l rooted plants. Schnitzer and Neyroud (1975) also extracted f a t t y acids from humic and f u l v i c acid preparations of a Black Chernozemic s o i l i n Central Alberta and of a poorly drained Podzol i n Prince Edward Island, r e s p e c t i v e l y . Carbon atoms of these f a t t y acids ranged from n-C. to n-C 0 0 with most being i n the C . -l z jo IH C22 range. These f a t t y acids had C-even preponderance. The f a t t y acids extracted from the two humic.preparations contained 60 and 80% r e s p e c t i v e l y of n-C, and n-C 1 0 acids, which suggested a m i c r o b i a l l b l o o r i g i n . I t i s probable that s a l t s of f a t t y acids are present i n mineral s o i l s of r e l a t i v e l y high pH. The presence of these f a t t y acid s a l t s are indicated by the f a c t that pretreatment of s o i l s with mineral acids usually r e s u l t s i n an increase i n the amount of material subsequently extracted'by l i p i d solvents, but the precise nature of t h i s a d d i t i o n a l material has never been studied.. Local concentrations of f a t t y acids and t h e i r i n s o l u b l e , p r i n c i p a l l y calcium, s a l t s are frequently found at b u r i a l s i t e s of human or other large animal corpses. These l o c a l accumulations of l i p i d s have been given the name "adipocire" and have been discussed i n some d e t a i l by Bergmann (1963). L i p i d preparations from s o i l s are l i a b l e to contain small amounts of aromatic acids such as benzoic, p-hydroxybenzoie or v a n i l l i c acids. Larger amounts of phenolic acids are chemically associated with humic substances and may be l i b e r a t e d , at least i n part, by acid or a l k a l i n e h y d r o l y s i s . Thus Schnitzer and Neyroud (1975) have released a number of phenolic acids from the humic and f u l v i c acid preparations. - 21 -This suggests that i n the humic substances f a t t y acids react with phenolic OH groups to form esters. 1.6.3 Hydrocarbons Higher alkanes are generally present i n s o i l s but, compared with waxes and acids, i n r e l a t i v e l y small q u a n t i t i e s . Normal p a r a f f i n i c hydrocarbons i n the C^^ to C ^ range have been found i n the nonsaponifiable f r a c t i o n of l i p i d extracts of s o i l by Schreiner and h i s associates (ref. i n Stevenson, 1966). Normal alkanes, n-hentriacontane, ^31' (Schreiner and Shorey, 1911b) and n-pentatriacontane, ^ 35' a n ( ^ n - t r i t r i a c o n t a n e , £33* (Titow, 1932) have been i s o l a t e d from a North Carolina organic s o i l and from a Russian peat, re s p e c t i v e l y , by using such c r i t e r i a as melting point and elementary a n a l y s i s . However, these early attempts to i s o l a t e i n d i v i d u a l components must be suspect, for i t i s only with the development of chromatographic methods that such separations have become r e a l l y f e a s i b l e . Stevens et_ a l . (1956) have found normal alkanes i n the to range i n several s o i l types, among which n-nonacosane (C^g) a n < * n-hentriacontane ( C ^ ) predominated. In an a l i p h a t i c hydrocarbon f r a c t i o n from a peat bitumen, G i l l H a n d and Howard (1968) have shown that the hydrocarbon f r a c t i o n consisted mainly of the C-odd n-alkanes, C^g (11%), (40%), and (34%), but i t contained also small amounts of a l l other n-alkanes from C^-j to ^rom a n A u s t r a l i a n s o i l , Butler et a l . (1964) obtained a hydrocarbon f r a c t i o n which gave peaks corresponding to n-alkanes from to with odd and even-carbon - 22 -numbers i n approximately equal amounts. The d i s t r i b u t i o n of n-alkanes i n t h i s A u s t r a l i a n s o i l was remarkably s i m i l a r to that of n-alkanes ranging from to i n an ancient sediment reported by Oro et a l . (1965). Furthermore the n-alkanes i n t h i s chert also showed no preference of C-odd to C-even carbon atoms. Morrison and Bick (1967) also obtained n-alkane f r a c t i o n s from both a garden s o i l and a peat which consisted l a r g e l y of alkanes from n-C i n and n-C„„ with about 87% odd-numbered. The main components iy 3 j i n the f r a c t i o n from the garden s o i l were n-C^ (21%), n-C^ (31%) and n-C 3 3 (15%). Recently Jones (1970) reported that the l i p i d extract from an upland moorland s o i l contained n-alkanes i n the to C„, range demonstrating a d e f i n i t e preponderance of odd-carbon JO numbered. Wang et a l . (1971) reported that p a r a f f i n i c hydrocarbons frequently appeared i n the s o i l they studied. Very recently Fustec-Mathon et a l . (1977) also reported that n-alkanes contained i n acid sandy s o i l s were i n the range from to C^^ with main components being C^y, ^29' ^31 a n ^ with preponderance of C-odd. Schnitzer and Neyroud (1975) extracted alkanes from two humic and f u l v i c preparations of a Black Chernozemic s o i l and a poorly drained Podzol, of which carbon numbers varied from C ^ to C^g, the majority being i n the str a i g h t - c h a i n C^g to C^^ range, and with an odd to even C r a t i o of 1.0. The d i s t r i b u t i o n of n-alkanes and the odd to even C r a t i o were s i m i l a r to those of microbial hydrocarbons reported by Jones (1969). - 23 -Polynuclear aromatic hydrocarbons have also been detected i n small quantities i n s o i l s . Kern (1947) i s o l a t e d chrysene (I) from a garden s o i l and the concentration estimated was 15 ppm. (IV) perylene (V) coronene Although Cooper and Lindsey (1953) suggested that the presence of chrysene i n s o i l s was due to atmospheric s e t t l i n g of dust p a r t i c l e s from combustion gases, i t seems l i k e l y that t h i s hydrocarbon may be indigenous to s o i l s , since the chrysene and other polynuclear aromatic hydrocarbons such as 3,4- and 1,2-benzpyrene, phenanthrene, fluoranthene ( I I ) , pyrene ( I I I ) , perylene (IV), anthanthrene, anthracene, triphenylamine benzanthracene, benzfluorene, 1,12-- 24 -benzperylene and coronene (V) were detected f r o m s o i l s of r u r a l areas d i s t a n t from major highways and industries (Blumer, 1961). Cheshire, jit al. (1967) have also i d e n t i f i e d many of these aromatic hydrocarbons as products of the d i s t i l l a t i o n of humic acids with zinc dust. Swan (1965) also i d e n t i f i e d dehydroabietene (VI) from a for e s t s o i l of B e l l a Coola, B r i t i s h Columbia. Wang et alL. (1971) indicated that p a r a f f i n i c hydrocarbons frequently appeared i n Taiwan s o i l s they studied and i n some s o i l s , an unknown nonpolar compound and d i o c t y l phthalate were also found. The p a r a f f i n i c hydrocarbons were i d e n t i f i e d by the t y p i c a l C-H stretching frequencies at 2853-2962 cm \ C-H deformation frequencies at around 1450 cm 1 and 1380 cm as well as s k e l e t a l v i b r a t i o n at 720 cm 1 i n the IR spectrum. (VI) dehydroabietene The p r i n c i p a l compounds i d e n t i f i e d from a "bound" l i p i d f r a c t i o n by Serra and Felbeck (1972) were a homologous serie s of normal alkanes. The bound l i p i d f r a c t i o n s they examined were obtained by extraction of the acid hydrolysis product of the organic matter from - 25 -a muck s o i l by using chloroform-methanol azeotrope. 1.6.4 Fats From the a l k a l i n e extract of a s i l t loam, Schreiner and Shorey (1911a) detected g l y c e r o l and a.mixture of u n i d e n t i f i e d f a t t y acids i n the product obtained by sa p o n i f i c a t i o n of the s o i l l i p i d s . Since then there have been no reports of the detection or determination of simple glycerides i n s o i l s u n t i l Wang e^ t a l . (1969) obtained t r i g l y c e r i d e s from s o i l l i p i d extracts using S i l i c a g e l .column chromatography. Although they did not mention the detection of the g l y c e r o l moiety of f a t s , they i d e n t i f i e d the f a t t y acids a f t e r s a p o n i f i c a t i o n of the s o i l l i p i d s . The s o i l t r i g l y c e r i d e s consisted of palmitic (C..,), p a l m i t o l e i c (CL,). and o l e i c (C, 0) acids together l o l o l o with docosanoic (C^)» 22-methyltrieosanoic ( C ^ ) a n c ^ 24-methyl-pentacosanoic (C^g) a°ids as t h e i r predominant components. Although reports on the presence of f a t s i n s o i l s are scarce, i t seems reasonable to believe that small amounts are usually present. 1.6.5 Phospholipids Considerable attention has been given to the occurrence of phospholipids i n s o i l because these compounds are p o t e n t i a l sources of phosphorus for plant growth. The presence of phospholipids i n s o i l has been inferred from the small amounts of phosphorus extracted from s o i l with l i p i d solvents. The amount of phosphorus thus extracted - 26 -i s small, usually 1 to 7 ppm, but 34 ppm has been reported by Black and Goring (1953). L i p i d phosphorus accounts for l e s s than 1% of the organic phosphorus i n s o i l s . Hance and Anderson (1963) obtained values of l i p i d phosphorus for f i v e s o i l s ranging from 0.31 to 0.70 mg phosphorus per 100 g s o i l , equivalent to only 0.6-0.9% of the t o t a l organic phosphorus. Simoneaux and Caldwell (1965), for nine s o i l extracts, reported l i p i d phosphorus contents ranging 0.24 ppm to 1.70 ppm. This represented from 0.06 to 1.02% of the organic phosphorus i n the selected s o i l s . The t o t a l l i p i d phosphate content of Chernozemic s o i l s of Southern Alberta ranged from 13 ppm i n the brown Ah horizon of a l a c u s t r i n e s o i l to 0.089 ppm i n the t h i n black Bm horizon of a l a c u s t r i n e s o i l (Domaar, 1970). Kowalenko and McKercher (1970a) reported that phospholipid contents of 20 Saskatchewan surface mineral s o i l s ranged from 0.6 to 14.5 ppm and averaged 3.4 ppm P. An L-H horizon contained 30.5 ppm P but subsurface horizons had. n i l to 0.6 ppm l i p i d P. I t was suggested from the r e s u l t s that most of the t o t a l phospholipid P may accumulate from the combined b a c t e r i a l and fungal biomass. Kowalenko and McKercher (1971b) also reported that phosphatidyl choline represented about 40% of t o t a l phospholipid P and phosphatidyl ethanolamine about 30%, for Saskatchewan s o i l s . - 27 -1.6.6 S t e r o i d s and T r i t e r p e n o i d s S i n c e s t e r o i d s and t r i t e r p e n o i d s a r e w i d e l y d i s t r i b u t e d i n p l a n t and a n i m a l t i s s u e s , t h e r e i s l i t t l e doubt t h a t t h e y o c c u r i n s o i l . However, t h e r e i s l i t t l e i n f o r m a t i o n on t h e n a t u r e and amount o f them i n s o i l . The o n l y s t e r o i d p o s i t i v e l y i d e n t i f i e d i n s o i l i s p - s i t o s t e r o l , a common s t e r o l of h i g h e r p l a n t s , w h i c h has been o b t a i n e d f r o m a g a r d e n s o i l (Bergman, 1963) and f r o m p e a t s ( G i l l i l a n d and Howard, 1968 and McLean e t a l . , 1 958). S c h r e i n e r and Shorey (1909 and 1911c) o b t a i n e d two p r e p a r a t i o n s from d i f f e r e n t s o i l s . One p r e p a r a t i o n f r o m a s o i l c o n t a i n e d a c r y s t a l l i n e compound w h i c h had c h e m i c a l p r o p e r t i e s t y p i c a l of t h e c h o l e s t e r o l g r o u p , but had a m e l t i n g p o i n t (237°C) d i s s i m i l a r t o any known s u b t a n c e i n t h i s g roup. T h i s compound was s u b s e q u e n t l y r e f e r r e d t o as " a g r o s t e r o l " . The o t h e r p r e p a r a t i o n w h i c h t h e y c a l l e d " p h y t o s t e r o l " had m e l t i n g p o i n t 135 C and was o b t a i n e d from a p e a t by a p r o c e s s i n v o l v i n g s a p o n i f i c a t i o n . . The f o r m e r , a g r o s t e r o l and t h e l a t t e r p h y t o s t e r o l m i g h t have been a t r i t e r p e n o i d and ?^ - s i t o s t e r o l , r e s p e c t i v e l y ( M o r r i s o n , 1969). T u r f i t t (1943) examined a number of E n g l i s h s o i l s f o r t h e p r e s e n c e of f r e e s t e r o l s and f o u n d t h a t t h e range of v a l u e s was from n i l t o 12 mg p e r k g of s o i l , t h e h i g h e s t v a l u e s were c h a r a c t e r i s t i c of p o o r l y a e r a t e d and a c i d i c s o i l s and t h e l o w e r v a l u e s c h a r a c t e r i s t i c o f a r a b l e s o i l s . On t h i s b a s i s f r e e s t e r o l c o u l d a c c o u n t f o r about 1% o f t h e s o i l l i p i d s . I v e s and O ' N e i l l (1958a and 1958b) r e p o r t e d t h e p r e s e n c e i n sphagnum p e a t moss of t h e s t e r o l s and t h e t r i t e r p e n o i d s - 28 -such as o(-amyrin, taraxerol and taraxerone. Meinschein and Kenny (1957), i n t h e i r study of benzene-methanol extracts of mineral subsoils, detected s t e r o l s and penta- and hexacyclic compounds which are thought to be t r i t e r p e n o i d s . The known occurrences of terpenes and s t e r o l s of g e o l o g i c a l i n t e r e s t are summarized by Bergmann (1963) and Breger (1966), r e s p e c t i v e l y . 1.6.7 Carotenoids and Chlorophylls Carotenoids and chlorophylls are, i n general, r e a d i l y oxidized i n the presence of a i r . In t h i s regard, Hoyt (1971) pointed out that chlorophylls are le s s stable i n s o i l environments than i n lakes. It i s reasonable to believe that carotenoids and chlorophylls are r e a d i l y degraded i n most s o i l s and are detected only i n small quantities i n arable s o i l s . Indeed no report seems to have been published on the occurrence of carotenoids and c h l o r o p h y l l d e r i v a t i v e s i n a g r i c u l t u r a l s o i l s , although the presence of degradation products o f . c h l o r o p h y l l (Vallentyne, 1957) i n anaerobic lake mud and i n non-calcareous surface woodland s o i l s (Gorham, 1959) has been known for many years. Gorham and Sanger (1967) pointed out that lake muds are very much r i c h e r than woodland s o i l s i n the contents of c h l o r o p h y l l d e r i v a t i v e s and carotenoids, c h i e f l y because of the anaerobic environment provided. Povoledo et a l . (1972) noticed a colored spot i n the bidimensional TLC of ethyl ether extract from a lake sediment humic acid preparation of which color, nonfluorescent feature and Rf values were c h a r a c t e r i s t i c of carotenoids. They also revealed the I - 29 -presence i n l a c u s t r i n e humus of chlor o p h y l l d e r i v a t i v e s , notably pheophytin a. 1.6.8 Ketones Morrison and Bick (1966), from a garden s o i l and a peat, obtained a f r a c t i o n c o n s i s t i n g of methyl ketones (n-alkan-2-ones) with carbon-chain length ranging from C^^-C^^ and with the odd-carbon numbered members predominating. This i s the f i r s t report of the occurrence of methyl ketones i n t h i s range as natural products; the lower members, up to C ^ , are well known as products of the action of microfungi on milk f a t t y acids i n c e r t a i n cheeses. Very recently Serra and Felbeck (1972) also suggested the presence of diketones i n the soluble f r a c t i o n i n chloroform-methanol azeotrope of acid hydrolysates•of the organic matter from a muck s o i l by means of gas chromatography, mass spectrometry and in f r a - r e d spectrometry. 1.6.9 Miscellaneous Many other substances of a l i p i d nature such as tocopherols and porphyrins from higher plants and polynuclear quinones of fungal o r i g i n are l i k e l y to be present i n s o i l s , but only i n very small amounts. Chlorinated i n s e c t i c i d e s and t h e i r degradation products, although not l i p i d s i n the s t r i c t sense, would be associated with them (Morrison, 1969) . - 30 -2. NATURALLY OCCURRING SULFOLIPIDS 2.1 Introduction; The s u l f o l i p i d s have been known since the c l a s s i c a l study of Thudichum (1874) on the chemical composition of brain. Their occurrence outside of brain tissue, however, was f i r s t reported by Benson et a l . (1959) only twenty years ago. Since t h i s f i r s t report, the s u l f o l i p i d s have been reported i n a v a r i e t y of l i v i n g systems, which include nearly the e n t i r e biosphere. According to the nomenclature of Haines (1971 and 1973b), the term s u l f o l i p i d denotes any sulfur-containing l i p i d . The s u l f a t i d e i s a s u l f o l i p i d i n which the s u l f u r occurs as a s u l f a t e ester and the s u l f o n o l i p i d i s used i n reference to s u l f o l i p i d s which contain s u l f u r i n the s u l f o n i c acid form. S u l f o l i p i d s with s u l f u r i n the reduced form are termed t h i o l i p i d s . Although no l i p i d has been reported containing the sulfoxide or sulfone states of s u l f u r , he suggested that sulfoxo-l i p i d and s u l f o n e l i p i d can be used i n reference to such compounds should they be i d e n t i f i e d . Mammalian s u l f a t i d e s include cerebroside s u l f a t e (Yamakawa jit al_., 1962), l a c t o s y l ceramide s u l f a t e (Martensson, 1966), ganglioside s u l f a t e (Leikola et a l . , 1969) and g l y c o l i p i d s u l f a t e (Ishizuka, et a l . , 1973). M i c r o b i a l s u l f a t i d e s include g l y c o l i p i d sulfates of h a l o p h i l i c • bacteria (Kates j2t a l . , 1967 and Marshall and Brown, 1968) and of Tubercle b a c i l l i s (Goren, 1970a and 1970b), a l k y l s u l fates and h a l o a l k y l s u l f a t e s (Haines and Block, 1962; Mayer and Haines, 1967; Elvoson and Vagelos, 1969; Haines et a l . , 1969) of Ochromonas danica, and - 31 -phosphatidylglycerosulfate of Halobactefium cutirubrum (Hancock and Kates, 1973). Another class of s u l f o l i p i d s are the s u l f o n o l i p i d s discovered by Benson et_ a l . (1959) . Reports of t h i o l i p i d s i n small amounts i n yeasts and plants have appeared (Chu et a l . , 1968). In ad d i t i o n to these s u l f o l i p i d s there are many reports of s u l f o l i p i d s which have not been followed up with f u l l c h a r a c t e r i z a tion. A s u l f o l i p i d s i m i l a r to that of the plant s u l f o n o l i p i d has been described i n the sea urchin (Isono and Nagai, 1966). There are several reports of s u l f o l i p i d s i n bacteria (Karlsson et a l . , 1971; Kates et a l . , 1968; Goren, 1971; Gangadharam et a l . , 1963; Roberts et a l . , 1955). A s u l f o l i p i d found i n diatoms (Kates and Tornabene, 1972) does not correspond to the plant s u l f o n o l i p i d . There are also several reports of fungal s u l f o l i p i d s ( C o l l i e r and Kennedy, 1963; Jack, 1964a and 1964b). A v a r i e t y of s u l f o l i p i d s have been reported i n insects ( G i r a l , 1941; G i r a l _et/_al., ' 1946) and chicken eggs (reference i n Haines, 1971). However, i t i s possible that these substances are not s u l f o l i p i d s i n the usual sense, but s t e r o i d s u l f a t e s or s u l f a t e esters of other organic molecules. S u l f o l i p i d s of diverse structure d i f f e r s i g n i f i c a n t l y i n t h e i r chemistry and biochemistry. The formation of s u l f a t i d e s i s c l e a r l y through an e n t i r e l y d i f f e r e n t biosynthetic route than that of the s u l f o n o l i p i d s , and t h e i r metabolic behavior should be r a d i c a l l y d i f f e r e n t . Their extraction, i s o l a t i o n and cha r a c t e r i z a t i o n should accordingly follow up with a v a r i e t y of techniques and methodologies. - 32 -2.2 Mammalian S u l f o l i p i d s U n t i l Benson e t a l . (1959) d i s c o v e r e d a s u l f o n o l i p i d , a s u l f o n i c a c i d e s t e r , i n p l a n t s a l l s u l f o l i p i d s were s u l f a t i d e s . Four mammalian s u l f a t i d e s have been c h a r a c t e r i z e d t o d a t e . These a r e c e r e b r o s i d e s u l f a t e i n b r a i n ( B l i x , 1933; Yamakawa e t a l . , 1962), l a c t o s y l c e r a m i d e s u l f a t e o f k i d n e y ( M a r t e n s s o n , 1966), g a n g l i o s i d e s u l f a t e i n h a r d t i s s u e s ( L e i k o l a e t a l . , 1969) and a g l y c o l i p i d s u l f a t e , s u l f o g l y c e r o g a l a c t o l i p i d s u l f a t e , from boar t e s t i s and spermatozoa ( I s h i z u k a e t a l . , 1973; Hatanaka e t a l . , 1975). C e r e b r o s i d e s u l f a t e ( V I I ) was t h e f i r s t s u l f a t i d e t o be d i s c o v e r e d i n 1874 (Thudichum). However, t h e e x i s t e n c e o f t h e s u l f a t i d e was n o t a s c e r t a i n e d u n t i l 1925 when L a n d s t e i n e r and Levene (1925) i s o l a t e d t h e s u l f a t i d e and e s t a b l i s h e d t h e s u b s t a n c e as a s u l f a t e - c o n t a i n i n g g l y c o l i p i d . A few y e a r s l a t e r , B l i x (1933) found s p h i n g o s i n e , c e r e b r o s i d e a c i d , g a l a c t o s e and s u l f a t e i n h i s p r e p a r a t i o n o f b r a i n s u l f a t i d e . He s u g g e s t e d t h a t t h e s u l f a t e group was on t h e g a l a c t o s e m o i e t y , p r o b a b l y i n t h e carbon-6 p o s i t i o n . T h i s p o s i t i o n of t h e s u l f u r i c e s t e r group on t h e carbon-6 o f t h e g a l a c t o s e m o i e t y was I N H O H - 0 V I I HO / I \ o - C H 2 - C H - C H - C H = C H - ( C H 2 ) I 2 - C H 3 o s o ; C H 2 O H ( V I I ) a c e r e b r o s i d e s u l f a t e - 33 -b e l i e v e d t o be c o r r e c t as r e c e n t l y as 1961 (Thannhauser e t a l . , 1955; J a n t z k e w i t z , 1958; J a n t z k e w i t z , 1960; G o l d b e r g , 1961). However, Yamakawa e t a l . (1962) s u g g e s t e d t h a t t h e s u l f a t e group was l o c a t e d on carbon-3 of t h e g a l a c t o s e and t h i s s t r u c t u r e has s i n c e been c o n f i r m e d by S t o f f y n and S t o f f y n ( 1 9 6 3 ) . The prop o s e d s t r u c t u r e was f u r t h e r c o n f i r m e d from a d i f f e r e n t p o i n t o f v i e w by T a k e t o m i and Yamakawa (1 9 6 4 ) . A second s u l f a t i d e , l a c t o s y l c e r a m i d e s u l f a t e ( V I I I ) , was i s o l a t e d and c h a r a c t e r i z e d by M a r t e n s s o n (1963b and 1966) and by Benson (1968). I n 1963 M a r t e n s s o n i s o l a t e d two d i f f e r e n t t y p e s o f s u l f o l i p i d s f r om human k i d n e y . . One o f them has t h e same c h e m i c a l c o m p o s i t i o n as b r a i n c e r e b r o s i d e s u l f a t i d e s . The o t h e r i s p r o b a b l y an a c y l - s p h i n g o s i n e -g l u c o s e - g a l a c t o s e s u l f a t e . T h i s second s u l f a t i d e appears t o be ab s e n t from b o v i n e k i d n e y ( K a r l s s o n 'et. a l . , 1 968), a l t h o u g h r a t b r a i n (Cumar e t a l . , 1968) appears t o c o n t a i n an enzyme f o r i t s b i o s y n t h e s i s . S u b s e q u e n t l y , M a r t e n s s o n (1966) e s t a b l i s h e d t h e p o s i t i o n o f t h e s u l f a t e group as g a l a c t o s y l - g l u c o s y l - c e r a m i d e w i t h t h e s u l f a t e group on t h e CH20H CH20H NH OH I I 0-CH 2-CH-CH-CH = CH-(CH2),2-( V I I I ) l a c t o s y l c e r a m i d e s u l f a t e - 34 -3 - p o s i t i o n o f g a l a c t o s e . The s t r u c t u r e of t h e s u l f a t i d e was t h e n c o n f i r m e d t o be t h e l a c t o s y l c e r a m i d e s u l f a t e by S t o f f y n e t a l . ( 1 9 6 8 ) . I n an i n v e s t i g a t i o n on human e p i d e r m i s , N i e m i n e n e t a l . (1967, i n H a i n e s , 1971) found a s u l f u r - c o n t a i n i n g l i p i d f r a c t i o n , w h i c h i n TLC m i g r a t e d s l i g h t l y ahead o f t h e c e r e b r o s i d e s u l f a t e e s t e r s . S i n c e t h e n a l i p i d f r a c t i o n w i t h s i m i l a r b e h a v i o r has been i s o l a t e d from human k i d n e y , h a i r and n a i l s , and from t h e w a l l s of b o v i n e and h o r s e s ' hooves ( L e i k o l a e t a l . , 1969). The s u l f o l i p i d was f ound t o c o n t a i n c e r a m i d e , s i a l i c a c i d , g a l a c t o s e , g a l a c t o s a m i n e , and s u l f a t e i n e q u i m o l a r amounts. They c o n c l u d e d t h a t t h e new l i p i d was a g a n g l i o s i d e s u l f a t e , f o r t h e s i a l i c a c i d i s a c h a r a c t e r i s t i c component of g a n g l i o s i d e s . The s u l f o l i p i d s r e p o r t e d by Maruyama (1962a and 1962b i n H a i n e s , 1971) i n c h i c k e n bone a r e a l s o l i k e l y t o b e l o n g i n t h i s g r o u p , a l t h o u g h t h e y were n o t w e l l c h a r a c t e r i z e d . I n a d d i t i o n t o s i x k i n d s o f s u l f u r - c o n t a i n i n g g l y c o l i p i d a l r e a d y c h a r a c t e r i z e d t o d a t e (Benson e t a l . , 1959; Yamakawa e t a l . , 1962; M a r t e n s s o n , 1966; K a t e s e t a l . , 1967; Goren, 1970b; Hancock and K a t e s , 1973), I s h i z u k a e t a l . (1973) r e p o r t e d t h e o c c u r r e n c e o f a n o v e l a l k y l e t h e r g l y c o l i p i d s u l f a t e as t h e major sugar c o n t a i n i n g l i p i d o f t e s t i s and spermatozoa o f boa r t o w h i c h t h e name " S e m i n o l i p i d " ( I X ) was g i v e n . S u b s e q u e n t l y t h e y found t h a t g u i n e a p i g t e s t i s a l s o c o n t a i n e d t h e s e m i n o l i p i d ( S u z u k i e t a l . , 1973). I t i s a l s o l i k e l y t h a t t h e s u l f o l i p i d s r e p o r t e d by K o r n b l a t t e t a l . (1972) b e l o n g i n t h i s group a l t h o u g h t h e y were n o t f u l l y c h a r a c t e r i z e d beyond t h e components - 35 -C H 2 o I II H C - O - C - ( C H 2 ) | 4 - C H 3 I H 2 C - 0 - ( C H 2 ) l s - C H 3 (IX) a seminolipid of the g l y c o l i p i d found to contain galactose, chimyl alcohol, palmitic acid and s u l f a t e . 2.3 Plant S u l f o l i p i d s The term "plant s u l f o l i p i d " (X) was f i r s t introduced by Benson and h i s coworkers (Benson et a l . , 1959 and 1960; Daniel _et _al., 1961; Lepage et a l . , 1961; Miyano and.Benson, 1962a and 1962b). I t i s the most widespread and best characterized s u l f o l i p i d to date. This s u l f o n o l i p i d has been found i n many green plants and i s not r e s t r i c t e d to the higher plants and green algae. I t has been reported i n red algae (Benson and Shibuya, 1962 and Radunz, 1969), and blue-green algae, brown algae and purple b a c t e r i a (Radunz, 1969). Highest concentrations of the plant s u l f o n o l i p i d appear to occur i n the marine red algae (Burwell, - 36 -CH 2 S0 3H J r . , 1945) although i t has been found i n barley, clover, spinach, chive and coleus (Benson et a l . , 1959), i n a l f a l f a (O'Brien et a l . , 1964) , i n maize, runner beans and Paul's s c a r l e t rose (Davies et a l . , 1965) , and i n a v a r i e t y of microbes (Benson et_ a l . , 1959; Davies et al_., Miyachi ^ t a l . , 1966). Nagai and Isono (1965) i s o l a t e d a s u l f o n o l i p i d from the sperm and eggs of the sea urchin, Pseudocentrotus depressus. Their s t r u c t u r a l studies suggest that the sugar moiety i s a 6-deoxyhexose-6-sulfonate such as sulfoquinovose of the plant s u l f o n o l i p i d . Although i t was not found i n the sperm of a s t a r f i s h , A s t e r i s amurensis (Isono, 1966) , or that of a mussel, Hypriopsis s c h e g l l i (Isono et a l . , 1967), the s u l f o n o l i p i d has been reported i n another sea urchin, Hemicentrotus  pulcherrimus by Isono (1967). - 37 -Haines (1971) pointed out that the d i f f i c u l t y . i n e s t a b l i s h i n g the structure of the s u l f o n o l i p i d i s i l l u s t r a t e d by the fa c t that i t took seven publications (Benson et a l . , 1959 and 1960; Peat et a l . , 1960; Daniel et a l . , 1961; Shibuya and Benson, 1961; Miyano and Benson, 1962a and 1962b). to complete the s t r u c t u r a l proof. The s u l f o n o l i p i d does not y i e l d s u l f a t e on hydrolysis, has not been reported i n mammalian tissues and i s among the most polar of the polar l i p i d s . Like the s u l f a t e esters, t h i s compound i s ionized at a l l pH values i n aqueous solutions. For these reasons, the l a t e appearance of these s u l f o n o l i p i d s i s e a s i l y explained and i t would remain i n the i n t r a c t a b l e residue of plant extracts. 2.4 M i c r o b i a l S u l f o l i p i d s Since the discovery of the plant s u l f o l i p i d i n green plants and microorganisms, C h l o r e l l a and Scendesmus, • by Benson and his coworkers (1959) the s u l f o n o l i p i d has been reported i n various organisms. I t has been found i n the sperm and eggs of the sea urchin (Nagai and Isono, 1965 and Isono, 1967). I t has also been reported i n a v a r i e t y of microbes, Euglena g l a c i l i s (Davies et^ al., 1965) and Ochromonas danica and Chamydomonas. (Miyachi et a l . , 1966). A report of i t s absence i n Ochromonas danica (Haines and Block, 1962) was found incorrect and due to growth conditions (Miyachi et a l . , 1966).. In 1959, Middlebrook jet ad. reported investigations providing evidence that the material responsible for the f i x a t i o n of neutral red, a s t a i n which distinguishes v i r u l e n t M. tuberculosis from - 38 -c e r t a i n a v i r u l e n t m u t a n t s , i s a new t y p e of s u l f o l i p i d . The a u t h o r s s u g g e s t e d t h a t t h i s s u l f o l i p i d was a s u l f o n i c a c i d . About one decade l a t e r , Mayer and H a i n e s (1967) s u g g e s t e d t h a t t h e s u l f o l i p i d was a s u l f a t e e s t e r and n o t a s u l f o n i c a c i d . T h i s s u g g e s t i o n was c o n f i r m e d by t h e s t r u c t u r a l s t u d i e s o f Goren (1970b). He has c h a r a c t e r i z e d t h e s u l f o l i p i d as t h e t e t r a e s t e r o f t r e h a l o s e s u l f a t e ( 2 , 3 , 6 , 6 ' - t e t r a e s t e r 2 ' - t r e h a l o s e s u l f a t e , X I ) . H a i n e s and B l o c k (1962) and H a i n e s (1965) r e p o r t e d a new s u l f o l i p i d f rom p h y t o f l a g e l l a t e s , Ochromonas d a n i c a , 0. mehamensis, and C h l o r e l l a p y r e n o i d o s a . The new s u l f o l i p i d was l a t e r c h a r a c t e r i z e d ( X I ) a t e t r a e s t e r o f t r e h a l o s e s u l f a t e as d o c o s a n e d i o l d i s u l f a t e ( X I I ) by Mayer and H a i n e s ( 1 9 6 7 ) . T h i s a l k y l s u l f a t i d e c o m p r i s e s t h e o n l y group of l i p i d m o l e c u l e s w h i c h c o n t a i n a p o l a r group a t b o t h ends of t h e m o l e c u l e . - 39 -o s o r I 3 C H 3 - ( C H 2 ) 7 — CH — (CH 2)| 2 — CH 2OS0 3~ (XII) docosanediol d i s u l f a t e The presence of c h l o r i d e i n the a l k y l s u l f a t i d e was f i r s t noticed by Elvoson and Vagelos (1969). Later on, these c h l o r o a l k y l s u l f a t i d e s were further characterized as a group of polychloro derivatives of 1,14-docosanediol (^22' XIII) and 1,15-tetracosanediol (C 2^) i n which the chloro-groups substitute f o r various hydrogens on 0 S 0 3 -I C H 3 - ( C H 2 ) 7 — C — CH - (CH2),, — CH 20S0 3-I CI (XIII) a chlorodocosanediol" d i s u l f a t e - 40 -the chain, but never more than s i x (Elvoson and Vagelos, 1969 and 1970; Haines, 1973b; Haines et a l . , 1969). These s u l f o l i p i d s are also unique as the only h a l o l i p i d s to be characterized i n natural f a t s . the absence of ch l o r i d e ion and i n the presence of bromide (references i n Haines, 1971 and 1973a). They include a hexabromo- and a monobromo-compound, both of which appear to be otherwise i d e n t i c a l to the chloro-compounds. Evidence for the occurrence of i o d o s u l f a t i d e was equivocal and f l u o r i d e was found to be toxic to the culture (Haines, 1973a). Halobacterium cutirubrum (Sehgal et a l . , 1962; Kates et a l . , 1965a and 1965b), characterized a s u l f o l i p i d as the s u l f a t e ester of g l y c o s y l -monogalactosyl diphytanyl g l y c e r o l (diphytanyl diether g l y c o s u l f a t e , XIV). The occurrence of t h i s s u l f o l i p i d i n one of the extreme A group of bromosulfatides was found i n c e l l s cultured i n Kates et _al. (1967), i n t h e i r expanded.studies of l i p i d s of HO 0 C H 2 ( C 2 0 H 4 i ) - 0 - C H HO (XIV) diphytanyl diether g l y c o s u l f a t e - 41 -h a l o p h i l e s , H a l o b a c t e r i u m h a l o b i u m was c o n f i r m e d by M a r s h a l l and Brown (1968) . 2.5 A n a l y t i c a l Methods f o r S u l f o l i p i d s The a s s a y o f s u l f o l i p i d s can be a c c o m p l i s h e d by a v a r i e t y o f methods as r e v i e w e d by H a i n e s (1971 ) . The c h o i c e o f a method w i l l depend upon t h e n a t u r e of t h e s u l f o l i p i d t o be s t u d i e d , t h e b i o l o g i c a l s o u r c e , t h e o c c u r r e n c e o f i n t e r f e r i n g s u b s t a n c e s i n t h e p r e p a r a t i o n , t h e p u r p o s e o f t h e a n a l y s i s , and t h e f a c i l i t i e s o f t h e l a b o r a t o r y . I n g e n e r a l , t h e a n a l y t i c a l p r o c e d u r e s a r e of two t y p e s . The f i r s t t y p e i n v o l v e s d e s t r u c t i o n o f t h e l i p i d m o i e t y and a n a l y s i s o f t h e l i b e r a t e d s u l f u r . The o t h e r a n a l y t i c a l p r o c e d u r e s r e l y upon some p h y s i c a l method w h i c h s e n s e s q u a n t i t a t i v e l y a p h y s i c a l p r o p e r t y o f t h e l i p i d o r a d e r i v a t i v e o f i t . The most common d e s t r u c t i v e method f o r t h e a s s a y of s u l f a t i d e s i s h y d r o l y s i s , a l t h o u g h t h e p r o c e d u r e i s p r o b a b l y t h e l e a s t r e l i a b l e . L e e s e t _ a l . (1959) s u g g e s t e d t h a t t h e p h o s p h o l i p i d s e x e r t a p r o t e c t i v e i n f l u e n c e on t h e h y d r o l y s i s o f s u l f a t i d e s . U n l i k e s u l f a t e e s t e r , s u l f o n i c a c i d o f t h e p l a n t s u l f o n o l i p i d s i s v e r y s t a b l e so t h a t e x c l u d e s t h e p o s s i b i l i t y o f h y d r o l y s i s under p r a c t i c a l l a b o r a t o r y c o n d i t i o n s . The r e d u c t i o n method o f M a r t e n s s o n (1963a) appears t o be r e l i a b l e f o r most p u r p o s e s f o r s u l f a t i d e s a l t h o u g h i t i s l e s s s e n s i t i v e and more cumbersome t h a n o t h e r methods. I t would a l s o seem p o s s i b l e t h a t t h e r e d u c t i v e method c o u l d be used f o r t h e p l a n t s u l f o n o l i p i d s , b u t t h i s t e c h n i q u e has n o t y e t been a p p l i e d t o s u l f o n o l i p i d s . - 42 -H a i n e s (1971) s u g g e s t e d t h a t a c o m b i n a t i o n o f S c h o n i g e r ' s c o m b u s t i o n p r o c e d u r e (1955) w i t h t h e f l a m e p h o t o m e t r i c method of R o b i n s o n (1960) w o u l d p r o b a b l y be a p o w e r f u l , d e s t r u c t i v e a n a l y t i c a l p r o c e d u r e f o r s u l f o l i p i d s . The a z u r e - A c o l o r i m e t r i c p r o c e d u r e of Kean (1968) can be used e f f e c t i v e l y on c r u d e p r e p a r a t i o n s of s u l f a t i d e s as w e l l as s c r a p e d s p o t s f r o m TLC. The o n l y known i n t e r f e r i n g s u b s t a n c e i n t h i s p r o c e d u r e was c a r d i o l i p i n . The a n t h r o n e method of Weenink (1963) appears t o be s p e c i f i c f o r 6-deoxy hexose s u l f o n i c a c i d s u c h as t h a t of t h e p l a n t s u l f o n o l i p i d and t h e sea u r c h i n s u l f o n o l i p i d . The m e t h y l e n e b l u e method o f Jones (1945) f o r t h e e s t i m a t i o n of a l k y l s u l f a t e s and t h e p r o c e d u r e by Kean (1968) u s i n g a z u r e - A f o r s u l f a t i d e i n mammalian t i s s u e s make t h e s e t e c h n i q u e s a v a i l a b l e f o r a l k y l s u l f o n i c a c i d s and s u l f o n o l i p i d s . The most v e r s a t i l e , s e n s i t i v e and g e n e r a l l y c o n v e n i e n t 35 o f t h e p h y s i c a l methods t o d e t e c t and e s t i m a t e the s u l f o n o l i p i d s i s S l a b e l l i n g . I t s o n l y l i m i t a t i o n , f o r example, i s t h a t human b r a i n l i p i d s 35 a r e n o t amenable t o S l a b e l l i n g . L i k e w i s e t h e a c t i v a t i o n a n a l y s i s of M cCandless ( 1 9 6 4 ) , t h e c r e s y l v i o l e t a s s a y o f Svennerholm ( 1 9 6 3 ) , and t h e a p p l i c a t i o n o f d e n s i t o m e t r y t o c h a r r e d p l a t e s a r e a l l a p p l i c a b l e t o s u l f o l i p i d s a f t e r t h e i r s e p a r a t i o n v i a chromatography. The most u s e f u l method f o r i d e n t i f i c a t i o n and s t r u c t u r a l s t u d i e s o f t h e s u l f o l i p i d s has been i n f r a r e d s p e c t r o p h o t o m e t r y . There . a r e f o u r m a j o r peaks c h a r a c t e r i s t i c o f i o n i c and c o v a l e n t s u l f a t e s f o u n d i n s u l f a t i d e s . The l a r g e s t due t o asymmetric v i b r a t i o n o f t h e S-0 bond a t 1210-1260 cm 1 i s u s e f u l t o c h a r a c t e r i z e i o n i c s u l f a t e . - 43 -(as distinguished from c y c l i c or dicovalent). A second band i s due to the symmetric v i b r a t i o n s of the S-0 bond i n the 1040-1080 cm 1 region. A t h i r d band i n the 935-1040 cm 1 region i s due to the C-0 bond of s u l f a t e esters and distinguishes primary from secondary su l f a t e s . A fourth band, due to the 0-S v i b r a t i o n a l modes, i d e n t i f i e s the substance as a s u l f a t e ester (as distinguished from a s u l f o n i c acid) and i s i n the 760-840 cm•1 region. The i n f r a r e d spectra of s u l f o n i c acids has been examined i n several laboratories (Haines, 1971). The asymmetric stretching of (SO2) was found to be 1350 cm 1 and the symmetric (SO2) stretching mode was assigned at 1160 cm 1 . The S-0 str e t c h was consistently at 900 cm An infrared spectrum of the plant s u l f o n o l i p i d has not been published while a spectrum of the sea urchin s u l f o l i p i d has been published (Isono and Nagai, 1966). To date there has not been an i n v e s t i g a t i o n of the nuclear magnetic resonance (NMR) spectra of organic s u l f a t e esters. One would expect that s u l f a t e esters would y i e l d NMR spectra e s s e n t i a l l y i d e n t i c a l to those of the alcohol from.which the s u l f a t e ester i s derived. The presence of electronegative s u l f a t e group should reduce the electron density around the protons attached to the sulfated carbon and produce a small downfield s h i f t . I t would, therefore, appear that the study of higher r e s o l u t i o n NMR spectra of t h i s problem would be very productive and very l i k e l y solve the problem of l o c a t i n g a s u l f a t e on a sugar (or any poly-hydroxyl compound). The mass spectra of s u l f a t e esters have not been explored due to the i n a b i l i t y to overcome the problem of v o l a t i l i t y . The only s u l f a t e esters that would appear to be open to i n v e s t i g a t i o n are the - 44 -the c y c l i c s u l f a t e s , which.with few exceptions have.not been reported to occur n a t u r a l l y (Haines, 1971). 2. 6 I s o l a t i o n of S u l f o l i p i d s The i s o l a t i o n of br a i n s u l f a t i d e has been the object of much e f f o r t and many pub l i c a t i o n s . R e l a t i v e l y large amounts of crude s u l f a t i d e were best extracted and i s o l a t e d by the procedure of Folch and coworkers (1957). The procedure would c e r t a i n l y be used as a f i r s t step i n any procedure used to i s o l a t e a quantity of cerebroside s u l f a t e . If small amounts of highly p u r i f i e d s u l f a t i d e are required the most rapid procedure would undoubtedly be. i s o l a t i o n of the crude s u l f a t i d e by the Folch extraction procedure followed by preparative t h i n layer chromatography (Wagner, et a l . , 1964; Haines, 1971). Cerebroside s u l f a t e has also been i s o l a t e d by zone electrophoresis using borate buffer (Svennerholm and Svennerholm, 1963). Martensson (1963a) extracted the l a c t o s y l ceramide s u l f a t e from l y o p h i l i z e d t i s s u e with chloroform-methanol (2:1). This extract was chromatographed on s i l i c i c acid columns, then DEAE c e l l u l o s e and the f r a c t i o n s containing s u l f a t i d e were separated on preparative t h i n layer plates of S i l i c a Gel G. In order to obtain pure s u l f o l i p i d i t was necessary to repeat the preparative t h i n layer chromatography twice on S i l i c a Gel G and then twice on F l o r i s i l . L e i k o l a e_t al. (1969) i s o l a t e d ganglioside s u l f a t e from the walls of horses' hooves using Folch' et a l . procedure (1957) and pre-parative t h i n layer chromatography. Seminolipid was also i s o l a t e d - 45 -from boar t e s t i s and spermatozoa (Ishizuka : et al.,,. 1973) and from guinea pig t e s t i s (Suzuki'et ' a l . , 1973) by the Folch et a l . procedure (1957) and subsequently by s i l i c i c acid and t h i n layer chromatography. Kates et a l . (1967) achieved the i s o l a t i o n of the g l y c o l i p i d s u l f a t e of h a l o p h i l i c b a c t e r i a by f i r s t i s o l a t i n g the i o n i c l i p i d s as an acetone insoluble p e l l e t which was dissolved i n a minimum volume of chloroform and p r e c i p i t a t e d with ten volumes of methanol. This p r e c i p i t a t e was subjected to chromatography on consecutive columns of S i l i c a using stepwise elutions of chloroform-methanol (4:1) i n t h e i r system. Middlebrook et al_. (1959) extracted the g l y c o l i p i d s u l f a t e of T u b e r c l e . b a c i l l i with hexane containing 0.05% decylamine. Using a procedure s i m i l a r to that of Jones (1945), the authors were able to follow the material during the i s o l a t i o n , by i t s hexane-soluble neutral red complex i n a water-hexane system. A more de t a i l e d procedure for the i s o l a t i o n has been published elsewhere (Gangadharam et a l . , 1963; Goren, 1969) . A l k y l s u l f a t i d e s (Haines, 1965; Mayer and Haines, 1967; Mayer et a l . , 1969; Haines and Block, 1962) and.the c h l o r o a l k y l s u l f a t i d e s (Elvoson and Vagelos, 1969; Haines et a l . , 1969; Elvoson and Vagelos, 1970) were extracted from broken c e l l s of Ochromonas danica with a chloroform-methanol (2:1) mixture; the crude s u l f a t i d e s were is o l a t e d using subsequent extraction with petroleum ether and butanol a f t e r s a p o n i f i c a t i o n of the chloroform-methanol extracts of the broken c e l l s . - 46 -Seven procedures (Benson et a l . ; 1959; Yagi and Benson, 1962) have been used for the i s o l a t i o n of the plant s u l f o n o l i p i d . For the i s o l a t i o n of a reasonable quantity of rather pure material the procedure of O'Brien and Benson (1964) i s the method of choice. The procedure begins with a crude l i p i d extract, which i s placed on a F l o r i s i l column and f r a c t i o n s are eluted with chloroform-methanol solvents which involve stepwise increases of p o l a r i t y . The solvents contain 5% (w/v) 2,2-dimethylpropane to maintain anhydrous conditions on the column during chromatography. The s u l f o n o l i p i d f r a c t i o n i s then placed on a DEAE-cellulose column which separates a pure product. The procedures are very well described including the methods used to prepare the supports and columns. Although no report on the use of Folch's extraction procedure (1957) i s a v a i l a b l e , i t would appear that t h i s procedure would be a good one f o r obtaining a crude preparation of the s u l f o n o l i p i d which might be further p u r i f i e d on DEAE c e l l u l o s e i f necessary. This procedure has a high p r o b a b i l i t y of success i n view of the polar nature of the s u l f o n o l i p i d on s i l i c i c a c i d inpregnated paper (Mumma and Benson, 1961). C l e a r l y small amounts of the s u l f o n o l i p i d can be i s o l a t e d by preparative t h i n layer chromatography (0'Brien jit a l . , 1964). Electrophoresis at low pH appears to be u s e f u l for small scale preparative work (Benson, 1963) . Nagai and Isono (1965) extracted the s u l f o n o l i p i d of the sea urchin with chloroform-methanol (1:1). The ether insoluble l i p i d s were then chromatographed on a s i l i c i c acid column. An anthrone p o s i t i v e g l y c o l i p i d f r a c t i o n was c o l l e c t e d and dried i n vacuo. The - 47 -r e s u l t i n g white powder was s o l u b l e i n chloroform c o n t a i n i n g a s m a l l amount of methanol and water. The f r a c t i o n was f u r t h e r p u r i f i e d according to the procedure of Radin et a l . (1956) to o b t a i n a phosphorus-free p r e p a r a t i o n . 2.7 S o i l S u l f o l i p i d s Although no r e p o r t s on the occurrence of s o i l s u l f o l i p i d s c ould be found, one might expect s u l f o l i p i d s , excreted by l i v i n g or returned i n decaying organisms, would be found i n s o i l s . This expectation i s supported by Haines (1964) who obtained a p o s i t i v e Jones methylene blue t e s t (Jones, 1945) from the n u t r i e n t medium of sorghum and white c l o v e r c u l t u r e d h y d r o p o n i c a l l y and from a sample of f o r e s t s o i l from Yonkers, New York. Lowe and DeLong (1961) found that i n a few Quebec s o i l s , a t h i r d of the organic s u l f u r was i n the form of s u l f a t e s u l f u r . Freney (1961) a l s o estimated the s u l f a t e ester content i n A u s t r a l i a n s o i l s to be about 52% of the organic s u l f u r . Thus appreciable q u a n t i t i e s of s u l f a t e e s t e r s i n s o i l s may a r i s e from the widespread d i s t r i b u t i o n of s u l f a t e e s t e r s as s t r u c t u r a l components and e x c r e t i o n products of organisms, of which components may be l i p i d s u l f a t e e s t e r . The f i n d i n g of s u l f o l i p i d s i n s o i l s , i f any, would help i n the b e t t e r understanding of both the s o i l s u l f u r c y c l e and the p o t e n t i a l of the s o i l environment as a source of plan t a v a i l a b l e s u l f a t e . - 48 -REFERENCES Benson, A.A. 1963. The p l a n t s u l f o l i p i d . I n advances i n L i p i d R e s e a r c h . R. P a o l e t t i and D. K r i t c h e v s k y , (Eds.) Academic P r e s s , New Y o r k , V o l . 1, pp. 387-394. Benson, A.A. 1964. P l a n t membrane l i p i d s . Ann. Rev. P l a n t P h y s i o l . 15: 1-13. Benson, A.A. and I . S h i b u y a . 1962. S u l f a c t a n t l i p i d s . I n P h y s i o l o g y and B i o c h e m i s t r y o f A l g a e . R.A. L e w i n , (ed.) Academic P r e s s , New Y o r k , pp. 371-383. Benson, A.A., H. D a n i e l and R. W i s e r . 1959. A s u l f o l i p i d i n p l a n t s . P r o c . N a t l . Acad. S c i . , U.S. 45: 1582-1587. Benson, A.A., R. W i s e r and B. Maruo. 1960. The s u l f o l i p i d s o f b r a i n and c h l o r o p l a s t s . P r o c . IV I n t e r n . Congr. of Biochem, V i e n n a , 1958. B i o c h e m i s t r y o f L i p i d s . G. P o p i a k , (ed.) Pergamon P r e s s , New Y o r k . 1960. pp.8-10. Bergmann, W. 1963. G e o c h e m i s t r y o f l i p i d s . I n O r g a n i c G e o c h e m i s t r y . I.A. B r e g e r , (ed.) Pergamon P r e s s , O x f o r d , pp. 503-542. B l a c k , C A . and C.A.I. G o r i n g . 1953. O r g a n i c phosphorus i n s o i l s . I n Agronomy. W.H. P i e r r e and A.G. Norman, (Eds.) M a r c e l D e k k e r , New Y o r k . No. 4, pp. 123-152. B l i x , G. 1933. Zur K e n t n i s d e r s c h w e f e l h a l t i g e n L i p o i d s t o f f e des G e h i r n s u b e r C e r e b r o n s c h w e f e l s a u r e . Z. P h y s i o l . Chem. H o p p e - S e y l e r ' s 219: 82-98. B l o o r , W.R. 1915. A method f o r t h e d e t e r m i n a t i o n of " l e c i t h i n " i n s m a l l amounts o f b l o o d . J . B i o l . Chem. 22:133-144. Blummer, M. 1961. Benzpyrenes i n s o i l s . S c i e n c e 134: 474-475. B r a i d s , O.C. and R.H. M i l l e r . 1975. F a t s , Waxes and R e s i n s i n S o i l . I n S o i l Components. J . G i e s e k i n g , (ed.) S p r i n g e r - V e r l a g , New Y o r k . V o l . 1, pp. 343-368. B r e g e r , I.A. 1966. G e o c h e m i s t r y o f l i p i d s . J . Am. O i l . C h e m i s t s ' Soc. 43: 197-202. B u t l e r , J.H.A., D.T. Downing and R . J . Swaby. 1964. I s o l a t i o n o f a c h l o r i n a t e d pigment f r o m g r e e n s o i l . A u s t r . J . Chem. 17: 817-819. - 49 -Burwell, J r . R.L. 1945. Racemization, a l k y l s u l f u r i c a c i d formation and exchange i n the rea c t i o n between s u l f u r i c acid-d2 and o p t i c a l l y a c t i v e 2-butanol. J . Am. Chem. Soc. 67: 220-223. Cheshire, M.V., P.A. Cranwell, C P . Falshaw, A.J. Floyd, and R.D. Haworth. 1967. Humic acid. I I . Structure of humic acids. Tetrahedron 23: 1669-1682. Chu, T.M., M.F. Mallette, and R.O. Mumma. 1968. I s o l a t i o n and charac t e r i z a t i o n of 5'-S-methyl-5'thiadenosine from Escherichia c o l i . Biochem. 7: 1399-1406. C o l l i e r , R. and G.Y. Kennedy. 1963. The s u l f o l i p i d s of plants. I. The d i s t r i b u t i o n of s u l f o l i p i d s and phospholipids among the major plant classes. J . Mar. B i o l . Ass. U.K. 43: 605-612. Cooper, R.L. and A.J. Lindsey. 1953. Atmospheric p o l l u t i o n by p o l y c y c l i c hydrocarbons. Chem. Ind. (London) pp. 1177-1178. Cumar, F.A., H.S. Barra, H.J. Maccioni, and R. Caputto. 1968. Sul f a t i o n of glycosphingolipids and re l a t e d carbohydrates by brain preparations from young r a t s . J . B i o l . Chem. 243: 3807-3816. Daniel, H., M. Miyano, R.O. Mumma, T. Yagi, M. Lepage, I. Shibuya, and A.A. Benson. 1961. The plant s u l f o l i p i d . I d e n t i f i c a t i o n of 6-sulfoquinovose. J. Am. Chem. Soc. 83: 1765-1766. Davies, W.H., E.I. Mercer and T.W. Goodwin. 1965. The occurrence and i n t r a c e l l u l a r d i s t r i b u t i o n of the plant s u l f o l i p i d i n maize, runner beans, plant ti s s u e cultures and Euglena g r a c i l i s . Phytochem. 4: 741-749. Dormaar, J.F. 1970. Phospholipids i n chernozemic s o i l s of Southern Alberta. S o i l S c i . 110: 136-139. Elovson, J. and P.R. Vagelos. 1969. A new class of l i p i d s : c h l o r o s u l -f o l i p i d s . Proc. Nat. Acad. S c i . U.S.A. 62: 957-963. Elovson, J . and P.R. Vagelos. 1970. Structure of the major species of c h l o r o s u l f o l i p i d s from Ochromonas danica. 2,2,11,13,15,16-hexa-chloro-N-docosane 1,14-disulfate. Biochem. 9: 3110-3126. Fehl, A.J. and W. Lange. 1965. S o i l s t a b i l i z a t i o n induced by growth of microorganisms on high c a l o r i e mold n u t r i e n t s . S o i l S c i . 100: 368-374. Folch, J . , M. Lees, and G.H. Sloanestanley. 1957. A simple method f o r the i s o l a t i o n and p u r i f i c a t i o n of t o t a l l i p i d s from animal t i s s u e s . J . B i o l . Chem. 226: 497-509. - 50 -F r e n e y , J.R. 1961. The n a t u r e of o r g a n i c s u l f u r compounds i n s o i l . A u s t r a l . J . A g r . Res. 12: 424-429. F r e n e y , J.R. 1967. S u l f u r c o n t a i n i n g o r g a n i c s . I n S o i l B i o c h e m i s t r y . A.D. McLaren and G.H. P e t e r s o n (Eds.) M a r c e l Dekker, I n c . , New Y o r k . P a r t I I , pp. 229-259. F r e n e y , J.R., F . J . S t e v e n s o n , and A.H. B e a v e r s . 1972. S u l f u r c o n t a i n i n g amino a c i d s i n s o i l h y d r o l y s a t e s . S o i l S c i . 114: 468-476. F r i d l a n d , Ye. V. 1976. L i p i d ( a l c o h o l - b e n z e n e ) f r a c t i o n o f o r g a n i c m a t t e r i n d i f f e r e n t s o i l g r o u p s . S o v i e t S o i l S c i . 8: 548-557. F u s t e c - M a t h o n , E., D. R i g h i , and P. Jambu. 1975. I n f l u e n c e des b i t u m e s e x t r a i t s de p o d z o l s humiques hydromorphes des Landes du Medoc s u r l a m i c r o f l o r e t e l l u r i q u e . Rev. E c o l . B i o l . S o l . 12: 393-404. F u s t e c - M a t h o n , E., P. Jambu, G. J o l l y , and R. J a c q u e s y . 1977. A n a l y s e e t r o l e des b i t u m e s dans l e s s o l s s a b l e u x a c i d e s . I n S o i l O r g a n i c M a t t e r S t u d i e s . V o l . I I , pp. 105-114, V i e n n a . P r o c e e d i n g s o f a Symposium, B r a u n s c h w e i g , 6-10 Sept. 1976. Gangadharam, P.R.J., M.L. Cohn and G. M i d d l e b r o o k . 1963. I n f e c t i v i t y , p a t h o g e n i c i t y and s u l f o l i p i d f r a c t i o n o f some I n d i a n and B r i t i s h s t r a i n s o f T u b e r c l e b a c i l l i . T u b e r c l e , Lond. 44: 452-455. G i l l i l a n d , M.R. and A . J . Howard. 1968. Some c o n s t i t u e n t s o f p e a t wax s e p a r a t e d by column chromatography. T r a n s . 2nd I n t e r n . P e a t Congr., L e n i n g r a d , 1963. V o l . I I , pp. 877-886. E d i n b u r g h : H.M.S.O., 1968. G i r a l , F. 1941. Sobre A c e i t e s de i n s e c t o s . I . T a e n i o p o d a a u r i c o r n i a . Rev. Soc. Mex. H i s t . N a t . 2: 243-250. G i r a l , J . , F. G i r a l , and M.L. G i r a l . 1946. F a t s o f i n s e c t s . IV. C o m p o s i t i o n o f t h e f a t o f M e l a n o p l u s a t l a n i s R i l e y . J . B i o l . Chem. 162: 55-59. G o l d b e r g , I.H. 1961. The s u l f o l i p i d s . J . L i p i d Res. 2: 103-109. Goren, M.B. 1969. S u l f o l i p i d s o f m y c o b a c t e r i u m t u b e r c u l o s i s : p u r i f i c a t i o n and p r o p e r t i e s . B a c t e r i o l . P r o c . 121, p.28. Goren, M.B.' 1970a. S u l f o l i p i d I o f M y c o b a c t e r i u m t u b e r c u l o s i s , S t r a i n H37Rv. I . P u r i f i c a t i o n and P r o p e r t i e s . B i o c h i m . B i o p h y s . A c t a . 120: 116-126. - 51 -Goren, M.B. 1970b. S u l f o l i p i d I of Mycobacterium tuberculosis, S t r a i n H37Rv. I I . S t r u c t u r a l studies. Biochim. Biophys. Acta 210: 127-138. Goren, M.B. 1971. Mycobacterial s u l f o l i p i d s : spontaneous d e s u l f a t i o n . L i p i d s 6: 40-46. Gorham, E. 1959. Chlorophyll derivatives i n woodland s o i l s . . S o i l S c i . 87: 258-261. Gorham, E. and J . Sanger. 1967. Plant pigments i n woodland s o i l s . Ecology 48: 306-308. Haines, T.H. 1964. A new s u l f o l i p i d i n microbes. Ph.D. Thesis. Rogers. The State University, New Brunswick, New Jersey. Haines, T.H. 1965. A microbial s u l f o l i p i d . I. I s o l a t i o n and P h y s i o l o g i c a l studies. J . Protozool. 12: 655-659. Haines, T.H. 1971. The chemistry of the s u l f o l i p i d s . In Progress i n the Chemistry of Fats and Other L i p i d s . R.T. Holman, (Ed.) Pergamon Press, Oxford. V ol. I I , pp. 297-345. Haines, T.H. 1973a. Halogen- and sulfur-containing l i p i d s of Ochromonas. Ann. Rev. M i c r o b i o l . 27: 403-411. Haines, T.H. 1973b. S u l f o l i p i d s and h a l o s u l f o l i p i d s . In L i p i d s and Biomembranes of Eukaryotic Microorganisms. J . Erwin, (Ed.) Academic Press, New York. pp. 197-232. Haines, T.H. and R.J. Block. 1962. Sulfur metabolism i n algae. I. Synthesis of metabolically i n e r t chloroform-soluble s u l f a t e esters by two chrysomonads and c h l o r e l l a pyranoidosa. J . Protozool. 9: 33-38. Haines, T.H., M. Pousada, B. Stern, and G.L. Mayer. 1969. M i c r o b i a l s u l f o l i p i d s : (R)-13-chloro-l-(R)-14-docosane-diol d i s u l f a t e and p o l y c h l o r o s u l f o l i p i d s i n Ochromonas danica. Biochem. J . 113: 565-566. Hance, R.T. and G. Anderson. 1963. Extraction and estimation of s o i l phospholipids. S o i l S c i . 96: 94-98. Hancock, A.J. and M. Kates. 1972. A s u l f a t e ester of phosphatidyl g l y c e r o l (diether analogue) from Halobacterium cutirubrum. Chem. Phys. L i p i d s 8: 87-90. Hancock, A.J. and Kates, M. 1973. Structure determination of the phosphatidyl gly c e r o s u l f a t e (diether analog) from Halobacterium  cutirubrum. J . L i p i d Res. 14: 422-429. - 52 -Hatanaka, H., Y. Ogawa, F. Egami, I. Ishizuka, and Y. Nagai. 1975. Sulf a t i d e and seminolipid as substrates of Charoriia lampas a r y l s u l f a t a s e . J . Biochem. 78: 427-429. Himes, F.L. and C. Bloomfield. 1967. Extraction of t r i a c o n t y l stearate from a s o i l . Plant S o i l 26: 383-384. Howard, A.J. and D. Hamer. 1960. The extraction and c o n s t i t u t i o n of peat wax. J . Am. O i l Chemists' Soc. 37: 478-481. Hoyt, P.B. 1971. Fate of chl o r o p h y l l i n s o i l . S o i l S c i . I l l : 49-53. Ishizuka, I., M. Suzuki and T. Yamakawa. 1973. I s o l a t i o n and char a c t e r i z a t i o n of a novel s u l f o g l y c o l i p i d , "Seminolipid", from boar t e s t i s and spermatozoa. J . Biochem. 73: 77-87. Isono, Y. 1966. G l y c o l i p i d s i n s t a r f i s h gametes. S c i . Pap. C o l l . Gen. Educ. Univ. Tokyo 16: 101-110. Isono, Y. 1967. Changes of g l y c o l i p i d s during early development of sea urchin embryos. Japan J . Exptl. Med. 37: 87-96. Isono, Y. and Y. Nagai. 1966. Biochemistry of g l y c o l i p i d s of sea urchin gametes. I. Separation and chara c t e r i z a t i o n of new type of s u l f o l i p i d and s i a l o g l y c o l i p i d . Japan J . E x p t l . Med. 36: 461-476. Isono, Y., H. Mohri, and Y. Nagai. 1967. E f f e c t of egg s u l f o l i p i d on r e s p i r a t i o n of sea urchin spermatozoa. Nature 214: 1336-1338. Ives, D.A.J, and A.N. O' N e i l l . 1958a. The chemistry of peat. Part 1. The s t e r o l s of peat moss (Sphagnum) Can. J . Chem. 36: 434-439. Ives, D.A.J, and A.N. O ' N e i l l . 1958b. The chemistry of peat. I I . The triterpenes of peat moss (Sphagnum). Can. J . Chem. 36: 926-930. Jack, R.C.M. 1964a. L i p i d metabolism i n fungi. I. L i p i d s of the conidia of Glomerella cingulata. Contrib. Boyce Thompson Inst. 22: 311-333. Jack, R.C.M. 1964b. L i p i d metabolism i n fungi. I I . Characterization of c o n i d i a l l i p i d s of Glomerella cingulata by i n f r a r e d s o l u t i o n spectra. Contrib. Boyce Thompson Inst. 22: 335-342. Jantzkewitz, H. 1958. Zwi Typen von cerebrosid-schwafelsaureestern a l s sog " P r a l i p o i d e " und Speichersubstanzen b e i der Leukodystrophie, Typ Scholoz (Metaehromatische Form der d i f f u s e n Sklerose). Z. P h y s i o l . Chem. 311: 279-282. Jantzkewitz, H. 1960. Cerebron- und Karasin-schwefelsaureester a l s Speichersubstanzen b e i der Leukodystrophie, Typ Scholoz (Metaehromatische Form der di f f u s e n Sklerose). Z. Ph y s i o l . Chem. 320: 134-148. - 53 -J e f f e r y , L.M. 1966. L i p i d s i n sea water. J . Am. O i l Chemists' Soc. 43: 211-214. Jones, J.H. 1945. General eolorimetric method f o r the determination of small quantities of sulfonated or sul f a t e d surface a c t i v e compounds. J . Assoc. Off. Agr. Chem. 28: 398-409. Jones, J.G. 1969. Studies on l i p i d s of s o i l micro-organisms with p a r t i c u l a r reference to hydrocarbons. J . Gen. M i c r o b i a l . 59: 145-152. Jones, J.G. 1970. The o r i g i n and d i s t r i b u t i o n of hydrocarbons i n an upland moorland s o i l and underlying shale. J . S o i l S c i . 21: 330-339. Karlsson, K.A., B.E. Samuelsson, and G.O. Steen. 1968. Structure and function of sphingolipids. 2. Differences i n sphin g o l i p i d concentration, e s p e c i a l l y concerning s u l f a t i d e s , between some regions of bovine kidney. Acta Chem. Scand. 22: 2723-2724. Karlsson, K.A., B_^E.+Samuelsson, and G.O. Steen. 1971. L i p i d pattern and Na -K -dependent adenosine triphosphatase a c t i v i t y i n the s a l t gland of duck before and a f t e r adaptation to hypertonic s a l i n e . J . Membrane B i o l . 5: 169-184. Kates, M. and T. Tonabene. 1972. Reference No. 117a. Cited by Haines, T.H. 1971. The chemistry of the s u l f o l i p i d s . In Progress i n the Chemistry of Fats and Other L i p i d s . R.T. Holman (Ed.), Pergamon Press, Oxford. V ol. XI, pp. 297-345. Kates, M., B. Palameta, and L.S. Yengoyan. 1965a. A l i p h a t i c diether analogs of glyceride-derived l i p i d s . I I . Synthesis of n a t u r a l l y occurring L-2,3-di-0-3',7',11',15'-tetramethyl-hexadecyl g l y c e r o l and i t s D isomer. Biochem. 4: 1595-1599. Kates, M., B. Palameta, M.P. Perry, and G.A. Adams. 1967. A new g l y c o l i p i d s u l f a t e ester i n Halobacterium cutirubrium. Biochim. Biophys. Acta 137: 213-216. Kates, M., M.K. Wassef, and D.J. Kushnev. 1968. Radioisotopic studies on the biosynthesis of the g l y c e r y l diether l i p i d s of Halobacterium cutirubrum. Can. J . Biochem. 46: 971-977. Kates, M., L.S. Yengoyan, and P.S. Sastry. 1965b. A diether analog of phosphatidyl glycerophosphate i n Halobacterium cutirubrum. Biochem. Biophys. Acta 98: 252-268. Kean, E.L. 1968. Rapid and s e n s i t i v e spectrophotometry method f o r quantitative determination of s u l f a t i d e s . J . L i p i d Res. 9: 319-327. - 54 -Kern, W. 1947. Uber des Vorkommen von Chrysen i n der Erde. Holv. Chim. Acta 30: 1595-1599. Kornblatt, M.J., H. Schachter and R.K. Murray. 1972. P a r t i a l c h a r a c t e r i z a t i o n of a novel g l y c e r o g a l a c t o l i p i d from rat t e s t i s . Biochim. Biophys. Res. Commun. 48: 1489-1494. Kowalenko, C.G. and R.B. McKercher. 1971a. Phospholipid P. content of Saskatchewan s o i l s . S o i l B i o l . Biochem. 3: 243-247. Kowalenko, C.G. and R.B. McKercher. 1971b. Phospholipid components extracted from Saskatchewan s o i l s . Can. J . S o i l S c i . 51: 19-22. Landsteiner, K. and P.A. Levene. 1925. Observations on the s p e c i f i c part of the heterogenic antigen. J . Immunol. 10: 731-733. Lees, M. 1956. Simple procedure f o r the i s o l a t i o n of brain s u l f a t i d e . Federation Proc. 15: 298. Lees, M., J . Folch, G.H. Stanley, and S. Carr. 1959. A simple procedure f o r the preparation of br a i n s u l f a t i d e s . J . Neurochem. 4: 9-18. Le i k o l a , E., E. Nieminen, and A.M. Teppo. 1969. New s i a l i c acid-containing s u l f o l i p i d : "unglic a c i d " . J . L i p i d Res. 10: 440-444. Lepage, M., H. Daniel, and A.A. Benson. 1961. The plant s u l f o l i p i d . I I . I s o l a t i o n and properties of s u l f o g l y c o s y l g l y c e r o l . J . Am. Chem. Soc. 83: 157-159. Lowe, L.E. and W.A. DeLong. 1961. Aspects of the s u l f u r status of three Quebec s o i l s . Can. J . S o i l S c i . 41: 141-146. Marshall, C L . and A.D. Brown. 1968. The membrane l i p i d s of Halobacterium  helobium. Biochem. J . 110: 441-448. Martensson, E. 1963a. Quantitative estimation of s u l f a t e i n l i p i d extracts. Biochim. Biophys. Acta 70: 1-8. Martensson, E. 1963b. On the s u l f a t e containing l i p i d s of human kidney. Acta Chem. Scand. 17: 1174-1176. Martensson, E. 1966. Sulfatides of human kidney: I s o l a t i o n , i d e n t i f i c a t i o n , and f a t t y acid composition. Biochim. Biophys. Acta 116: 521-531. Martin, J.P., J.O. Erv i n and R.A. Shepherd. 1959. Decomposition and aggregating e f f e c t s of fungus c e l l material i n s o i l . S o i l S c i . Soc. Am. Proc. 23: 217-220. Mayer, G.L. and T.H. Haines. 1967. A microbial s u l f o l i p i d . I I . Str u c t u r a l studies. Biochem. 6: 1665-1671. - 55 -Mayer, G.L., M. Pousada, and T.H. Haines. 1969. M i c r o b i a l s u l f o l i p i d s . ITT.' The d i s u l f a t e of (+)-l, 14-docosanedioI i h Ochromonas  danica. Biochem. 8: 2981-2986. McCandless, E.L. 1964. Determination of s u l f u r i n polysaccharides by neutron a c t i v a t i o n a n a l ysis. Anal. Biochem. 7: 357-365. McLean, J . , G.H. R e i t t i e and F.S. Spring. 1958. Triterpenoids from peat. Chem. Ind. (London) 1515-1516. Meinschein, W.G. and G.S. Kenny. 1957. Analysis of a chromatographic f r a c t i o n of organic extracts of s o i l s . Anal. Chem. 29: 1153-1161. Middlebrook, G. , CM. Coleman and W.B. Schaefer. 1959. S u l f o l i p i d from v i r u l e n t tubercle b a c i l l u s . Proc. N a t l . Acad. S c i . U.S. 45: 1801-1804. Miyachi, S., S. Miyachi and A.A. Benson. 1966. S u l f o l i p i d i n Ochromonas danica. J . Protozool. 13: 76-78. Miyano, M. and A.A. Benson. 1962a. The plant s u l f o l i p i d . VI. Configuration of the g l y c e r o l moiety. J . Am. Chem. Soc. 84: 57-59. Miyano, M. and A.A. Benson. 1962b. The plant s u l f o l i p i d . VII. Synthesis of 6-sulfo- -D-quinouopyranosyl-~(l- 1')-glycerol and radiochemical synthesis of s u l f o l i p i d s . J . Am. Chem. Soc. 84: 59-62. Morrison, R.I. 1969. S o i l l i p i d s . In Organic Geochemistry. Methods and Results. G. Eglinton and M.T.J. Murphy, (Eds.) Springer-Verlag, Berlin-Heidenberg. pp. 558-575. Morrison, R.I. and W. Bick. 1966. Long-chain methyl ketones i n s o i l s . Chem. Ind. (London) 596-597. Morrison, R.I. and W. Bick. 1967. The wax f r a c t i o n of s o i l s : separation and determination of some components. J . S c i . Fd. A g r i c . 18: 351-355. Mumma, R.O. and A.A. Benson. 1961. L i p i d chromatography on anion exchange paper. Biochem. Biophys. Res. Commun. 5: 422-423. Nagai, Y. and Y. Isono. 1965. Occurrence of animal s u l f o l i p i d i n the gametes of sea urchins. Japan. J . Exptl. Med. 35: 315-318. Nazir, D. and G. Rauser. 1966. Removal of water-soluble contaminants from l i p i d extracts of heart. L i p i d s 1:159. O'Brien, J.S. and A.A. Benson. 1964. I s o l a t i o n and f a t t y acid composition o f the plant s u l f o l i p i d and g a l a c t o l i p i d s . J . L i p i d Res. 5: 432-436. - 56 -O'Brien, J.S., D.L. F i l l e r u p , and J.F. Mead. 1964. Brain l i p i d s : I. Q u a n t i f i c a t i o n and f a t t y acid' composition of cerebroside s u l f a t e i n human cerebral gray and white matter. J . L i p i d Res. 5: 109-116. Oro, J . , D.W. Nooner, A. Z l a t k i s , S.A. Wikstrom, and E.S. Barghoorn. 1965. Hydrocarbons of" b i o l o g i c a l o r i g i n i n sediments about two b i l l i o n years o l d . Science 148: 77-79. Peat, S., J.R. Turvey, M.J. Clancy, and T.P. Williams. 1960. Sulphate of monosaccharides and t h e i r d e r i v a t i v e s . Part I. Preparation. J. Chem. Soc. 4761-4766. Parker, P.L. and R.F. Leo. 1965. Fatty acids i n blue-green a l g a l mat communities. Science 148: 373-374. Povoledo, D., D. Murray and M. P i t z e . 1972. Pigments and l i p i d s i n the humic acids of some Canadian lake sediments. Proc. Intern. Meet. Humic substances, Nieuwersluis, Pudoc, Wageningen. pp. 233-258. Radin, N.S., J.R. Brown and F.B. Lavin. 1965. The preparative i s o l a t i o n of cerebrosides. J . B i o l . Chem. 219: 977-983. Radunz, A. 1969. Uber das Sul f o c h i n o v o s y l - d i a c y l g l y c e r i n aus Hoheren Planzen, Algen und Purpurbakterien. Hoppe-seyler's. Z. Physiol. Chem. 350: 411-417. Rewald, B. 1944. The f a t s and phosphatides i n grass. O i l and Soap 21: 50-52. Roberts, R.B., P.H. Abelson, D.B. Cowie, E.T. Bolton, and R.J. B r i t t e n . 1955. Sulfur metabolism. In Studies of Biosynthesis i n Escherichia c o l i . Carnegie I n s t i t u t i o n of Washington Pu b l i c a t i o n No. 607, Washington, D.C. pp. 318-405. Robinson, G.A. 1960. A micromethod f o r the determination of sulphate by flame photometry. Can. J . Biochem. Ph y s i o l . 38: 643-648. Rovira, A.D. 1962. Plant-root exudates i n r e l a t i o n to the rhizosphere m i c r o f l o r a . S o i l s F e r t i l i z e r s 25: 167-172. Sallans, H.R., J.M. S n e l l , H.W. MaeKinney, and R.R. McKibbin. 1937. Water soluble acid substances i n the raw humus of podzol s o i l s . Schnitzer, M. 1975. Chemical, spectroscopic, and thermal methods f or the c l a s s i f i c a t i o n and cha r a c t e r i z a t i o n of humic substances. In Humic Substances, Their Structure and Function i n the . Biosphere. E.D. Povoledo and H.L. Golterman, (Eds.) Centre for A g r i c u l t u r a l Publishing and Documentation, Wageningen. pp. 293-310. - 57 -Schnitzer, M. and J.A. Neyroud. 1975. Alkanes and f a t t y acids i n humic substances. Fuel 54: 17-19. Schoniger, W. 1955. Eine mikroanalytische Schnellbestimmung von Halogen i n organischen Substanzeh. Mikrochim. Acta 1: 123-129. Schreiner, 0. andE.C. Shorey. 1908. The i s o l a t i o n of dihydroxystearic acid from s o i l s . J . Am. Chem. Soc. 30: 1599-1607. Schreiner, 0. and E.C. Shorey. 1909. The presence of c h o l e s t e r o l substances i n s o i l s : Agrosterol. J . Am. Chem. Soc. 31: 116-118. Schreiner, 0. and E.C. Shorey. 1910. Some acid constituents of s o i l humus. J . Am. Chem. Soc. 32: 1674-1680. Schreiner, 0. and E.C. Shorey. 1911a. Glycerides of f a t t y acids i n s o i l s . J . Am. Chem.-Soc. 33: 78-80. Schreiner, 0. and E.C. Shorey. 1911b. P a r a f f i n hydrocarbons i n s o i l s . J . Am. Chem. Soc. 33: 81-83. Schreiner, 0. and E.C. Shorey. 1911c. Cholesterol bodies i n s o i l s : phytosterol. J . B i o l . Chem. 9: 9-12. S c i a c o v e l l i , 0., N. Senes, V. Solinas, and C. T e s t i n i . 1977. Spectroscopic studies of s o i l organic f r a c t i o n s . I. IR and NMR spectra. S o i l B i o l . Biochem. 9: 287-293. Segal, S.N., M. Kates and N.E. Gibbons. 1962. L i p i d s of Halobacterium  cutirubrum. Can. J . Biochem. Ph y s i o l . 40: 69-81. Serra, J.M. and G.T. Felbeck, J r . 1972. Fr a c t i o n a t i o n and p a r t i a l analysis of the "bound" l i p i d f r a c t i o n of muck s o i l organic matter. Proc. Intern. Meet Humic Substs., Nieuwersluis, Pudoc, Wageningen. pp. 311-319. Shewan, J.M. 1938. The proximate analysis of the organic constituents i n Northeast S c o t t i s h s o i l s . J . Agr. S c i . 28: 324-340. Shibuya, I. and A.A. Benson. 1961. Hydrolysis of 6^-sulphoquinovosides by -galactosidase. Nature 192: 1186-1187. Simoneaux, B.J. and A.G. Caldwell. 1965. Phospholipids i n selected s o i l s . Agron. Abst., Annual Mtg. Amer. Soc. Agron. La, Agr i c . Ex. Sta. p. 77. Smith, L.M. and N.K. Freeman. 1959. Analysis of milk phospholipids by chromatography and i n f r a r e d spectrophotometry. J . Dairy S c i . 42:. 1450-1462. Stevens, N.P., E.E. Bray, and E.D. Evans. Hydrocarbons i n sediments of Gulf of Mexico. B u l l . Amer. Assoc. P e t r o l Geo. 40: 975-983. - 58 -Stevenson, F.J. 1966. Lipids in s o i l . J. Am. O i l Chemists' Soc. 43: 203-210. Stoffyn, P.J. 1966. The structure and chemistry of sulfatides. J. Am. Oil Chemists' Soc. 43: 69-74. Stoffyn, S. and A. Stoffyn. 1963. Structure of sulfatides. Biochim. Biophys. Acta 70: 218-220. Stoffyn, A. and Stoffyn, P.J. and E. Martensson. 1968. Structure of kidney ceramide dihexoside sulfate. Biochim. Biophys. Acta. 152: 353-357. Suzuki, A., I. Ishizuka, N. Ueta, and T. Yamakawa. 1973. Isolation and characterization of seminolipid (1-0-alkyl- -0-acyl-3- 3'-sulfogalactosyl glycerol) from guinea pig testis and incorporation of 35S-sulfate into seminolipid in sliced testis. Japan. J. Exptl. Med. 43: 435-442. Svennerholm, L. 1963. Chromatographic determination of sulfatides. Acta Chem. Scand. 17: 1170-1172. Svennerholm, E. and L. Svennerholm. 1963. The separation of neutral blood-serum glycolipids by thin-layer chromatography. Biochim. Biophys. Acta 70: 432-441. Svennerholm, L. and H. Thorin. 1962. Quantitative isolation of brain sulfatides. J. Lipid Res. 3: 483-485. Swan, E.P. 1965. Identity of a hydrocarbon found in a forest s o i l . Forest Product J. 15: 110. Taketomi, T. and T. Yamakawa. 1964. Further confirmation on the structure of brain cerebroside sulfuric ester. J. Biochem. 55: 87-89. Thannhauser, S.J., J. Felling, and G. Schmidt. 1955. The structure of cerebroside sulfur ester in beef brain. J. Biol. Chem. 215: 211-216. Thudichum, J.L.W. 1874. Researches on the chemical constitution of the brain. Rep. Med. Officer Privy Council. London (N.S.) 3, Append. 5, 134-247. Cited by T.H. Haines. Ann Rev. Microbiol. 27: 403-411 (1973). von Titow, N. 1932. Uber die Bitumina des Sphagnumtorfes. Brennstoff-Chem. 13: 266-269. Turfitt, G.E. 1943. The microbial degradation of sterols. I. The sterol content of soils. Biochem. 37: 115-117. - 59 -Vallentyne, J.R. 1957. The molecular nature of organic matter in lakes and oceans, with lesser reference to sewage and te r r e s t r i a l s o i l s . J. Fisheries*Res. Board Canada 14: 33-82. Wagner, G.H. and E.I. Muzorewa. 1977. Lipids of microbial origin in s o i l organic matter. In Soil Organic Matter Studies. Vol. II, pp. 99-104, Vienna, 1977. Proceedings of a symposium. Braunschweig, 6—10 Sept. 1976. Wagner, H., L. Horhammer, and P. Waeff. 1964. Dunnschichtchromatographie von Phosphatiden und Glycolipiden. Biochem. Zeitschrift 334: 175-184. Waksman, S.A. "and I.J. Hutching. 1935. Chemical nature of organic matter in different s o i l types. Soil Sci. 40: 347-363. Waksman, S.A. and K.R. Stevens. 1930. A c r i t i c a l study of the methods for determining the nature and abundance of s o i l organic matter. Soil Sci. 30: 97-116. Wang, T.S.C., Y.C. Liang, and W.C. Shen. 1969. Method of extraction and analysis of higher fatty acids and triglycerides in soi l s . Soil Sci. 107: 181-187. Wang, T.S.C, P.T. Hwang and C.Y. Chen. 1971. Soil l i p i d s under various crops. Soil Sci. Soc. Am. Proc. 35: 584-587. Weenink, R.O. 1963. Reaction of sulfoquinovose from red-clover l i p i d s with anthrone. Nature 197: 62-63. Wells, M.A. and J.C. Dittmer. 1965. A preparative method for the isolation of brain cerebroside, sulfatide and sphingomyelin. J. Chromatog. 18: 503-511. Wollrab, V. and M. Streibl. 1969. Earth waxes, peat, montan wax and other organic brown coal constituents. In Organic Geochemistry, Methods and Results. G. Eglinton and M.T.J. Murphy, (Eds.). Springer-Verlag, Berlin-Heidelberg, pp. 576-598. Yagi, T. and A.A. Benson. 1962. Plant sulfolipid. V. Lysosulfolipid formation. Biochim. Biophys. Acta 57: 601-603. Yamakawa, T., N. Kiso, S. Handa, A. Makita, and S. Yokoyama. 1962. On the structure of brain cerebroside sulfuric ester and ceramide dihexoside of erythrocytes. J. Biochem. 52: 226-227. - 60 -CHAPTER 3 THE DISTRIBUTION OF SULFUR IN LIPID EXTRACTS OF SOILS INTRODUCTION Although most of the sulfur in non-calcareous soils of humid regions usually occurs in organic combination, relatively l i t t l e is yet known of the exact chemical forms involved. A large number of organic sulfur compounds have been isolated from plants, animals and microorganisms (Freney, 1967), but few have so far been found in soils. The amino acids, cystine and methionine, have been isolated from s o i l , and.their derivatives, cysteic acid, methionine sulfoxide and methionine sulfone have been identified in s o i l hydrolysates. In two Australian soils, Freney et a l . (1972) reported that.amino acid sulfur accounted for 21 and 30% of the total organic sulfur. Approximately 60% of the amino acid sulfur was cystine sulfur. The remainder of the organic sulfur remains largely unknown, except to the extent that a major portion is thought to occur in ester sulfate form. Although the presence of sulfolipids in s o i l has not yet been reported, the presence of sulfolipids in a l l living matter (Haines, 1971 and 1973) indicates that a continual input to the s o i l system i s likely, and complexes with clay, protein and carbohydrates, or their presence in microbial tissue could contribute to an accumulation in the s o i l . However, i t is expected that only a small portion of s o i l - 61 -organic sulfur i s . l i k e l y to be present as sulfolipid since these forms are likely to be susceptible to rapid microbial degradation. There are no reports on the content of l i p i d sulfur (the organic sulfur present in the l i p i d extract) in s o i l at a l l and no other authors have related phospholipid ,P (li p i d P), contents with other s o i l characteristics, except for the report of Kowalenko and McKercher (1971b) on organic P. The purpose of this study was to determine the content of l i p i d sulfur in a range of soils of British Columbia and to relate these values to other s o i l characteristics, particularly with s o i l l i p i d contents as a whole and l i p i d phosphorus. METHODS AND MATERIALS Soils The thirty-seven s o i l horizon samples used in this study were from various places in the Province of British Columbia. Eighteen of the samples, representing three different horizon types, were forest soils. Nine of them were organic soils and ten of them were surface horizons of either virgin or agricultural grassland soils. Origin and gross chemical composition of the soils are given in Table 3.1. A l l samples were air dried at room temperature, crushed, and passed through a 2 mm sieve. For the chemical analyses the samples were further crushed and passed through a 100 mesh (0.149 mm) sieve. Table 3.1 Or i g i n and chemical c h a r a c t e r i s t i c s of s o i l samples (analysis expressed on oven dry basis). Dominant Vegetation ( L o c a t i o n / C l a s s i f i c a t i o n Horizon LI % Sitka spruce (Port Renfrew) F 85.4 ii II II H 81.7 Western hemlock (Jordan River) F 80.7 II II n H 82.5 Lodgepole (Manning Park) F 71.9 ti II H 52.5 Western red cedar (Victoria) F 61.7 Douglas-fir (Diamond Head) F 69.9 ii II H 83.3 Aspen (Beatton River) F 55.9 Hemlock-Cedar (Mt. Seymour) F 47.1 II II H 71.4 Western red cedar (Victoria) Ah 14.4 Aspen (Beaton River) Ah x 27.0 n II Ah, 9.48 Oak (Upland Park) Ah^ 23.1 Douglas-fir (Stamp F a l l s Park) Ah 1 14.3 Maple (Stamp F a l l s Park) Ah 16.7 Subalpine grass (Mt. Kobau) Ah 10.2 Grass (St.Ft. John/Solodic Black) Ah 11.4 II II II Ah 19.7 " /Eluv. Black) Ah 13.6 " (Oxbow/Orthic Black) Ah 4.53 " (Cloverdale/Humic Eluv. Gleysol) Ap 11.4 " (Delta/Saline Humic Gleysol) Ap 6.26 " (Langley/Hutnic Eluv. Gleysol) Ah 20.7 " (Prest/Orthic Humic Gleysol) Ah 9.03 " (Hazelwood/ " Ah 10.8 Grass/sedge (Kamloops/Mesisol) Om 55.8 n II Om 75.5 " (Lulu Island/Humisol) Oh 77.7 " (Metchosin/Humisol) Oh 51.4 " (Whipsaw Creek/Mesisol) Om 80.8 " ( " /Humisol) Oh 37.3 ( " /Mesisol) Om 65.7 ( " /Humisol) Oh 38.0 Subalpine grass (Church Mt.) Om 29.8 PH C Li p i d Total-S HI-S Lipid-S Total-P L i p i d - ! Ln H 20 % % ppm ppm ppm ppm ppm 3.7 57.5 2.57 2328 533 24.2 1099 25.5 3.6 54.8 2.22 2117 461 22.8 815 16.4 3.9 55.7 4.01 1750 469 37.5 1005 44.4 3.0 57.0 4.24 2016 579 37.5 637 15.9 4.6 50.5 5.33 936 200 14.8 862 22.3 4.7 33.8 5.24 887 187 30.8 921 22.6 5.5 42.9 3.92 1015 264 13.8 830 21.5 3.8 53.6 2.93 1063 207 14.7 683 17.3 3.5 63.5 3.01 1405 281 14.8 599 13.5 5.9 36.7 1.61 1726 337 16.6 1624 29.3 3.6 31.7 1.77 985 219 13.8 364 10.3 3.4 49.6 2.22 1354 364 11.0 402 8.15 6.1 7.74 0.333 375 144 5.33 709 2.77 5.6 16.2 0.610 704 217 7.13 1749 11.3 5.6 4.27 0.115 162 74.9 1.59 535 4.34 5.5 15.9 0.368 1219 394 7.04 1642 5.81 5.5 7.51 0.350 213 75.2 4.48 809 3.25 5.7 8.00 0.294 543 193 3.94 3668 8.84 5.9 5.28 0.289 542 252 . 4.17 1612 3.96 6.6 5.70 0.146 421 208 2.85 1308 4.76 5.9 11.8 0.357 858 374 7.81 1190 5.95 5.1 7.33 0.250 926 291 5.15 1415 4.27 8.5 1.92 0.061 286 142 2.50 578 1.45 5.5 5.47 0.239 684 361 9.65 1324 4.49 5.8 3.24 0.237 581 219 12.9 1458 2.83 5.2 12.0 0.268 928 564 6.54 1592 4.13 5.2 4.15 0.155 367 155 3.30 1240 2.95 5.2 4.78 0.278 510 259 9.19 1515 4.13 5.2 39.6 3.40 2330 772 39.0 1281 21.9 5.7 57.5 2.59 18321 5259 192.0 954 25.3 3.3 51.4 2.01 23053 7707 150.0 777 10.1 5.2 36.6 3.19 7193 1995 181.0 1849 7.10 5.0 55.3 3.32 3172 1597 68.3 583 26.1 3.7 28.1 1.71 30431 22758 291.0 1040 12.9 4.3 47.4 4.95 3716 1695 46.5 1817 10.9 4.1 31.7 1.43 14135 8477 146.0 1387 10.2 4.6 17.8 1.30 1122 397 25.4 1680 15.9 - 63 -Extraction Extraction of s o i l lipids was carried out by a modification of the method of Bligh and Dyer (1959) without any pretreatment. The Bligh and Dyer method was adapted in this study, since the merit of the method as examined by Kowalenko and McKercher (1970) rests in i t s ease of adaptability to bulk extraction for qualitative work and quantitative as well. The air dried soils were suspended in sufficient water to make the water content of the suspension 80 + 1%. One volume of the suspension was homogenized with three volumes of methanol-chloroform (2:1, v/v) in a Waring blender for 2 minutes. One volume of chloroform was then added while blending, and blending continued for a further 30 seconds.' Finally one volume of d i s t i l l e d water was added and blending continued for another 30 seconds. After f i l t e r i n g with Whatman No. 1 f i l t e r paper with slight pressure (the homogenate was centrifuged before f i l t r a t i o n , i f necessary, depending on the clay content of sample), the f i l t r a t e was transferred into a graduated cylinder and allowed to separate for 30 minutes to an hour. The volume of the chloroform layer was then recorded. After removing the alcoholic layer by aspiration, the chloroform layer, containing total lipids (Bligh and Dyer, 1959), was reduced to a known volume at 35-40°C in vacuo. The extraction of total li p i d s was repeated for each sample and the chloroform layer of each extract was combined u n t i l the sulfur content in the concentrated total li p i d s was sufficient to permit satisfactory analysis. The concentrated total li p i d s contained in the volumetric - 64 -flask were stored in a freezer and a portion of this solution was used for analysis. Analytical Methods Soil pH was determined with a Radiometer Model PHM 62 standard pH mater using a soil:water ratio of 1:5. Total organic carbon content was determined by the Walkley-Black wet-combustion method (Allison, 1965) . Samples of 0.02-0.3 g were used depending on the carbon content. Loss on ignition (LI) was. determined "by" igniting in a furnace at 450°C for three -hours. Total sulfur content was measured on the s o i l samples using the method outlined by Tabatabai and Bremner (1970) using the colorimetric determination described by Kowalenko and Lowe (1972). Hydriodic acid reducible sulfur (HI-S) was determined by the bismuth colorimetric finish as described by Kowalenko and Lowe (1972). Sulfur content in l i p i d extracts (lipid-S) was determined on an aliquot of the l i p i d extract (1 to 10 ml), which was taken to dryness under an IR lamp in a stream of nitrogen gas using the methods for total sulfur as described above. Total phosphorus was measured on the s o i l samples using the method of Dick and Tabatabai (1977). The procedure for oxidation of phosphorus was identical to that of total sulfur except for the temperature of the sand bath (260-280°C). - 65 -Phosphorus content in l i p i d extracts (lipid-P) was determined by the method of Dick and Tabatabai (1977) for total phosphorus after an aliquot of the l i p i d extract was taken to dryness as described for the determination of lipid-S. The determination of l i p i d contents followed the procedure of Bligh and Dyer (1959). The procedure was as follows: a portion of the l i p i d extract containing 100 to 200 mg l i p i d was transferred into a tared flask; the extract was evaporated to dryness under an IR lamp at 40-50°C under a stream of nitrogen; after drying over phosphoric anhydride in a vacuum -desiccator, the dried residue was weighed. RESULTS AND DISCUSSION Organic Carbon and Total Phosphorus The organic carbon contents for the thirty-seven soils f e l l in the range 1.92 to 63.5% with a mean of 29.0%, and the total phosphorus content varied from 364 to 3,688 ppm with a mean of 1,177 ppm (Table 2). The mean value of the total phosphorus content was considerably higher 1 than the means found by Walker and- Adams (1959) for New Zealand soils (488 ppm), Williams et a l . (1960) for Scottish soils (1,080) and Neptune jit a l . (1975) for Iowa (542 ppm) and Brazil (381 ppm) soils. This was. probably due to the relatively large number of organic samples included. - 66 -When the s o i l s were grouped into organic and mineral s o i l s as shown i n Table 3.2 the mean organic carbon contents of organic samples were, as expected, s i g n i f i c a n t l y higher than those of the mineral s o i l s , but the t o t a l phosphorus contents tended to be lower i n the organic s o i l s . When the s o i l s are grouped according-to vegetation, drainage and horizon type, the mean organic carbon contents of samples of poorly drained grass-sedge organic s o i l s and well drained forest organic horizons also appeared to be s i g n i f i c a n t l y higher than those of the mineral horizons of poorly and well drained grassland and of well drained f o r e s t , while the mean t o t a l phosphorus contents tended to be lowest i n the well drained forest organic horizons. The mean values for the rest of four groups were s i m i l a r . There was a close r e l a t i o n s h i p between the organic carbon and the pH, as i l l u s t r a t e d by the highly s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t (r = -0.687***) for a l l s o i l samples; there was no s i g n i f i c a n t c o r r e l a t i o n (r = 0.259) between the t o t a l phosphorus and pH values (Table 3.3). Other factors such as s o i l texture and differences i n a g r i c u l t u r a l and manurial p r a c t i c e were doubtless also involved and contributed to the v a r i a t i o n s within groups. The t o t a l phosphorus content was s i g n i f i c a n t l y correlated with organic carbon content (r = -0.371*), but only at the 5% l e v e l . This low c o r r e l a t i o n may indicate that inorganic phosphorus of s o i l can be a predominant component of the t o t a l phosphorus present. Table 3.2 t Relationship between organic carbon and t o t a l phosphorus contents and pH values among s o i l gtoups. S o i l Group Number of Samples PH (H20) Mean (Range) Organic C (%) Mean (Range) t o t a l P (ppm) Mean (Range) A l l samples 37 4.94 (3.03-8.49) 29.0 (1.92-63.5) 117"7 (364-3668) Organic (LH, Om and Oh) Mineral (Ah and Ap) 21 16 4.29 (3.03-5.85) 5.80 (5.10-8.49) 45.4 (17.8-63.5) 7.58 (1.92-16.2) 1010 (364-1849) 139(3 (535-3668) Well Drained Forest LH Poorly Drained Grassland Om/Oh Well Drained Forest Ah Well Drained Grassland Ah Poorly Drained Grassland Ah/Ap 12 9 6 5 5 4.09 (3.03-5.85) 4.57 (3.32-5.77) 5.67 (5.53-6.11) 6.39 (5.10-8.49) 5.35 (5.15-5.76) 48.9 (31.7-63.5) 40.6 (17.8-57.5) 9.94(4.27-16.2) 6.41(1.92-11.8) 5.93(3.24-12.0) 82b (364-1624) 126*3 (583-1849) 15lS (535-3668) 1221 (578-1612) 1426 (1240-1592) Table 3.3 Correlation matrix of some chemical characteristics of soil samples. pH C L TS HI-S LS TP LP LS/TS LS/L pH 1.000 C -0.687*** 1.000 L -0.528*** 0.827*** 1.000 TS -0.299 0.282 0.118 1.000 HI-S -0.272 0.137 0.044 0.924*** 1.000 LS -0.272 0.372* 0.233 . 0.935*** 0.873*** 1.000 TP 0.259 -0.371* -0.267 0.037 -0.045 -0.029 1.000 LP -0.367* 0.733*** 0.704*** 0.114 0.044 0.178 -0.152 1.000 LS/TS -0.129 0.165 0.486** -0.160 -0.153 0.050 -0.128 0.335* 1.000 LS/L 0.Q13 -0.091 -0.163 0.816*** 0.842*** 0.794*** 0.017 -0.135 -0.088 1.000 C = Organic Carbon; L = Lipid; TS = Total Sulfur; HI-S = Hi-reducible Sulfur; LS = Lipid Sulfur TP = Total Phosphorus; LP = Lipid Phosphorus; * = Significant (95%); ** = Highly Significant (99%) *** = Very Highly Significant (99.9%). - 69 -S u l f u r The t o t a l s u l f u r c o n t e n t s v a r i e d between 162 ppm and 30,431 ppm w i t h a mean o f 3,524 ppm ( T a b l e 3.4). The range f o r t h e t o t a l s u l f u r was v e r y w i d e and t h e mean v a l u e was s u b s t a n t i a l l y h i g h e r t h a n t h o s e r e p o r t e d by Rehm and C a l d w e l l (1968) f o r M i n n e s o t a s o i l s (501 ppm), T a b a t a b a i and Bremner (1972) f o r Iowa s o i l s (294 ppm), B e t t a n y e t a l . (1973) f o r Saskatchewan s o i l s (284 ppm), Neptune e t a l . (1975) f o r B r a z i l (166 ppm) and Iowa (319 ppm), and W i l l i a m s (1975) f o r N o r t h West P e m b r o k e n s h i r e s o i l s (710 ppm). S i n c e t h e t o t a l s u l f u r c o n t e n t s of s o i l s can v a r y o v e r an e x t r e m e l y w i d e range f r o m 0.002 t o 3.5% (Whitehead, 1964), t h e d i s a g r e e m e n t i n t h e v a l u e s i s not u n e x p e c t e d , c o n s i d e r i n g t h e p r o p o r t i o n o f h i g h l y o r g a n i c samples i n c l u d e d i n the s t u d y . As shown i n t h e T a b l e 3.4, t h e ran g e s of t h e t o t a l s u l f u r c o n t e n t s f o r w e l l d r a i n e d f o r e s t m i n e r a l h o r i z o n s and b o t h p o o r l y and w e l l d r a i n e d g r a s s l a n d m i n e r a l h o r i z o n s a r e s i m i l a r t o t h e v a l u e s d e t e r m i n e d by Rehm and C a l d w e l l (1968) f o r a g r i c u l t u r a l t o p s o i l s o f M i n n e s o t a (131 t o 940 ppm w i t h a mean o f 510 ppm). The t o t a l s u l f u r c o n t e n t s were g r e a t e r i n o r g a n i c h o r i z o n s t h a n i n m i n e r a l h o r i z o n s o f t h e same w e l l d r a i n e d f o r e s t s o i l s as shown i n T a b l e 3.4. The t r e n d s a r e i n agreement w i t h t h e r e p o r t by Lowe (1964) and Jones e t a ] - . (1972), and T a b a t a b a i and Bremner (1972) and Levesque (1974), r e s p e c t i v e l y . T h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the.mean t o t a l s u l f u r c o n t e n t s between w e l l and p o o r l y d r a i n e d g r a s s l a n d m i n e r a l h o r i z o n samples. T h i s r e s u l t i s i n c o n t r a s t t o t h e Table 3.4 Relationships of t o t a l s u l f u r with the Hi-reducible sulfur (HI-S) and carbon-bonded sul f u r (C-S) for s o i l groups. S o i l Group A l l Samples Organic (LH, Om and Oh) Mineral (Ah and Ap) Well Drained Forest LH Poorly Drained Grassland Om/Oh Well Drained Forest Ah Well Drained Grassland Ah Poorly Drained Grassland Ah/Ap Total S (ppm) Mean (Range) 3,524 (162-30,431) 5,765 (887-30,431) 582 (162-1,219) 1,465 (887-2,328) 11,497 (1,122-30,431) 536 (162-1,219) 607 (286-926) 614 (367-928) HI-S (ppm) Mean (Range) 1,587 (74.9-22,758) 2,609 (187-22,758) 245 (74.9-564) 345 (187-579) 5,629 (397-22,758) 183 (74.9-394) 253 (142-374) 312 (155-564) HI-S (% of t o t a l S) Mean (Range) 36.5 (19.5-74.8) 31.7 (19.5-74.8) 42.7 (30.8-60.8) 23.1 (19.5-28.7) 43.2 (27.7-74.8) 36.4 (30.8-38.4) 44.1 (31.4-49.7) 48.9 (37.7-60.8) C-S (% of t o t a l S) Meari (Range) 63.5 (25.2-80.5) 68.3 (25.2-80.5) 57.3 (39.2-69.2) 76.9 (71.3-80.5) 56.8 (25.2-72.3) 63.6 (53.8-69.2) 55.9 (50.3-68.6) 51.1 (39.2-62.3) - 71 -r e s u l t s o f Lowe ( 1 9 6 9 ) , who found c o n s i d e r a b l y h i g h e r l e v e l s o f t o t a l s u l f u r i n G l e y s o l i c p r o f i l e s t h a n i n Chernozems and P o d z o l s . However, i n each c a s e t o t a l S t e n d s t o i n c r e a s e w i t h o r g a n i c c a r b o n c o n t e n t ( T a b l e 3.2). The t o t a l s u l f u r c o n t e n t s o f t h e s o i l s were found t o be n o t s i g n i f i c a n t l y c o r r e l a t e d w i t h o r g a n i c c a r b o n ( r = 0.282), t o t a l phosphorus ( r = 0.037), o r pH ( r = -0 . 2 9 9 ) , as shown i n T a b l e 3.3. The poor c o r r e l a t i o n w i t h o r g a n i c c a r b o n i s c o n t r a s t t o t h e r e s u l t o f B e t t a n y e t a l . (1973) who found a v e r y h i g h c o r r e l a t i o n w i t h o r g a n i c c a r b o n ( r = 0.91***). The c l o s e r e l a t i o n s h i p i n d i c a t e s t h e dominance and i m p o r t a n c e o f t h e o r g a n i c s u l f u r i n t h o s e s o i l s . The poor c o r r e l a t i o n s f ound i n t h i s s t u d y were p r i m a r i l y due t o much more d i v e r s e samples, and p a r t i c u l a r l y due t o some samples from o r g a n i c s o i l s w h i c h have e x t r e m e l y h i g h s u l f u r c o n t e n t s b u t r e l a t i v e l y low o r g a n i c c a r b o n and t o t a l phosphorus c o n t e n t s . The H l - r e d u c i b l e s u l f u r f r a c t i o n o f s o i l s i n c l u d e s t h e i n o r g a n i c s u l f a t e p l u s a p a r t o f t h e o r g a n i c s u l f u r t h ought t o c o n s i s t o f o r g a n i c s u l f a t e ( F r e n e y , 1961). The H l - r e d u c i b l e s u l f u r c o n t e n t s ranged from 74.9 t o 22,758 ppm w i t h a mean of 1,587 ppm ( T a b l e 3.4). The H l - r e d u c i b l e s u l f u r c o n t e n t s were h i g h l y c o r r e l a t e d ( r = 0.924***) w i t h t h e t o t a l s u l f u r c o n t e n t s ( T a b l e 3.3) and c o n s e q u e n t l y gave a s i m i l a r r e l a t i o n s h i p s among s o i l groups ( T a b l e 3.4). When t h e H l -r e d u c i b l e s u l f u r c o n t e n t s were e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l s u l f u r , i t c a n be seen t h a t t h e mean v a l u e s o b t a i n e d f o r t h e o r g a n i c s o i l s (31.7%) were c o n s i d e r a b l y l o w e r t h a n t h o s e f o r m i n e r a l s o i l s ( 4 2 . 7 % ) . The mean v a l u e o b t a i n e d from t h e samples o f w e l l d r a i n e d - 72 -f o r e s t o r g a n i c h o r i z o n s (23.1%) were s i g n i f i c a n t l y l o w e r t h a n t h o s e from p o o r l y and w e l l d r a i n e d g r a s s l a n d (44.1% and 48.9%, r e s p e c t i v e l y ) and w e l l d r a i n e d f o r e s t (36.4%) m i n e r a l h o r i z o n s , and from o r g a n i c s o i l s ( 4 3 . 2 % ) . S i m i l a r t r e n d s but s l i g h t l y h i g h e r v a l u e s were r e p o r t e d f o r v i r g i n s o i l s o f A l b e r t a by Lowe (1965) who f o u n d t h a t 32.5% and 64.1% of t h e t o t a l s u l f u r i n Grey Wooded LH and C h e r n o z e r m i c Ah h o r i z o n s , r e s p e c t i v e l y , c o u l d be a c c o u n t e d f o r as H l - r e d u c i b l e s u l f u r . The v a l u e s r e p o r t e d f o r Saskatchewan s o i l s (50% f o r C h e r n o z e r m i c and 36% f o r Grey Wooded s o i l s ) by B e t t a n y ( 1 9 7 3 ) , and f o r B r a z i l (51.1%) and Iowa (54.5%), s o i l s by Neptune _et a l . (1975) a r e i n c l o s e r agreement t o t h o s e f o r w e l l and p o o r l y d r a i n e d g r a s s l a n d s o i l s . The p r o c e d u r e f o r t h e d i r e c t d e t e r m i n a t i o n of carbon^bonded s u l f u r (C-S) i n s o i l by Raney n i c k e l r e d u c t i o n i n t h e p r e s e n c e of a l k a l i (DeLong and Lowe, 1962),has been c r i t i c i z e d by F r e n e y e t a l . ( 1 9 7 0 ) . They s u g g e s t e d t h a t a b e t t e r e s t i m a t e o f carbon-bonded s u l f u r r e s u l t e d from t h e d i f f e r e n c e between t o t a l and H l - r e d u c i b l e s u l f u r . C o n s e q u e n t l y t h i s a n a l y s i s was n o t p e r f o r m e d . The mean v a l u e s f o r carbon-bonded s u l f u r o b t a i n e d by t h e d i f f e r e n c e as a p e r c e n t of t o t a l ; s u l f u r a r e shown i n T a b l e 3.4. The t r e n d s d e s c r i b e d f o r t h e H l -r e d u c i b l e s u l f u r a r e r e v e r s e d w i t h t h e s o i l s f rom o r g a n i c h o r i z o n s o f w e l l d r a i n e d f o r e s t samples c o n t a i n i n g a h i g h e r p e r c e n t a g e o f t o t a l s u l f u r as carbon-bonded s u l f u r . L i p i d and L i p i d P hosphorus The l i p i d c o n t e n t s o f s o i l s v a r i e d o v e r a v e r y w i d e r a n g e , - 73 -from 0.061 to 5.33% with a mean of 1.82%. (Table 3.5). The similar wide range for the values but with a very low mean (0.161% for surface mineral soils) were reported very recently by Siem jit a l . (1975) for North Vietnam soils. The authors pointed out that the l i p i d contents of North Vietnam soils were very low, about one-tenth that of the soils of USSR. Moreover, their values included the l i p i d contents of subsurface soils which, in general, contain l i t t l e l i p i d except in the case of humic podzols. Fridland (1976) reported that the contents of lipids in the Horizon of most soils ranged within. 1.4 to 0.06%, 0.5 to 0.2% being most common. As would be expected, the mean l i p i d contents (3.00%) for organic soils were much higher than those (0.271%) for mineral soils (Table 3.5). It also was seen that well drained forest mineral horizons had slightly higher mean l i p i d content than the corresponding poorly and well drained grassland mineral horizons. These values are in contrast to the values of Jones (1970) who found much higher l i p i d content in mineral horizon (A.,) samples (5.80% at around 20 cm depth and 3.72% at around 40 cm depth) than in organic horizon (A^) samples (0.76% at around 5 cm depth). However, the organic matter contents of mineral horizons (80% and 60% at 20 cm and 40 cm depths, respectively) and that of organic horizon (15% at 5 cm depth) show a similar trend in l i p i d content. When the l i p i d contents were expressed as a percentage of the organic matter (OM) contents (1.724 multiplied by organic carbon contents) as shown in Table 3.5, i t can be seen that the mean value obtained from Table 3.5 The distribution of l i p i d and lipid phosphorus in soils. Soil Group All Samples Organic (LH, Om and Oh) Mineral (Ah and Ap) Well Drained Forest LH Poorly Drained Grassland Om/Oh Well Drained Forest Ah Well Drained Grassland Ah Poorly Drained Grassland Ah/Ap Lipid (%) Mean (Range) 1.82 (0.061-5.33) 3.00 (1.30-5.33) 0.271(0.061-0.61) 3.26 (1.61-5.33) 2.26 (1.30-4.95) 0.345(0.115-0.610) 0.221 (0.061-0.357) 0.235 (0.155-0.278) Lipid (% of OM) Mean (Range) 3.22 (1.30-8.99) 3.95 (2.27-8.99) 2.27 (1.30-4.20) 4.01 (2.35-8.99) 3.87 (2.27-6.06) 2.07 (1.34-2.70) 2.05 (1.49-3.18) 2.71 (1.30-4.20) Lipid P (ppm) Mean (Range) 12.5 (1.45-44.4) 18.5 (7.10-44.4) 4.70 (1.45-11.3) 20.6 (8.15-44.4) 15.6 (7.10-26.1) 6.05 (2.77-11.3) 4.08 (1.45-5.95) 3.71 (2.83-4.49) Lipid P (% of total P) Mean (Range) 1.36 (0.194-4.48) 2.11 (0.334-4.48) 0.363(0.194-0.811) 2.53 (1.81-4.42) 1.56 (0.384-4.48) 0.474 (0.241-0.811) 0.333 (0.246-0.500) 0.261 (0.194-0.339) - 75 -the organic soils (3.95%) was considerably higher than that for mineral soils (2.27%). The mean value from the samples of well drained forest organic horizons (4.01%) was similar to the value of poorly drained grassland organic soils (3.87%), but was considerably higher than those of poorly and well drained grassland (2.05% and 2.71%) and well drained forest (2.07%) mineral horizons. Similar trends but somewhat higher values (5.45% for peat and 2.69% for mineral soils) were reported by Morrison and Bick (1967). In a review ar t i c l e , Stevenson (1966) also pointed out that the range for the l i p i d content of humus of most agriculturally important soils of world f e l l between 1.2 to 6.3%. The l i p i d contents up to 6.4% of the lake sediment humic acids have been observed by Povoledo^et ail. (1972). The l i p i d content of the soils was highly correlated with organic carbon (r = 0.827***) and pH of s o i l (r = -0.528***) for a l l samples as shown in Table 3.6. These results indicate that high l i p i d content w i l l be found in.the soils with a higher organic carbon content and a lower pH value as pointed out by Stevenson (1966) and Fridland (1976) in their review articles. Although i t is uncertain whether the high l i p i d content of organic matter in acidic soils results from inability of microorganisms to decompose completely the lipids occurring in plant remains, or larger quantities of lipids are synthesized by microorganisms (Stevenson, 1966), the maximum l i p i d content is believed to be confined to soils with low biological activity (Fridland, 1976). The l i p i d content was highly correlated with organic carbon content (r = 0.806***) only for mineral soils, when soils were grouped into two groups, i.e., organic and mineral groups. Similarly when Table 3.6 Correlation c o e f f i c i e n t s between l i p i d content and other s o i l properties. S o i l Group PH Organic C Total S HI-S L i p i d F A l l Samples Organic (LH, Om and Oh) Mineral (Ah and Ap) -0.528*** 0.125 -0.395 0.827*** 0.344 0.806*** 0.118 -0.388 0.439 0.044 -0.369 0.219 0.704*** 0.283 0.717** ON Well Drained Forest LH Poorly Drained Grassland Om/Oh Well Drained Forest Ah Well Drained Grassland Ah Poorly Drained Grassland Ah/Ap 0.127 0.346 -0.025 -0.811 0.035 0.071 0.536 0.820* 0.860 0.431 -0.350 -0.477 0.470 0.802 0.638 -0.213 -0.465 0.404 0.943* 0.608 0.287 0.119 0.620 0.800 0.652 - 77 -the samples were separated into five groups (Table 3.6), a significant correlation (r = 0.820*) between l i p i d and organic carbon was found only for well drained forest Ah horizons. Furthermore, l i p i d contents were not correlated with s o i l pH at a l l , when soils were grouped either into two or five groups. These results were, not expected and they deserve further investigation. The l i p i d contents were also not significantly correlated with either total or Hl-reducible sulfur contents except for HI-reducible sulfur content of well drained grassland Ah samples (r = 0.943*). These poor correlations are probably due to the organic soils which have very high total sulfur contents and consequently very high HI-reducible sulfur contents. Lipid phosphorus includes those phosphorus-containing materials which are components of lipids and consequently represents phospholipid. Hance and Anderson (1963b) provided evidence that the phosphorus in alkaline hydrolysis products of s o i l l i p i d phosphorus extracts occurred probably as phospholipid, primarily phosphatidyl choline. Kowalenko and McKercher (1971a) also identified phosphatidyl choline and phosphatidyl ethanolamine as the major components. Dormaar (1970) and Simoneaux and Caldwell (1965) also showed the presence of these phospholipids. Anderson and Malcom (1974) provided evidence of the possible presence of phosphoinositide lipids in a s o i l extract. Lipid phosphorus represents a small percentage of the total phosphorus in soils. The l i p i d phosphorus contents were between 1.45 and 44.4 ppm with a mean of 12.5 ppm (Table 3.5). The range was also wide and the mean value was considerably higher than the values - 78 -reported by Hance and Anderson (1963a) for soils of Great Britain ranging from 3.1 to 7.0 ppm with a mean of 4.9 ppm; by Simoneaux and Caldwell (1965) for U.S.A. soils ranging from 0.24 to 1.70 ppm; by Dormaar (1970) for Alberta soils from 0.32 to 1.30 ppm with a mean of 7.32 ppm; by Kowalenko and McKercher (1971b) for Saskatchewan soils from 0.6 to 14.5 ppm with a mean of 3.4 ppm. The l i p i d phosphorus is significantly correlated with the organic carbon content (r = 0.733***) and l i p i d content (r = 0.704***) as shown in Table 3.3 and Table 3.6 Therefore the trends w i l l be similar to l i p i d content distribution of s o i l groups (Table 3.5). The high correlation of the l i p i d phosphorus with the organic carbon content and the poor correlation with total phosphorus were in agreement with the result of Kowalenko and McKercher (1971b). Although the l i p i d phosphorus content was highly correlated with the l i p i d content, when a l l samples were considered, there was no significant correlation between l i p i d phosphorus and l i p i d content when soils were classed into five different groups on the basis of vegetation and drainage condition. However, there was significant correlation between them (r = 0.717**) for mineral soils alone, excluding organic so i l s . The poor correlation for organic soils is likely to be due to the poor correlation between l i p i d and organic carbon content, which is d i f f i c u l t to interpret : as described previously. - 79 -The D i s t r i b u t i o n of t h e L i p i d S u l f u r i n S o i l s The l i p i d s u l f u r i n c l u d e s any s u l f u r w h i c h i s a s s o c i a t e d w i t h l i p i d components i n s o i l s and may t h e r e f o r e i n c l u d e a v a r i e t y of f o r m s , such as s u l f a t e e s t e r , s u l f o n i c e s t e r and t h i o l s u l f u r . Not much i s known about t h i s l i p i d s u l f u r f r a c t i o n i n s o i l s . Kowalenko (1973) f o u n d t h a t s u l f u r i n o x i d i z e d ( H i - r e d u c i b l e ) and r e d u c e d (carbon-bonded) form were p r e s e n t i n c h l o r o f o r m e x t r a c t s of two o r g a n i c s o i l s u s i n g t h e B l i g h and Dyer (1959) method and t h e s e two forms o f s u l f u r r e p r e s e n t e d 0.3 and 0.6% of t h e t o t a l s u l f u r . The l i p i d s u l f u r c o n t e n t s of t h e t h i r t y - s e v e n samples had an e x t r e m e l y w i d e r a n g e , f r o m 1.59 t o 291 ppm w i t h a mean of 40.1 ppm ( T a b l e 3.7). There was a s i g n i f i c a n t d i f f e r e n c e i n t h e c o n t e n t s of l i p i d s u l f u r between o r g a n i c and m i n e r a l s o i l s . When r e l a t i n g t h e l i p i d s u l f u r c o n t e n t s t o h o r i z o n t y p e s , t h e p o o r l y d r a i n e d m i n e r a l h o r i z o n s of g r a s s l a n d had s l i g h t l y h i g h e r mean v a l u e t h a n t h e c o r r e s p o n d i n g h o r i z o n s o f more f r e e l y d r a i n e d g r a s s l a n d samples. There d i d not seem t o be a c l e a r - c u t d i s t r i b u t i o n o f t h e l i p i d s u l f u r between s o i l s of f o r e s t and g r a s s l a n d samples f o r t h e same m i n e r a l h o r i z o n s . The d i f f e r e n c e was s m a l l and t h e r a n g e s and s t a n d a r d d e v i a t i o n s were l a r g e . However, i t c a n be s a i d t h a t t h e l i p i d s u l f u r c o n t e n t s i n m i n e r a l h o r i z o n s of f o r e s t samples a r e l o w e r t h a n t h o s e i n w e l l d r a i n e d f o r e s t o r g a n i c h o r i z o n s , and t h e l i p i d s u l f u r i s c o n c e n t r a t e d i n o r g a n i c s o i l s . T a b l e 3.7 a l s o shows t h e d i s t r i b u t i o n of t h e l i p i d s u l f u r c o n t e n t s e x p r e s s e d as p e r c e n t a g e o f t o t a l s u l f u r . I n c o n t r a s t t o t h e l i p i d s u l f u r c o n t e n t s measured i n ppm, t h e p e r c e n t . l i p i d s u l f u r d i d n o t Table 3.7 The d i s t r i b u t i o n of l i p i d sulfur i n s o i l s . S o i l Group L i p i d S (ppm) Mean + SD a (Range) L i p i d S (% of t o t a l S) Mean (Range) L i p i d S (ppm of l i p i d ) Mean (Range) A l l Samples 40.1 + 65.5 (1.59-291) 1.33 (0.556-3.47) 2,455 (278-17,018) Organic (LH, Om and Oh) Mineral (Ah and Ap) 66.3 + 77.3 (11.0-291) 5.85 + 3.02(1.59-12.9) 1.51 (0.651-3.47) 1.10 (0.556-2.22) 2,523 (278-17,018) 2,364 (1,169-5,489) Well Drained Forest LH Poorly Drained Grassland Om/Oh Well Drained Forest Ah Well Drained Grassland Ah Poorly Drained Grassland Ah/Ap 21.0 + 9.50 (11.0-37.5) 127 + 88.7 (25.4-291) 4.92 + 2.09(1.59-7.13) 4.50 + 2.13(2.50-7.81) 8.32 + 3.60(3.30-12.9) 1.51 (0.813-3.47) 1.51 (0.651-2.52) 1.14 (0.578-2.10) 0.757 (0.556-0.910) 1.41 (0.704-2.22) 692 (278-1,031) 4,965 (939-17,018) 1,448 (1,169-1,913) 2,348 (1,443-4,098) 3,481 (2,129-5,489) SD = Standard Deviation - 81 -show a significant difference between organic and mineral soils. Furthermore, the mean values for well drained forest organic horizons and organic soils were identical. The l i p i d sulfur contents of the well drained forest and poorly drained grassland mineral horizons were significantly higher than the values for well drained grassland mineral horizons. The low values for well drained grassland samples were probably due to the higher valuers for to t a l sulfur content but lower l i p i d sulfur contents. There were highly significant correlations between l i p i d sulfur and total sulfur (r = 0.935***) and Hl-reducible sulfur (r = 0.873***), but the l i p i d sulfur correlated with organic carbon (0.327*) significantly only at the 5% level (Table 3.8). On the other hand, the relative l i p i d sulfur content was significantly correlated only with l i p i d content (r = 0.486**) when the l i p i d sulfur was expressed as per cent of total sulfur, and also was significantly correlated only with total sulfur (r = 0.816***) when the l i p i d sulfur was expressed as ppm of l i p i d (Table 3.9). The l i p i d sulfur contents of mineral soils were not significantly correlated with any other s o i l factors whether expressed as ppm of s o i l , as % of total sulfur or as ppm of l i p i d ; However, the l i p i d sulfur contents of organic soils were highly significantly correlated with total sulfur and Hl-reducible sulfur when expressed as ppm of s o i l , and only with total sulfur when expressed as ppm of l i p i d , but significantly only at the 5% level with l i p i d contents when expressed as % of total sulfur (Table 3.8 and 3.9). The correlationships were not consistent with those of a l l samples and organic or mineral samples when Table 3.8 Correlation c o e f f i c i e n t s for l i p i d sulfur versus some s o i l properties. S o i l Group pH Organic C Correlation C o e f f i c i e n t ; L i p i d Sulfur versus L i p i d Total S HI-S L i p i d P A l l Samples -0.272 0.327* 0.233 0.935*** 0.873*** 0.178 Organic (LH, Om and Oh) Mineral (Ah and Ap) 0.090 -0.339 -0.232 0.180 -0.303 0.382 0.925*** 0.468 0.871*** 0.458 -0.252 0.085 Well Drained Forest LH • -0.243 Poorly Drained Grassland Om/Oh -0.215 Well Drained Forest Ah 0.047 Well Drained Grassland Ah -0.624 Poorly Drained Grassland Ah/Ap 0.808 0.204 -0.010 0.922** 0.943* -0.314 0.438 -0.381 0.870* 0.888* 0.554 0.468 0.885** 0.768 0.850 0.176 0.585* 0.836** 0.729 0.970** -0.029 0.543 -0.262 0.396 0.744 0.040 Table 3.9 Correlation coefficients for Lipid S/Total S and Lipid S/Lipid versus some soil properties. Correlation Coefficient; Lipid S as % of Total S Correlation Coefficient; Lipid S as ppm of l i p i d Soil Group pH Organic C Lipid Total S pH Organic C Lipid Total S A l l Samples -0.129 0.165 0.486** -0.160 0.013 -0.091 -0.163 0.816**' Organic (LH, Om and Oh) 0.252 -0.279 0.507* -0.379 0.003 -0.238 -0.332 0.905**' Mineral (Ah and Ap) -0.118 -0.328 0.096 -0.396 0.264 -0.470 -0.390 0.035 Well Drained Forest LH -0.105 -0.331 0.731** -0.332 -0.156 0.022 -0.476 0.790** Poorly Drained Grassland Om/Oh 0.531 -0.273 0.184 -0.743* -0.314 -0.085 -0.359 0.862** Well Drained Forest Ah 0.163 -0.396 0.028 -0.651 0.157 0.253 -0.212 0.616 Well Drained Grassland Ah 0.546 0.076 0.045 -0.306 0.860 -0.524 -0.715 -0.498 Poorly Drained Grassland Ah/Ap 0.695 -0.686 0.285 -0.309 0.937* -0.497 0.262 -0.002 - 84 -samples are grouped into five different horizon types. If a l l correlations had been significant i t would have been inferred that a l l those factors show a linear mathematical relationship with the distribution of l i p i d sulfur. On the contrary the simple regression analysis did not lead to such a conclusion. This provides evidence that the chemistry of l i p i d sulfur in s o i l is complex and hence most of the factors affecting l i p i d sulfur equilibrium cannot be isolated and discussed without consideration of the other factors, since they are a l l interrelated. For example, the fact that there was no significant correlation between l i p i d sulfur as ppm of s o i l and l i p i d contents,while l i p i d sulfur as % of total sulfur was highly correlated with l i p i d content,means that a l l s o i l factors must be considered simultaneously in an examination of such relationships. A stepwise elimination multiple regression analysis was employed to find which of the s o i l factors were the best predictors of the l i p i d sulfur. The output of the computer analysis is summarized in Table 3.10. The stepwise elimination analysis of the thirty-seven samples showed 2 that the R of the relationship between the l i p i d sulfur as ppm of s o i l and a l l s o i l factors combined were 0.904. The dependence of the l i p i d sulfur values on l i p i d and total sulfur values is shown in the 2 equation representing the f i n a l stage and the R value was 0.889. 2 Although the R value was slightly decreased, as much as 89% of the variation in the l i p i d .sulfur could be explained by only these two factors. Thus the l i p i d content which had not been correlated significantly with l i p i d sulfur as ppm of s o i l on simple regression analysis, now turned out to be one of the best predictors of l i p i d 2 Table 3.10 Regression equations and coefficient of determination (R ) for relationships between li p i d sulfur as ppm of s o i l , as % of total sulfur, and as ppm of lipid and soil factors of samples of some British Columbian soils. Regression Equation 100 x R (%) Lipid S (ppm of soil) Lipid S (ppm of soil) Lipid S (% of total S) -51.9 + 7.15 pH + 0.224 C + 7.72 L + 0.00753 TS + 0.00280 HI-S + 0.00820 TP - 0.354 LP -0.191 + 4.931 L + 0.00890 TS 2.27 - 0.140 pll - 0.0352 C + 0.402 L - 0.0000205 HI-S - 0.000185 TP + 0.0233 LP 90.4 88.9 49.9 oo Lipid S (% of total S) = 1.21 - 0.0203 C + 0.435 L Lipid S (ppm of lipid) = -58.5 + 453 pH - 24.9 C + 0.230 TS + 0.334 HI-S - 0.188TP - 9.90 LP Lipid S-(ppm of lipid) = 2459 - 50.5 C + 0.415 TS 41.6 80.7 77.8 First and second equations for l i p i d S represent the i n i t i a l and final stages, respectively of the stepwise elimination procedures; TS = Total Sulfur; HI-S = Hl-reducible Sulfur; TP - Total Phosphorus; C = Organic Carbon; L = Lipid; LP = Lipid Phosphorus; LS = Lipid Sulfur. - 86 -sul f u r along with t o t a l s u l f u r . 2 Table 3.10 also shows that the R of regression of the l i p i d s u l f u r as % of t o t a l s u l f u r values on a l l s o i l factors combined was 2 only 0.49 and the R values of regression of the l i p i d s u l f u r on a l l s o i l factors combined and on organic carbon and t o t a l s u l f u r were 0.807 and 0.778 re s p e c t i v e l y , when the l i p i d s u l f u r was expressed as ppm of l i p i d . These r e s u l t s indicate that organic carbon contents of s o i l s could be one of the best predictors whether l i p i d s u lfur contents were expressed as % of t o t a l s u l f u r or as ppm of l i p i d contents of s o i l s . In view of the number of factors included and the lim i t e d number of samples i n each s o i l group," i t might not be useful to a r r i v e at mathematical r e l a t i o n s h i p s through s t a t i s t i c a l procedures. Moreover, such r e l a t i o n s h i p s would have l i m i t e d v a l i d i t y to the understanding of the chemical and b i o l o g i c a l transformations or o v e r a l l turnover of s u l f u r i n s o i l s . CONCLUSION L i p i d s u l f u r was found i n a l l s o i l s examined and the amount was very v a r i a b l e . The values varied from as low as 1.59 ppm found i n an Ah horizon of a forest s o i l to nearly two hundred times the amount (291 ppm) found i n a humisol sample. When s o i l s are grouped according to the horizon types, the l i p i d s u l f u r contents were higher i n the organic horizons than i n the mineral horizons, and poorly drained s o i l samples had higher l i p i d s u l f u r than f r e e l y drained s o i l samples. The - 87 -l i p i d s u l f u r was c o n c e n t r a t e d i n p o o r l y d r a i n e d g r a s s l a n d o r g a n i c s o i l s . The l i p i d s u l f u r a c c o u n t e d f o r s m a l l p e r c e n t a g e o f t o t a l s u l f u r r a n g i n g from 0.56% found i n an Ah h o r i z o n o f E l u v i a t e d B l a c k Chernozem t o 3.47% found i n an H h o r i z o n o f f o r e s t s o i l . The t r e n d s f o r t h e d i s t r i b u t i o n o f l i p i d s u l f u r e x p r e s s e d as p e r c e n t a g e of t o t a l s u l f u r were s i m i l a r t o t h o s e e x p r e s s e d as ppm of s o i l e x c e p t f o r o r g a n i c h o r i z o n s o f p o o r l y d r a i n e d g r a s s l a n d and w e l l d r a i n e d f o r e s t s o i l s . The l i p i d s u l f u r c o n t e n t s o f p o o r l y d r a i n e d g r a s s l a n d o r g a n i c and w e l l d r a i n e d f o r e s t o r g a n i c h o r i z o n s were i d e n t i c a l . The l i p i d s u l f u r a l s o a c c o u n t e d f o r a s m a l l p a r t o f t h e t o t a l l i p i d c o n t e n t o f s o i l , r a n g i n g from 278 ppm found i n a F h o r i z o n of a f o r e s t s o i l t o 17,018 ppm found i n a h u m i s o l . O r g a n i c h o r i z o n s had s l i g h t l y h i g h e r l i p i d s u l f u r t h a n m i n e r a l h o r i z o n s . The p o o r l y d r a i n e d g r a s s l a n d s o i l s had s i g n i f i c a n t l y h i g h e r (around seven t i m e s ) l i p i d s u l f u r c o n t e n t t h a n t h e w e l l d r a i n e d f o r e s t o r g a n i c h o r i z o n s . F o r m i n e r a l h o r i z o n s , t h e p o o r l y and w e l l d r a i n e d g r a s s l a n d m i n e r a l h o r i z o n s had n e a r l y t w i c e as much l i p i d s u l f u r as t h e w e l l d r a i n e d f o r e s t m i n e r a l h o r i z o n s . The l o w e r l i p i d s u l f u r c o n t e n t s i n w e l l d r a i n e d f o r e s t m i n e r a l h o r i z o n s as ppm of t o t a l c o n t e n t s was due t o t h e lo w e r l i p i d s u l f u r b u t t h e h i g h e r l i p i d c o n t e n t s i n t h e s e samples. L i p i d phosphorus was a l s o f ound i n a l l s o i l s examined. The o v e r a l l l i p i d s u l f u r c o n t e n t s were n e a r l y t h r e e t i m e s h i g h e r t h a n t h e o v e r a l l l i p i d phosphorus c o n t e n t s of s o i l s . I n o r g a n i c h o r i z o n s , t h e l i p i d s u l f u r c o n t e n t s were a l s o around t h r e e t i m e s h i g h e r t h a n t h e l i p i d phosphorus c o n t e n t s w h i l e . t h e v a l u e s were about t h e same i n - 88 -mineral horizons. Both l i p i d sulfur and l i p i d phosphorus contents were higher in organic horizons than in mineral horizons. The l i p i d sulfur contents were around eight times higher than the l i p i d phosphorus in poorly drained grassland organic horizons, but slightly over twice as high in poorly drained grassland Ah horizons, while the l i p i d sulfur contents were about the same as the l i p i d phosphorus contents in well drained forest organic and grassland Ah horizons but slightly lower in well drained forest Ah horizons. For overall samples, the l i p i d sulfur was highly significantly correlated only with total and Hl-reducible sulfur but also correlated with organic carbon significantly only at 5% level. The l i p i d sulfur as % of total sulfur was correlated significantly only with l i p i d . However, the l i p i d sulfur as ppm of l i p i d was significantly correlated only with total sulfur. The distribution of l i p i d sulfur in soils can be best explained . (89% of the variation in the l i p i d sulfur content) by two so i l factors, total l i p i d content and total sulfur content when the l i p i d sulfur was expressed as ppm of s o i l . When the l i p i d sulfur was expressed as % of total sulfur, the distribution of the l i p i d sulfur can be explained by organic carbon and total l i p i d contents • but only 42% of the variation in the l i p i d sulfur content can be explained. On the other hand, as much as 78% of the variation in the l i p i d content can be explained by organic carbon and total sulfur when the l i p i d sulfur was expressed as ppm of l i p i d . It is therefore imperative to use a suitable expression for the l i p i d sulfur distribution, so that the s o i l factors can be chosen accordingly. - 89 -REFERENCES Allison, L.E. 1965. Organic Carbon. In Methods of Soil Analysis. C A . Black, (Ed.) Amer. Soc. Agron. Monograph No. 9. Madison, Wisconsin. Part II, pp. 1367-1378. Anderson, G. and R.E. Malcolm. 1974. The nature of alkali-soluble s o i l organic phosphates. J. Soil Sci. 25: 282-297. Bettany, J.R., J.W.B. Stewart, and E.H. Halsted. 1973. Sulfur fractions and carbon, nitrogen, and sulfur relationships in grassland, forest, and associated transitional s o i l s . Soil Sci. Soc. Am. Proc. 37: 915-918. Bligh, E.G. and W.J. Dyer. 1959. A rapid method of total l i p i d extraction and purification. Can. J. Biochem. Physiol. 37: 911-917. DeLong, W.A. and L.E. Lowe. 1962. Note on carbon-bonded sulfur i n s o i l . Can. J. Soil Sci. 42: 223.' Dick, W.A. and M.A. Tabatabai. 1977. An alkaline oxidation method for determination of total phosphorus in soi l s . Soil Sci. Soc. Am. J. 41: 511-514. Dormaar, J.F. 1970. Phospholipids in chernozemic soils of southern Alberta. Soil Sci. 110: 136-139. Freney, J.R. 1967. Sulfur-containing organics. In Soil Biochemistry. A.D. McLaren and G.H. Peterson (Eds.) Marcel Dekker, Inc., New York. Part II, pp. 229-259. Freney, J.R., G.E. Melville, and CH. Williams. 1970. The determination of carbon-bonded sulfur in s o i l . Soil Sci. 109: 310-318. Freney, J.R., F.J. Stevenson, and A.H. Beavers. 1972. Sulfur-containing amino acids in s o i l hydrolysates. Soil Sci. 114: 468-476. Fridland, Ye. V. 1976. Lipid (alcohol-benzene) fraction of organic matter in different s o i l groups. (Soviet Soil Sci. 8: 548-557. Haines, T.H. 1971. The chemistry of the sulfolipids. In Progress in the Chemistry of Fats and Other Lipids. R.T. Holman, (Ed.) Pergamon Press, Oxford. Vol. XI, Part 3, pp. 297-345. Haines, T.H. 1973. Sulfolipids and halosulfolipids. In Lipids and Bio-membranes of Eukaryotic Microorganisms. J. Erwin, (Ed.) Academic Press, New York. pp. 197-232. - 90 -Hance, R.J. and G. Anderson. 1963a. Extraction and estimation of s o i l phospholipids. Soil Sci. 96: 94-98. Hance, R.J. and G. Anderson. 1963b. Identification of hydrolysis products of s o i l phospholipids. Soil Sci. 96: 157-161. Jones, J.G. 1970. The origin and distribution of hydrocarbons in an upland moorland s o i l and underlying shale. J. Soil Sci. 21: 330-339. Jones, L.H.P., D.W. Cowling, and D.R. Lockyer. 1972. Plant available and extractable sulfur i n some soils of England and Wales. Soil Sci. 114: 104-114. Kowalenko, C.G. 1973. Mineralization of s o i l sulfur and i t s relation to s o i l carbon, nitrogen and phosphorus. Ph.D. Thesis, University of British Columbia. Kowalenko, C.G. and L.E. Lowe. 1972. Observations on the bismuth sulfide colorimetric procedure for sulfate analysis in s o i l . Comm. Soil Sci. Plant Anal. 3: 79-86. Kowalenko, C.G. and R.B. McKercher. 1970. An examination of methods for extraction of s o i l phospholipids. Soil Biol. Biochem. 2: 269-273. Kowalenko, C.G. and R.B. McKercher. 1971a. Phospholipid components extracted from Saskatchewan soils. Can. J. Soil Sci. 51: 19-22. Kowalenko, C.G. and R.B. McKercher. 1971b. Phospholipid P content of Saskatchewan soils. Soil Biol. Biochem. 3: 243-247. Levesque, M. 1974. Relationship of sulfur and selenium in some Canadian s o i l profiles. Can. J. Soil Sci. 54: 333-335. Lowe, L.E. 1964. An approach to the study of the sulfur status of soils and i t s application to selected Quebec soils. Can. J. Soil Sci. 44: 176-179. Lowe, L.E. 1965. Sulphur fractions of selected Alberta s o i l profiles of the Chernozemic and Podzolic orders. Can. J. Soil Sci. 45: 297-303. Lowe, L.E. 1969. Sulfur fractions of selected Alberta profiles of the Gleysolic order. Can. J. Soil Sci. 49: 375-381. Morrison, R.I. and W. Bick. 1967. The wax fraction of s o i l s : Separation and determination of some components. J. Sci. Fd. Agric. 18: 351-355. - 91 -. Neptune, A.M.L., M.A. Tabatabai, and J.J. Hanway. 1975. Sulfur fractions and carbon-nitrogen-phosphorus-sulfur relationships in some Brazilian and Xowa soils. Soil Sci. Soc. Amer. Proc. 39: 51-55. Povoledo, D., D. Murray and M. Pitze. 1972. Pigments and lipids in the humic acids of some Canadian lake sediments. Proc. Int. Meet. Humic Substances, Nieuwersluis, Pudoc, Wageningen. pp.233-258. Rehm, G.W. and A.C. Caldwell. 1968. Sulfur supplying capacity of soils and the relationship to s o i l type. Soil Sci. 105: 355-361. Siem, N.T., D.S. Orlov, and Ya. M. Ammosova. 1975. A study of alcohol-benzene extracts of the principal soils of North Vietnam. Moscow University Soil Science Bulletin 30: 9-12. Simoneaux, B.J. and A.G. Caldwell. 1965. Phospholipids in selected soils. Agron. Abstr., Annual Mtg. Amer. Soc. Agron. p. 77. Stevenson, F.J. 1966. Lipids in s o i l . J. Am. Oil Chemist's Soc. 43: 203-210. Tabatabai, M.A. and J.M. Bremner. 1970. An alkaline oxidation method for determination of total sulfur in soils. Soil Sci. Soc. Am. Proc. 34: 62-65. Tabatabai, M.A. and J.M. Bremner. 1972. Distribution of total and available sulfur in selected soils and s o i l profiles. Agron. J. 64: 40-44. Wagner, G.H. and E.I. Muzorewa. 1977. Lipids of microbial origin in s o i l organic matter. In Soil Organic Matter Studies. Proceedings of a Symposium, Braunschweig, 6-10 Sept. 1976. Jointly Organized IAEA and FAO in Cooperation with Agrochimica. Walker, T.W. and A.F.R. Adams. 1959. Studies on s o i l organic matter: 2. Influence of increased leaching at various stages of weathering on levels of carbon, nitrogen, sulfur, and organic and total phosphorus. Soil Sci. 87: 1-10. Whitehead, D.C. 1964. Soil and plant-nutrition aspects of sulfur cycle. Soil Fert. 27: 1-8. Williams, C. 1975. The distribution of sulfur in the soils and herbage of North West Pembrokeshire. J. Agric. Sci., Camb. 84: 445-452. Williams, C.H., E.G. Williams and N.M. Scott. 1960. Carbon, nitrogen, sulphur, and phosphorus in some Scottish s o i l . J. Soil Sci. 11: 334-346. - 92 -CHAPTER 4 THE COLUMN CHROMATOGRAPHIC FRACTIONATION OF TOTAL LIPIDS AND L I P I D SULFUR IN SOME SELECTED BRITISH COLUMBIAN SOILS INTRODUCTION A p r e v i o u s r e p o r t ( C h a p t e r 3) e s t a b l i s h e d t h e i m p o r t a n c e o f u n d e r s t a n d i n g t h e d i s t r i b u t i o n o f l i p i d s u l f u r i n r e l a t i o n t o t h e d i f f e r e n c e i n t h e dominant v e g e t a t i o n , d r a i n a g e c o n d i t i o n s , and h o r i z o n t y p e s . I t was shown t h a t l i p i d s u l f u r was found i n a l l s o i l s examined a l t h o u g h t h e v a l u e s were v e r y v a r i a b l e . I t was a l s o found t h a t t h e s o i l l i p i d s u l f u r , c o m p r i s e d o n l y a s m a l l p o r t i o n of s o i l t o t a l s u l f u r and was most c o n c e n t r a t e d i n t h e p o o r l y d r a i n e d g r a s s l a n d o r g a n i c s o i l s . No r e p o r t s have e v e r been found i n t h e l i t e r a t u r e f r o m o t h e r l a b o r a t o r i e s t h a t d e s c r i b e any s e r i o u s a t t e m p t t o s e p a r a t e t h e s o i l l i p i d and l i p i d s u l f u r i n t o i n d i v i d u a l l i p i d c l a s s e s w i t h q u a n t i t a t i v e r e c o v e r y . The o b j e c t i v e of t h i s s t u d y was t o i n v e s t i g a t e t h e methods f o r t h e q u a n t i t a t i v e f r a c t i o n a t i o n of s o i l l i p i d s and l i p i d s u l f u r by means of s i l i c i c a c i d column chromatography. I t was a l s o hoped t h a t t h i s a p p r o a c h would p r o v i d e u s e f u l i n f o r m a t i o n about t h e a s s o c i a t i o n s of l i p i d s u l f u r w i t h each c l a s s of l i p i d s , and t h i s a s s o c i a t i o n would be f u n d a m e n t a l t o u n d e r s t a n d t h e c h a r a c t e r i s t i c s of t h e l i p i d s u l f u r i n s o i l s . - 93 -METHODS AND MATERIALS The eight s o i l samples used in this study were selected from thirty-seven soils reported previously (Chapter 3). The soils represent eight profile types including two forest organic horizons, three organic soils and three surface mineral horizons of forest and grassland so i l s . The samples were chosen so as to give a range of so i l characteristics representing s o i l groups and yet to have high ratios of l i p i d sulfur to total l i p i d contents to minimize problems in sulfur analysis. Some chemical analyses of s o i l samples are given in Table 4.1. The sample preparation, extraction of l i p i d and analytical methods used in this study were those reported previously. The column chromatographic fractionation was carried out using the procedure described by Rouser et^ a l . (1967a). Bio-Sil A (100-200 mesh, Bio-Rad Laboratories, Richmond, California) was used without additional washing or other preliminary treatment except the heat activation at 120°C. A chromatographic column, 2.0 cm i.d. and 40 cm long equipped with a 500 ml solvent reservoir and a Teflon stopcock and fri t t e d disc was used. A bed, 8.0 cm high, was prepared by pouring a slurry of about 15 g of the s i l i c i c acid in chloroform into the column, and the bed was prewashed with three column volumes of chloroform. The solvent level was allowed to descend to the top of the bed and 4 ml to 10 ml aliquots of the reduced extracts were applied. Any suspended solid was transferred along with the soluble material; quantitative transfer was ensured by thorough rinsing of a l l glasswares with chloroform. If any insoluble film remained on the Table 4.1 Some chemical analyses of soil samples. Soil Number Dominant Vegetation (Location/Classification) Drainage Horizon pH in H20 Organic C % Total S ppm Lipid S ppm Lipid % Western hemlock Free (Jordan River) Western hemlock Free (Jordan River) Subalpine grass Imperfect (Church Mt.) Western red cedar Free (Victoria) Grass Free (Ft.St. John/Solodic Black) Grass Imperfect (Delta/Saline Humic Gleysol) Grass-Sedge ' Imperfect (Whipsaw Cr./Humisol) Grass-Sedge Imperfect (Lulu Island/Humisol) Om Ah Ah Ah Oh Oh 3.9 3.0 4.6 6.1 6.6 5.8 3.7 3.3 55.7 57.0 17.8 7.74 5.70 3.24 28.1 51.4 1750 2016 1122 375 421 581 30431 23053 37.5 37.5 25.4 5.33 2.85 12.9 291.0 150.0 4.01 4.24 1.30 0.333 0.146 0.237 1.71 2.01 - 95 -sample container after vigorous shaking with chloroform, the film was treated with each of the solvents to be used for elution of the column and the dissolved solids were applied to the column just prior to addition of the bulk of each eluting solvent. Neutral l i p i d s (Fraction 1) were eluted with 175 ml of chloroform. Glycolipids were eluted into two separate fractions; one fraction of the glycolipids containing monoglycosyl diglycerides (Fraction 2) was eluted with 90 ml of chloroform-acetone (1:1, v/v) mixture; the other fraction of glycolipids containing diglyceride diglycosides and others (Fraction 3) was eluted with 700 ml of acetone. Polar lipi d s (Fraction 4) were eluted with 175 ml of methanol. Elution was accomplished at a flow rate of 3 ml per minute, and bulk fractions of the indicated volumes were collected. The solvent was evaporated on the flash evaporator to reduce to a small volume. The l i p i d was washed out of the flask with the desired pure solvent or solvent mixture and diluted to a known volume in a .-volumetric flask with a glass-stopper. The weight of the l i p i d in each fraction was determined by weighing a small aliquot that was discarded afterwards, as described by Bligh and Dyer (1959). RESULTS AND DISCUSSION With the l i p i d extracts, the elution of a s i l i c i c acid column using the sequence of chloroform, acetone, and then methanol provides an essentially quantitative separation into three groups; less polar (so called neutral) l i p i d s , glycolipids, and phosphatides (Rouser - 96 -e t a l , 1967a). V o r b e c k and M a r i n e t t i (1965) d e m o n s t r a t e d t h a t mono-and d l g l y c o s y l d i g l y c e r l d e s a r e s e p a r a b l e f r o m each o t h e r and o t h e r l i p i d c l a s s e s by e l u t i o n w i t h c h l o r o f o r m - a c e t o n e ( 1 : 1 , v/v) m i x t u r e f o l l o w e d by a c e t o n e . Rouser et^ a l . (1967b) t h e n d e m o n s t r a t e d t h e e l u t i o n o f c e r e b r o s i d e s u l f a t e , p l a n t s u l f o l i p i d , and cer a m i d e p o l y h e x o s e s w i t h a c e t o n e . These o b s e r v a t i o n s p r o v i d e d t h e b a s i s f o r a u s e f u l s e p a r a t i o n p r o c e d u r e e m p l o y i n g s i l i c i c a c i d t h a t i s e s s e n t i a l l y t h e o r i g i n a l p r o c e d u r e o f B o r g s t r o m (1952) c o u p l e d w i t h t h e a c e t o n e p r o c e d u r e o f S m i t h and Freeman (1959). I t was shown by Rouser e t a l . (1967b) t h a t t h e p r o c e d u r e i s p a r t i c u l a r l y u s e f u l f o r b r a i n and s p i n a c h l e a f l i p i d e x t r a c t s , i n w h i c h g l y c o l i p i d i s h i g h and d i p h o s p h a t i d y l g l y c e r o l i s a v e r y m i n o r component. I t was found i n t h e c o u r s e o f a p r e l i m i n a r y l i p i d f r a c t i o n -a c t i o n s t u d y t h a t a f a i r l y l a r g e amount o f l i p i d s appeared i n t h e f r a c t i o n e l u t e d w i t h a c e t o n e . T h e r e f o r e t h e p r o c e d u r e was adapt e d i n t h i s s t u d y f o r f r a c t i o n a t i o n o f s o i l l i p i d s . U s i n g t h e scheme d e v e l o p e d by Rouser e t a l . (1967a), t h e l i p i d c o m p o s i t i o n o f t h e f o u r chromato-g r a p h i c f r a c t i o n s was i n t e r p r e t e d as f o l l o w s : F r a c t i o n 1. The c h l o r o f o r m e l u a t e c o n t a i n s t h e l e s s p o l a r l i p i d s i n c l u d i n g s t e r o l s , s t e r o l e s t e r s , mono-, d i - , and t r i g l y c e r i d e s , h y d r o c a r b o n s , and f r e e f a t t y a c i d s . F r a c t i o n 2. The c h l o r o f o r m - a c e t o n e ( 1 : 1 , v/v) s o l v e n t m i x t u r e s e p a r a t e s m o n o g l y c o s y l d i g l y c e r i d e f r o m d i g l y c o s y l d i g l y c e r i d e w h i c h w i l l be e l u t e d w i t h a c e t o n e . C e r e b r o s i d e s a r e a l s o a l m o s t c o m p l e t e l y s e p a r a t e d f r o m s u l f a t i d e s w i t h t h i s s o l v e n t m i x t u r e . O t h e r somewhat l e s s p o l a r l i p i d s a r e e l u t e d a l o n g w i t h m o n o g l y c o s y l - 98 -Table 4.2 Lipid sulfur and l i p i d distributions in fractions of S i l i c i c acid column chromatography. Content (% of total applied) Soil Lipid S/Lipld Number Fraction Lipid S Lipid (ppm) 1 1 . 24.2 33.4 677 2 37.3 57.6 606 3 5.47 8.13 628 4 18.3 3.52 4875 Whole Soil 935 2 • 1 12.9 22.5 506 . ' 2 36,8 64.6 503 3 4.08 12.1 297 4 16.6 5.28 2768 Whole Soil 884 3 1 17.1 34.8. 960 2 30.0 54.6 1074 3 4.80 9.15 1025 4 36.90 4.59 15709 Whole Soil 1954 4 1 21.6 42.3 815 2 35.1 48.9 1149 3 7.30 7.63 1529 4 26.6 4.08 10471 Whole Soil 1601 5 1 24.9 37.3 1457 2 30.6 53.7 1249 3 6.18 7.60 1777 4 36.1 5.00 15715 Whole Soil 805 6 1 36.2 44.3 4506 2 45.2 47.3 5291 3 5.54 6.33 4789 4 7.31 4.14 9692 Whole Soil 5443 7 1 52.9 38.8 23202 2 32.4 53.6 10287 3 5.26 8.13 11072 4 3.61 3.98 15393 Whole Soil 17018 8 1 47.2 36.3 9683 2 14.1 51.2 2047 3 20.4 10.5 14500 4 10.0 3.24 23034 Whole Soil 7463 - 97 -diglycerides with spinach leaf l i p i d extracts. However this solvent mixture i s not as useful with animal organ extracts. Cerebrosides are almost completely separated from sulfatides by elution with this solvent mixture. Fraction 3. Acetone elutes diglycosyl diglycerides and sulfolipid with plant l i p i d extracts. With bacterial lipi d s both neutral and acidic glycosyl glycerides are eluted, although these are not present in extracts from a l l microorganisms. With l i p i d extracts of animal organs (particularly brain) sulfatides and ceramide poly-hexosides are eluted with acetone. With fecal l i p i d extracts a large number of uncharacterized substances devoid of phosphorus are eluted with acetone. No more than traces of phosphorus are found in acetone eluates except with samples that contain a large amount of diphosphatidyl glycerol that i s eluted in part with acetone. Fraction 4. The methanol eluate from animal organ extracts, spinach leaves, and some bacteria contains phosphatide with, at most, minute traces of glycolipids. Fecal l i p i d extracts contain a variety of substances devoid of phosphorus that are eluted with methanol along with phosphatides. The distribution of l i p i d sulfur and l i p i d in fractions eluted from s i l i c i c acid column are shown in Table 4.2. A l l eight soils studied gave similar elution patterns for the li p i d s . The highest amount of l i p i d was recovered in the Fraction 2, varying from 47.3 to 64.6% of the total l i p i d ; Fraction 4 contained the least l i p i d , varying from 3.24 to 5.28%. The Fraction 1 contained from 22.5 to 44.3% and the Fraction 3 from 6.33 to 12.1% of the total l i p i d s . For - 99 -the l i p i d sulfur elution, however, no consistent similarity in elution pattern was observed between corresponding fractions of the eight s o i l s . Thus the amount of l i p i d sulfur eluted in the Fraction 2 was the highest for the soils 1, 2, 4 and 6, varying from 35.1 to 45.5%, but the highest amount of l i p i d sulfur (36.9% and 36.1% respectively) was in the Fraction 4 for s o i l 3 and 5, and in the Fraction 1 (52.9% and 47.2%, respectively) for soils 7 and 8. In contrast to the distribution of l i p i d , the Fraction 3 contained the least amount of l i p i d sulfur ranging from 4.08 to 7.30% except for soils 7 and 8, of which l i p i d sulfur were the least in the Fraction 4. When the distributions of three general classes of lipids (Rouser et a l . , 1967a) are considered, the Glycolipids (Fractions 2 and 3 together) comprise more than half of the total s o i l l i p i d s , ranging from 53.6 to 76.7% with a mean of 62.6%, while the neutral or less polar lipids (Fraction 1) were around one-third of the total ranging from 22.5 to 44.3% with a mean of 36.2%. The mean value for the polar l i p i d (Fraction 4) was only 4.23% with a range of 3.24 to 5.28% (see Table 4.3). Although these distributions of l i p i d in the fractions are inconsistent with the distribution pattern of a bacterial l i p i d for a l l fractions reported by Langworthy et a l . (1974), the highest percentage (53.6%) of the total l i p i d was in the glycolipid fraction (acetone fraction). Unfortunately i t i s not possible to compare this result with others so as to understand the s o i l l i p i d component in general, because there are no reports on the s o i l l i p i d fractionation by the same or similar procedure used in this study. - 100 -Table A.3 also shows that relatively higher proportions of s o i l l i p i d sulfur (from 34.5 to 51.0% of total l i p i d sulfur) were associated with glycolipid component of the s o i l l i p i d s . However, for s o i l 3, as much as 36.9% of the total l i p i d sulfur was associated with polar lipids (mostly phosphatides), while 52.9% and 47.2% of the total l i p i d sulfur was associated with neutral or less polar lipids for soils 7 and.8 respectively. Virtually the association pattern of s o i l l i p i d sulfur with three general classes of s o i l l i p i d s differ from s o i l to s o i l and the association pattern i s not l i k e l y to be closely related to the s o i l factors. Whether the association pattern for s o i l l i p i d sulfur i s correlated with the l i p i d sulfur composition of dominant vegetation or with that of microorganisms in soils remains unclear, un t i l the compositions of l i p i d sulfur in vegetation and microorganisms on and in soils are characterized. The l i p i d sulfur expressed as ppm of l i p i d in each fraction and in whole soils are also shown in Table 4.2. The value for Fraction 4 of s o i l 7 was slightly lower but very similar to that for the whole s o i l . In contrast, the values for Fraction 4 of other seven soils were always higher than those for whole soils, and remaining fractions had lower values than the values for whole soils except for Fraction 1 of s o i l 7 and Fractions 1, 3 and 4 of s o i l 8. The recoveries of l i p i d sulfur and l i p i d from the s i l i c i c acid column as percentage of total amounts applied were shown in Table 4.3. Although, from a l l soils, the recoveries of l i p i d were over 100%, the recoveries of the l i p i d sulfur were, in a l l cases, below 100%. It was noticed that in most cases every eluate of - 101 -Table 4.3 Association with l i p i d classes of s o i l l i p i d s u l f u r and t o t a l recovery of l i p i d and l i p i d s u l f u r . % of t o t a l applied S o i l L i p i d Number Class ' L i p i d S L i p i d LS 24.2 33.4 G 42.8 65.7 P 18.3 3.52 TR 85.3 102.7 LS ' 12.9 22.5 G 40.9 76.7 P 16.6 5.28 TR 70.4 104.5 LS 17.1 34.8 G 34.8 63.8 P 36.9 4.59 TP 88.8 103.1 LS 21.6 42.3 G 42.4 56.5 P 26.6 4.08 TR 90.6 102.9 LS 24.9 37.3 G 36.8 61.3 P 36.1 5.00 TR 97.8 103.6 LS 36.2 44.3 G 51.0 53.6 P 7.31 4.14 TR 94.6 102.1 LS 52.9 •;"<''* ! 38.8 G 37.7 61.7 P •- 3.61 3.98 TR 94.2 104.5 LS 47.2 . 36.3 G 34.5 61.7 P 10.0 3.24 TR 91.7 101.2 LS = Less Polar; G = G l y c o l i p i d ; TR = Total Recovery. P = Polar; - 102 -f r a c t i o n s c o l l e c t e d and r e d u c e d i n t h e f l a s h e v a p o r a t o r had a s i g n i f i c a n t amount o f w h i t e m a t e r i a l w h i c h p r o b a b l y was f i n e p a r t i c l e s of s i l i c i c a c i d come a l o n g w i t h t h e e l u t i o n s o l v e n t t h r o u g h f r i t t e d g l a s s d i s k . I t was a l s o n o t i c e d t h a t , i n a l l c a s e s , t h e r e were v i s i b l e c o l o r remained a f t e r e l u t i o n o f a l l f o u r f r a c t i o n s w h i c h was p r e s u m a b l y l i p i d m a t e r i a l r e m a i n i n g on t h e column. T h e r e f o r e t h e h i g h e r r e c o v e r i e s of l i p i d were p r o b a b l y due t o t h e s i l i c i c a c i d e l u t e d from column and t h e l o w e r r e c o v e r i e s o f l i p i d s u l f u r were p r i m a r i l y due t o t h e l o s s o f l i p i d m a t e r i a l by b e i n g a d s o r b e d on column and t h e s i l i c i c a c i d e l u t e d a l o n g w i t h e l u t i n g s o l v e n t d i d n o t i n f l u e n c e t h e l i p i d s u l f u r c o n c e n t r a t i o n on measurement. CONCLUSION The f r a c t i o n a t i o n o f s o i l t o t a l l i p i d s i n t o t h r e e g e n e r a l c l a s s e s on t h e s i l i c i c a c i d column has shown t h a t t h e d i s t r i b u t i o n p a t t e r n s o f t h e t h r e e c l a s s e s o f l i p i d s were s i m i l a r f o r a l l e i g h t s o i l s s t u d i e d r e g a r d l e s s o f s o i l t y p e s . G l y c o l i p i d s were t h e dominant c l a s s o f s o i l l i p i d s and t h e y were more t h a n h a l f o f t h e t o t a l . The n e u t r a l l i p i d s were around o n e - t h i r d o f t h e t o t a l l i p i d s and p o l a r l i p i d s were o n l y 4% o f t h e t o t a l b e i n g t h e s m a l l e s t component o f t h e s o i l l i p i d s . The u n i f o r m i t y o f t h e d i s t r i b u t i o n i n each f r a c t i o n o f t h e column chromatography ( i . e . , each l i p i d c l a s s ) s u g g e s t s t h a t t h e l i p i d s i n t h e s e s o i l s were s i m i l a r i n t y p e and o r i g i n o r t h a t t h e i r d i s t r i b u t i o n s were a f f e c t e d by and made more u n i f o r m t h r o u g h i n t e r a c t i o n of m i c r o o r g a n i s m s w i t h o t h e r s o i l e n v i r o n m e n t a l f a c t o r s . - 103 -The s o i l l i p i d sulfur had no consistent similarity in the distribution pattern between corresponding classes for eight soils studies, differing from s o i l to s o i l . Perhaps the most significant finding of the study was that significant amounts of sulfur were, in a l l cases, recovered in the less polar (neutral), glycolipid and polar l i p i d fractions, with these three fractions accounting on average for 29%, 42% and 19% of the l i p i d sulfur, respectively. This finding clearly suggests that s o i l l i p i d sulfur i s present in a variety of forms. The ratios of the l i p i d sulfur to l i p i d as ppm of the l i p i d in each corresponding fraction and the ratios for whole soils have shown that the values for Fraction 4 (polar l i p i d class) of the seven soils except for s o i l 7 were always higher than those for whole soils. The values for the remaining fractions (1, 2 and 3) had lower values than the values for whole soils except for Fraction 1 of s o i l 7 and Fraction 1, 3 and 4 of s o i l 8. Fraction 1 (less polar l i p i d class) of s o i l 7 and 8, thus, provides a useful fraction for further investigation of the characteristics of the s o i l l i p i d sulfur since this fraction particularly for s o i l 7 and 8 contained the highest amounts of the l i p i d sulfur and yet the contents of the l i p i d sulfur in this fraction as ppm of l i p i d are higher than those in whole soi l s . - 104 -REFERENCES Borgstrom, B. 1952. Investigation on l i p i d separation methods. Separation'of phospholipids from neutral fat and fatty acids. Acta Physiol. Scand. 25: 101-110. Langworthy, T.A. 1974. Long-chain glycerol diether and polyol dialkyl glycerol triether lip i d s of sulfolobus acidocaldarius. J. Bacteriol. 119: 106-116. Rouser, G., G. Kritchevsky, and A. Yamamoto. 1967a. Column chromatographic and associated procedure for separation and determination of phosphatides and glycolipids. In Lipid Chromatographic Analysis. G.V. Marinetti, (Ed.) Marcel Dekker, Inc., New York. Vol. 1, pp. 99-162. Rouser, G., G. Kritchevsky, G. Simon, and G.J. Nelson. 1967b. Quantitative analysis of Brain and spinach leaf l i p i d s employing S i l i c i c acid column chromatography and acetone for elution of glycolipids. Lipids 2: 37-40. Smith, L.M. and N.K. Freeman. 1959. Analysis of milk phospholipids by chromatography and infrared spectrophotometry. J. Dairy Sci. 42: 1450-1462. Vorbeck, M.L. and G.V. Marinetti. 1965. Separation of glycosyl diglycerides from phosphatides using S i l i c i c acid column chromatography. J. Lipid Res. 6: 3-6. - 105 -CHAPTER 5 OBSERVATIONS ON THE GAS LIQUID AND THIN LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS IN TWO SELECTED SOILS INTRODUCTION I n a p r e v i o u s r e p o r t ( C h a p t e r 4) i t has been shown t h a t t h e d i s t r i b u t i o n p a t t e r n s o f t h e t h r e e c l a s s e s o f l i p i d s ( i . e . , l e s s p o l a r l i p i d s , g l y c o l i p i d s and p o l a r l i p i d s ) were s i m i l a r f o r e i g h t s o i l s s t u d i e d , r e g a r d l e s s o f s o i l t y p e . The s o i l l i p i d s u l f u r , however, had no c o n s i s t e n t s i m i l a r i t y i n d i s t r i b u t i o n s between c o r r e s p o n d i n g l i p i d c l a s s e s , d i f f e r i n g f rom s o i l t o s o i l . I t was a l s o shown t h a t F r a c t i o n 1 ( l e s s p o l a r l i p i d c l a s s ) o f two H u m i s o l samples c o n t a i n e d t h e h i g h e s t amounts of l i p i d s u l f u r among e i g h t s o i l s and f o u r f r a c t i o n s . I n a d d i t i o n t h e r a t i o s o f l i p i d s u l f u r t o l i p i d i n t h i s f r a c t i o n e x p r e s s e d as p a r t p e r m i l l i o n were h i g h e r t h a n t h o s e i n whole s o i l s . These r e s u l t s s u g g e s t e d t h a t F r a c t i o n 1 c o u l d be a u s e f u l one f o r a f u r t h e r q u a n t i t a t i v e i n v e s t i g a t i o n on l i p i d s u l f u r i n t h e s e o r g a n i c s o i l s . S i n c e F r a c t i o n 1 r e p r e s e n t s t h e l e s s p o l a r l i p i d c l a s s , and f a i r l y h i g h amounts o f l i p i d s u l f u r were p r e s e n t i n t h i s f r a c t i o n , t h e l i p i d s u l f u r a s s o c i a t e d w i t h t h e l e s s p o l a r l i p i d c l a s s may i n c l u d e s u c h s u l f u r forms as t h i o l s and t h i o e t h e r s , o f w h i c h b o i l i n g p o i n t s a r e g e n e r a l l y l o w enough t o work w i t h g a s - l i q u i d chromatography - 106 -( R y l a n d and Tamele, 1970). T h i s c h a p t e r d e s c r i b e s a s t u d y t h a t examines t h e p o s s i b i l i t y o f i s o l a t i n g and c h a r a c t e r i z i n g t h e l i p i d s u l f u r i n t h e f r a c t i o n s o f t h e l e s s p o l a r l i p i d c l a s s f r a c t i o n a t e d f r o m two s e l e c t e d H u m i s o l s by a p p l y i n g t h i n - l a y e r and g a s - l i q u i d chromatography. METHODS AND MATERIALS S o i l s The two s o i l s u sed i n t h i s s t u d y were s e l e c t e d from t h e e i g h t s o i l s r e p o r t e d i n C h a p t e r 4. These two s o i l s a r e H u m i s o l s sampled f r o m two d i f f e r e n t l o c a t i o n s r e p r e s e n t i n g Oh h o r i z o n s o f o r g a n i c s o i l s . The samples were chosen because o f t h e i r h i g h c o n t e n t s of t o t a l l i p i d s u l f u r and a l s o t h e w i d e s t l i p i d s u l f u r t o l i p i d r a t i o s i n F r a c t i o n 1 as shown i n C h a p t e r 4, so t h a t F r a c t i o n 1 c o u l d be u s e d f o r f u r t h e r a t t e m p t s t o i s o l a t e and c h a r a c t e r i z e i t s components. The H u m i s o l f r o m Whipsaw c r e e k was r e s a m p l e d f o r t h e b u l k e x t r a c t i o n s t u d y , and has shown s l i g h t l y d i f f e r e n t c h e m i c a l p r o p e r t i e s f r o m p r e v i o u s l y r e p o r t e d d a t a . The sample p r e p a r a t i o n s were t h o s e r e p o r t e d p r e v i o u s l y . O r i g i n and some c h e m i c a l a n a l y s e s a r e g i v e n i n T a b l e 5.1. A n a l y t i c a l Methods The methods used f o r t h e e x t r a c t i o n , measurement and d e t e r m i n a t i o n o f s o i l pH, t o t a l o r g a n i c c a r b o n , t o t a l s u l f u r , t o t a l T a b l e 5 . 1 Son.e c h e m i c a l a n a l y s e s o f s o i l s a m p l e s . %. o f oven ppm of o v e r d r i e d s o i l d r i e d s o i l ; — S o i l Dominant V e g e t a t i o n Number ( L o c a t i o n / C l a s s i f i c a t i o n ) 1 G r a s s ( L u l u I s l a n d / H u m i s o l ) 2 G r a s s (Whipsaw C r . / H u m i s o l ) pH O r g a n i c i n H 2 0 C L i p i d 3 . 3 5 1 . 4 2 .01 4 . 0 3 0 . 6 0 .836 T o t a l T o t a l L i p i d • S H I - S S 2 3 , 1 0 0 7 ,710 150 2 9 , 0 0 0 1 7 , 4 0 0 156 - 108 -H l - r e d u c i b l e s u l f u r , t o t a l l i p i d and l i p i d s u l f u r i n s o i l s were t h o s e r e p o r t e d p r e v i o u s l y . Column Chromatography T o t a l l i p i d e x t r a c t s were f r a c t i o n a t e d i n t o f o u r f r a c t i o n s ( F r a c t i o n s 1, 2, 3 and 4) on a s i l i c i c a c i d column a c c o r d i n g t o t h e p r o c e d u r e o f Rouser e t a l . (1967a) as d e s c r i b e d i n C h a p t e r 4. F r a c t i o n 1 was f u r t h e r p u r i f i e d on a s i l i c i c a c i d column i n t o s i x f r a c t i o n s ( F r a c t i o n s A, B, C, D, E and F) u s i n g p e t r o l e u m e t h e r , e t h e r , m e t h a n o l , and c h l o r o f o r m , and t h e i r m i x t u r e s a c c o r d i n g t o t h e m o d i f i e d method o f H a i n e s and B l o c k (1962). B i o - S i l A, s i l i c i c a c i d (100-200 mesh), was graded i n m e t hanol a t 5 m i n u t e s i n t e r v a l s t o remove t h e f i n e s u n t i l t h e s u p e r n a t a n t was c l e a r . The graded m a t e r i a l was suspended i n m e t h a n o l and packed i n a column 2.0 cm i n d i a m e t e r t o a h e i g h t o f 7 cm. I t was washed w i t h 2 volumes (50 ml) o f m e t h a n o l , 3 volumes (75 ml) o f a c e t o n e , 3 volumes o f e t h e r and 3 volumes o f p e t r o l e u m e t h e r . The l i p i d e l u t e d f r o m t h e f i r s t s i l i c i c a c i d column ( i . e . , F r a c t i o n 1) was c o n c e n t r a t e d n e a r l y t o d r y n e s s and t a k e n up i n p e t r o l e u m e t h e r w i t h enough c h l o r o f o r m t o s o l u b i l i z e t h e l i p i d s ( t o t a l volume was n o t more t h a n 750 pi). The f o l l o w i n g f r a c t i o n s e l u t e d w i t h 50 m l o f each e l u a n t were t a k e n : p e t r o l e u m e t h e r ( F r a c t i o n A ) , 50% (v/v) e t h e r i n p e t r o l e u m e t h e r ( F r a c t i o n B ) , e t h e r ( F r a c t i o n C ) , 50% m e t h a n o l i n e t h e r ( F r a c t i o n D ) , m e t h a n o l ( F r a c t i o n E ) , and c h l o r o f o r m ( F r a c t i o n F ) . - 109 -Thin-Layer Chromato graphy Chromatograms, precoated Eastman Chromagram Sheets, consisting of a 100-micron layer of s i l i c a gel coated onto a flexible support of solvent-resistant polyethylene terephthalate with fluorescent indicator incorporated in the active layer, were used without activation. The developing solvents of various combinations were attempted for one-dimensional, two-dimensional and two-dimensional mapping thin-layer chromatography (TLC). Visualization of separated components was carried out by exposing the developed sheets to iodine vapor in a closed chamber. Attempts at identification of individual spots by visualization with various spray reagents for specific functional groups were unsuccessful. Two-dimensional mapping TLC of four fractions (Fractions 1 through 4) were performed by using the developing solvents; chloroform-methanol-water (65:25:4, v/v/v) for the f i r s t solvent (vertical) and chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1) for the second solvent (horizontal) (see Figures 5.1 and 5.2). For the multiple development TLC of the four fractions and the six fractions on second column chromatography (i.e., Fraction A through F); chloroform-methanol-water (65:25:4) and hexane-ether (4:1) were used as the f i r s t and the second solvent system, respectively (Figures 5.3, 5.4 and 5.5). For one-dimensional TLC of the six fractions, the chromato-graphic sheet was cut out into several 2x6 cm mini sheets and developed in the mini developing tank. Various combinations of petroleum ether, - n o -ether, methanol, chloroform, acetic acid and water were used as developing solvent systems, in the hope that the best resolution of components could be achieved. The best solvent systems for the six fractions were as follows: for Fraction A, petroleum ether-acetic acid (40:10:1); Fraction B, petroleum ether-ether-methanol-acetic acid (45:5:5:1.5); Fractions C and D, chloroform-methanol-ether-acetic acid (20:20:30:1.5); Fraction E, methanol-ether-acetic acid (25:35:1.5); Fraction F, chloroform-methanol-ether-acetic acid (20:10:40:1) (see Figure 6). Gas-Liquid Chromatography The equipment used included a Micro Tek MT-200 Gas Chromatograph, dual channel solid state electrometer, a solid state regulated 750 volt power supply, and a Melpar flame photometric detector (Brody and Chaney, 1966) with a 394 mu f i l t e r for sulfur analysis. Two gas liquid chromatographic columns, one of 6'x"%" i.d. glass column containing 10% EGSS-X on 100-120 mesh Gas Chrom P (Applied Science Labs. Ltd., State College, Pa.) and the other one of 6'xV i.d. Stainless steel column packed with 5% OV-1 on 80-100 mesh Chromosorb W, were used. The column packed with 10% EGSS-X was conditioned overnight at 225°C and the one packed with 5% OV-1 was conditioned overnight at 350°C before use. - I l l -For these studies, the carrier gas was nitrogen. Hydrogen, oxygen and air were used as the burner gas mixture. Gas chromatographic conditions for the columns used were adjusted to gain optimal conditions. For the glass column packed with 10% EGSS-X, column temperature was programmed from 100 to 180°C at the increasing rate of 5°C per minute. Detector base temperature and inlet temperature were 195 C and 125 C, respectively. Nitrogen carrier gas, hydrogen, oxygen and air flow rates were held at 80, 120, 18 and 38 ml per minute, respectively. On the other hand, for the stainless steel column with 5% OV-1, column temperature was programmed from 100 to 200°C at the same rate as for the glass column. Detector base temperature and inlet temperature were held at 210°C and 125°C, respectively. Gas flow rates were held at 80, 125, 29 and 38 ml per minute for nitrogen, hydrogen, oxygen and a i r , respectively. Chart speed was set at 5 mm per minute for a l l chromatograms. RESULTS AND DISCUSSION Some of the chemical characteristics of the two s o i l samples used in this study are shown in Table 5.1. The samples were chosen to examine further i f these soils could give a different response or results on the TLC and GLC although they have similar l i p i d and l i p i d sulfur distribution on s i l i c i c acid column chromatography. Also as described in a previous report (Chapter 3), s o i l 1 (Lulu muck) was the identical s o i l sample reported in Chapter 3. However s o i l 2 (Whipsaw Creek) was resampled from the same location where the s o i l was sampled - 112 -for the previous report. The distribution of the l i p i d and l i p i d sulfur on the s i l i c i c acid column of the s o i l 2 resampled was very similar to that of the f i r s t sample as shown in Table 5.2. Thin-Layer Chromatography For monitoring column chromatography and to compare the thin-layer chromatographic behavior of each column chromatographic fraction of two soils, two-dimensional mapping and one-dimensional multiple developing TLC were carried out. Several attempts to visualize spots on the basis of specific reactive groups failed due to insignificant response of spray reagents on the specific spots. Reagents tested included: copper acetate-Rhodamine B for lipophilic alkyl sulfonates, copper sulfate for sulfur-containing glycosides, and resorcinol-ammonia for sulfonic acids (Kates, 1972). Therefore, only TLC visualized by exposing to iodine vapor are shown in Figures 5.1 through 5.6. To obtain overall developing patterns two dimensional mapping TLC of the four fractions was carried out and the results are shown in Figures 5.1 and 5.2 for s o i l 1 and 2, respectively. As shown in the figures, none of the corresponding four fractions (three general l i p i d classes) from each s o i l gave similar chromatograms. These dissimilarities indicate that the individual components of the one general l i p i d class fractionated from one s o i l are different from the same l i p i d class from the other s o i l . In other words, although the column chromatographic distribution of total lipi d s i s similar for the two soils, the nature of individual l i p i d components differ from one another. - 113 -Table 5.2 Lipid and lipid sulfur distributions in fractions from • S i l i c i c acid column. Soil Number Fraction Content (% of total applied) Lipid Lipid S 1 2 3 A Recovery 36.3 51.4 10.5 3.24 101.2 47.2 14.1 20.4 10.0 91.7 1 2 3 4 Recovery 40.6 53.9 9.68 3.42 107.6 55.8 27.8 4.70 2.51 90.8 - 114 -4 'i * 3 / 1yj, * - -*. * \ D \ i; / '.' ; r \* i J >' > ( II ( v 'i i. i' \ J ^ ' -/ ) ' •1 N d i \ \ \ 1 <• '.1 > l l V i *x * ' ' 1 1 t 1 1 1 1 , — - , _ l I % - ' " " " " I * Qy-'"""'" -* *. < 1 2 II—* Figure 5.1 Two-dimensional mapping TLC of Fractions 1, 2, 3 and 4 eluted from S i l i c i c acid column of total l i p i d extract of s o i l 1. First solvent (I, vertical), chloroform-methanol-water (65:25:4); second solvent (II, horizontal), chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1). Detection by exposing in the iodine vapor. - 115 -V 4 A i w D 3 1 ^ i O 2 •—•II Figure 5.2 Two-dimensional mapping TLC of Fractions 1, 2, 3 and 4 eluted from S i l i c i c acid column of total l i p i d extract of s o i l 2. First solvent (I, vertical), chloroform-methanol-water (65:25:4); second solvent (II, horizontal), chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1). Detection by exposing in the iodine vapor. - 116 -The differences in the composition of the corresponding l i p i d class of two soils were also shown in the one-dimensional multiple development thin-layer chromatograms in Figures 5.3 and 5.4. In comparison with two dimensional mapping chromatograms, the multiple development chromatograms gave fewer but better resolved spots. These fewer but well resolved spots in the chromatograms suggest that the multiple developing technique may only be used for preparative TLC. It may also suggest that a second column chromatography must be applied prior to the current TLC. Multiple development chromatograms of the fractions, from A to F, eluted from a second s i l i c i c acid column of Fraction 1 of s o i l 1 are shown in Figure 5.5. The chromatograms indicate that the l i p i d components in Fraction A are different from those in the other fractions and that Fraction B, C and F, and Fractions D and E have similar composition, respectively. In other words, for Fraction 1, only petroleum ether eluted d i s t i n c t i v e l i p i d s from the s i l i c i c acid column. Although Fractions D and E contained some different components of l i p i d s which were not shown in Fractions B, C and F, these fractions contained some components common also to Fractions B, C and F. One-dimensional mini TLC of the Fractions, from A to F, was carried out in the hope that the best developing solvents could be obtained. Various different solvent ratios of different solvent mixtures had been applied to each fraction and the TLC developed with the best resolving eluents for each fraction were shown in Figure 5.6. Except for Fraction B, a l l other fractions failed to give better resolution using single eluent on one-dimensional TLC. Furthermore, - 117 -0 o Figure 5.3 Multiple development TLC of Fractions 1, 2, 3 and 4 eluted from S i l i c i c acid column of total l i p i d extract of s o i l 1. First solvent, chloroform-methanol-water (65:25:4); second solvent, hexane-ether (4:1). Detection by exposing in the iodine vapor. - 118 -• i « . 1 2 3 4 Figure 5.4 Multiple development TLC of Fractions 1, 2, 3 and 4 eluted from S i l i c i c acid column, of total l i p i d extract of s o i l 2. First solvent, chloroform-methanol-water (65:25:4); second solvent, Hexane-ether (4:1). Detection by exposing in the iodine vapor. - 119 -Figure 5.5 Multiple development TLC of Fractions A, B, C, D, E, and F eluted from a second S i l i c i c acid column of Fraction. 1 from the f i r s t S i l i c i c acid column of s o i l 1. First solvent, chloroform-methanol-water (65:25:4); second solvent, hexane-ether (4:1). Detection by exposing in the iodine vapor. - 120 -One-dimensional mini-TLC of the Fractions A-F eluted from S i l i c i c acid columns of s o i l 1. Solvent systems: Fraction A, pet.ether-ether-acetic acid (40:10:1); B, pet. ether-ether-methanol-acetic acid (45:5:5:1.5); C and D, chloroform-methanol-ether-acetic acid (20:20: 30:1.5); E, methanol-ether-acetic acid (25:35:1.5); F, chloroform-methanol-ether-acetic acid (20:10:40:1). - 121 -Fraction B, C and F giving a similar pattern on the multiple development TLC, gave different behavior on one-dimensional TLC. Gas-Liquid Chromatography The major virtue of the gas-liquid chromatographic analysis of sulfur using the Melpar flame photometric detector is the high response specificity of the detector. It responds to sulfur-containing compounds with great sensitivity, e.g., sensitive to subnanogram quantities as low as 5 ng (Bowman and Beroza, 1966a) and 0.05 ng (Bremner and Banwart, 1974), although the detector i s an order of magnitude more sensitive to phosphorus than to sulfur , (Stevens, 1967) and the response in the sulfur analysis is not proportional to concentration (Brody and Chaney, 1966). The detector also i s so insensitive to extraneous material in a raw extract of biological tissue that compounds other than sulfur have virtu a l l y no photometric response as long as the concentration of the non-sulfur contaminant in the sample injected does not exceed 20 jig (Stevens, 1967). As a result, the high specificity of the detector should require fewer sample preparation steps to determine the organosulfur content in extracts of natural product (Bowman and Beroza, 1966b). The results of gas-liquid chromatography (GLC) using the Melpar flame photometric detector are shown in Figures 5.7 through 5.19. The resolution of GLC was poor when 3% OV-1 and 10% SE-30 were used as column packing materials for the total lipids and fractions on the column chromatography of both s o i l 1 and 2. Resolution was better when 19 I RESPONSE N5 to 100 120 140 160 180 TEMPERATURE ( °C ) 200 Figure 5.7 GLC of concentrated reagent grade chloroform. Linear temperature program as shown. Mikro Tek MT-200 equipped with a Melpar flame photometric detector with a 394 mu f i l t e r using stainless steel column of 6' x V i.d. packed with 5%.OV-1 on Chromosorb W (80-100 mesh). Nitrogen, hydrogen, oxygen and air flow rates held at 80, 120, 18 and 38 ml/min, respectively. Detector base and inlet temperatures held at 210 and 125°C, respectively. 5 p i of sample (ca. 95.2 ppm S) in chloroform. RESPONSE Figure 5.8 GLC of total l i p i d extracted from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.7. 5 JLII of sample (ca. 37.3 ppm S) in chloroform. 19 I Figure 5.9 GLC of Fraction 1 of l i p i d extracted from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other operating conditions as in Figure 5.7. 5 yl of sample (ca. 43.2 ppm S) in chloroform. - 125 -5% OV-1 was used for s o i l 1 and 10% EGSS-X for s o i l 2. Therefore two different column packing materials were used for the lipids from two different s o i l s . Although GLC of total li p i d s and a l l fractions from the f i r s t s i l i c i c acid column of soils 1 and 2, and fractions from the second s i l i c i c acid column of s o i l 1 were carried out, only GLC of the total lip i d s and Fraction 1 of s o i l 1 and 2 and six fractions (i.e., Fractions A through F) are presented because the results of GLC were not specific for other fractions and were quite similar to each other. GLC of total l i p i d and Fraction 1 of s o i l 1 on 5% OV-1 are shown in Figures 5.8 and 5.9, respectively. Those of s o i l 2 on 10% EGSS-X are shown in Figures 5.11 and 5.12. GLC of the total l i p i d extracted from s o i l 1 showed at least 5 distinctive peaks (peak numbers: 8, 9, 12, 19 and 23) of sulfur-containing compounds (Figure 5.8), and that of Fraction 1 showed at least 6 peaks (peak numbers: 3, 8, 9, 12, 16 and 19) in Figure 5.9. GLC of total l i p i d and Fraction 1 of s o i l 2 also showed 10 peaks (peak numbers: 2, 4, 5, 6, 7, 11, 12, e and f in Figure 5.11) and 6 peaks (peak numbers: 1, 2, 3, 4, 8 and 9 in Figure 5.12), respectively. At f i r s t , i t was thought that GLC could be used to monitor the sulfur-containing compounds in the l i p i d extracts and fractions of column chromatography, since there were several distinctive peaks in the chromatogram and certain peaks in the GLC of total lipids did show in the GLC of Fraction 1. However, this hope disappeared when GLC of the concentrated extraction-solvent blank was run by GLC (Figures 5.7 and 5.10). There were more than 24 peaks in the chromatogram of 5% 0V-1 separation of the extraction-solvent blank (Figure 5.7) and 17 peaks in the GLC R E S P O N S E 6 7 11 f—1 • <y> • _L 100 120 140 160 T E M P E R A T U R E ( 180 •C) Figure 5.10 GLC of concentrated extraction blank. Linear temperature program as shown. Micro Tek MT-200 equipped with a Melpar flame photometric detector with a 394 mu f i l t e r using glass column of 6' x V i.d. packed with 10% EGSS-X on Gas Chrom P (100-200 mesh). Nitrogen, hydrogen, oxygen and air flow rates held at 80, 120, 18 and 38 ml/min, respectively. Detector base, inlet temperatures held at 195 and 125°C, respectively. 5 }il of sample (ca. 92.5 ppm S) in chloroform. RESPONSE to _ L _ I ; I I I 100 120 140 160 180 TEMPERATURE ( °C ) Figure 5.11 GLC of total l i p i d extracted from s o i l 2. On 10% EGSS-X. Temperature program as shown. Instrument and other operating conditions as in Figure 5.10. 5 p.1 of sample (ca. 18.7 ppm S) in chloroform. RESPONSE TEMPERATURE ( °C ) ure 5.12 GLC of Fraction 1 of total l i p i d extracted from s o i l 2. On 10% EGSS-X. Temperature program as shown. Instrument and other operating conditions as in Figure 5.10. ^.jil of sample (ca. 12.8 ppm S) in chloroform. - 129 -on the 10% EGSS-X of the same extraction-solvent blank (Figure 5.10). For s o i l 1, the peaks in GLC of total lipids (Figure 5.8) and in that of Fraction 1 (Figure 5.9) were a l l identical to the peaks of the corresponding numbers in the GLC of the concentrated extraction-solvent blank (Figure 5.7). For s o i l 2, seven out of 10 peaks in GLC of total l i p i d extract (Figure 5.11) and a l l 6 peaks in GLC of Fraction 1 (Figure 5.12) were identical to the peaks of the corresponding numbers in the GLC of the concentrated extraction-solvent blank (Figure 5.10). The three distinctive peaks (peaks d, e and f) in the GLC of total l i p i d extract (Figure 5.11) were not shown in the GLC of the concentrated extraction-solvent blank in Figure 5.10. Therefore those three peaks in the GLC of total l i p i d extract of s o i l 2 could represent sulfur-containing compounds extracted from the s o i l . However i t remains uncertain whether these peaks represent a l l the sulfur-containing lipi d s in the s o i l or simply represent sulfur-containing organic compounds which could be extracted in the current extraction solvent. GLC of Fraction 1 on the f i r s t column chromatography and of Fraction A through F on the second column chromatography of Fraction 1 from s o i l 1 were shown in Figures 5".13 and 5.14 through 5.19, respectively. In these chromatograms quantities of sulfur injected are not shown since quantitation of the sulfur in each fraction requires large volumes of the fractions from which sulfur determination can be made. Therefore small portions of the fractions were concentrated under a stream of nitrogen gas, at which concentration the best resolution of peaks could be obtained. 1 RESPONSE i-1 o IOO 120 140 160 TEMPERATURE ( 180 200 Figure 5.13 GLC of Fraction 1 of l i p i d extracted from s o i l 1. Linear temperature program as shown. Mikro Tek MT-200 equipped with a Melpar flame photometric detector with a 394 mu f i l t e r using stainless steel column of 6' x V i.d. packed with 5% OV-1 on Chromosorb W (80-100 mesh). Nitrogen, hydrogen, oxygen and air flow rates held at 80, 120, 20 and 38 ml/min, respectively. Detector base and inlet temperatures held at 195 and l25°C, respectively. Figure 5.14 GLC of Fraction A from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.13. Figure 5.15 GLC of Fraction B from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.13. 1 Figure 5.16 GLC of Fraction C from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.13. RESPONSE CO 1 i IOO I20 I40 I60 I80 200 TEMPERATURE ( °C ) Figure 5.17 GLC of Fraction D from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.13. Figure 5.18 GLC of Fraction E from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as in Figure 5.13. RESPONSE lOO 120 140 160 180 200 TEMPERATURE ( ° C ) Figure 5.19 GLC of Fraction F from s o i l 1. On 5% OV-l. Temperature program as shown. Instrument and other conditions as in Figure 5.13. - 137 -While GLC of Fraction 1 showed 14 peaks (Figure 5.13), Fractions A, B, C, D, E and F showed 4, 9, 11, 1, 7 and 5 peaks, respectively. Among six fractions, Fractions B (peaks a and b in Figure 5.15), C (peaks a and b in Figure 5.16), E (peak a in Figure 5.18), and F (peak a in Figure 5.19) showed additional peaks which were not shown in the GLC of Fraction 1. It i s also uncertain whether these peaks represent sulfur-containing lipi d s of the s o i l or not. Further, i t i s not known whether these peaks represent sulfur-containing compounds which were detected after further fractionation on column (i.e., clean up) or simply sulfur-containing compounds which were contaminants of the different elution solvents employed for the second column chromatography such as petroleum ether and ether, since the concentrated pure solvents of these two had shown many gas-liquid chromatographic peaks. CONCLUSION Thin-layer chromatographic behavior of the corresponding four fractions of two soils were not similar to each other, although column chromatographic behavior of total l i p i d s and l i p i d sulfur of two soils were similar to each other. These dissimilarities may indicate that the individual components of one general l i p i d class fractionated from one s o i l differ from the same l i p i d class of the other s o i l even though the column chromatographic distribution of total li p i d s was similar between two soils. - 138 -Since the multiple development and one-dimensional mini TLC of the Fractions A through F of Fraction 1 of s o i l 1 showed a few common spots in each fraction, i t indicates that the second s i l i c i c acid column chromatography did not help very much in further fractionation of Fraction 1. Instead, the multiple development technique with the current developing solvent system could be used as a preparative TLC without applying the second column chromatography. Although the sulfur-containing contaminants in the solvents used for l i p i d extraction and for elution from column chromatography masked gas-liquid chromatographic monitoring of sulfur-containing compounds in l i p i d extracts and fractions, i t was suggested that the GLC could be used in monitoring the sulfur-containing compounds in l i p i d extracts and fractions of s o i l , especially for s o i l 2 with 10% EGSS-X column for example. However in the absence of complete characterization i t i s uncertain whether the monitored sulfur-containing compounds represent the sulfur-containing lipids extracted from s o i l or simply represent sulfur-containing organic compounds extracted in the current extraction-solvent system. It is now apparent that future experiments should be careful-ly designed with regard to the effect of solvent contaminants, the kind of organic solvents used, the use of column and thin-layer chromatographies before application of GLC, and the proper choice of type of GLC columns. Since there i s no report of work on characterization of sulfur-containing lipids in s o i l , future experiments should be planned in the light of the results observed in this study. Clearly solvents of higher purity w i l l be needed to take advantage of GLC techniques in such studies. - 139 -In conclusion, this last phase of the investigation, involving attempts to separate and characterize individual sulfur-containing l i p i d components by chromatographic methods, failed to significantly advance our knowledge of individual l i p i d constituents, partly because of technical problems, and partly because of lack of time. However i t served to re-emphasize the complexity both of s o i l l i p i d fractions in general, and of their sulfur-containing constituents in particular. It also indicated some of the more promising lines of investigation for future studies. - 140 -REFERENCES Bowman, M.C. and M. Beroza. 1966a. Gaschromatographic determination of trace amounts of the insect chemosterilants Tepa, Metepa, Methiotepa, Hempa, and Apholate and the analysis of Tepa in insect tissue. J. Ass. Off. Anal. Chem. 49: 1046-1052. Bowman, M.C. and M. Beroza. 1966b. Pesticide Residue. Determination of Imidan and Imidoxon in sweet corn by gas chromatographic detection. J. Ass. Off. Anal. Chem. 49: 1154-1157. Bremner, J.M. and W'.L. Banwart. 1974. Identifying volatile S . compounds. Sulfur Instit. J. Spring 1974, pp. 6-9. Brody, S.S. and J.E. Chaney. 1966. The application of a specific detector for phosphorus and for sulphur compounds-sensitive to sub-nanogram quantities. J. Gas Chromat. 4: 42-46. Haines, T.H. and R.J. Block. 1962. Sulfur metabolism in algae. I. Synthesis of metabolically inert chloroform-soluble sulfate esters by two Chrysomonads and Chlorella pyrenodosa. J. Protozool. 9: 33-38. Kates, M. 1972. Techniques of lipidology. Isolation, analysis and identification of l i p i d . American Elsevier Co., Inc., New York, pp. 610. Rouser, G., G. Kritchevsky, and A. Yamamoto. 1967a. Column chromatographic and associated procedures for separation and determination of phosphatides and glycolipids. In Lipid Chromatographic Analysis. G.V. Marinetti, (Ed.) Marcel Dekker, Inc., New York. Vol. 1, pp. 99-162. Rouser, G., G. Kritchevsky, G. Simon, and G.J. Nelson. 1967b. Quantitative analysis of brain and spinach leaf lipids employing S i l i c i c acid column chromatography and acetone for elution of glycolipids. Lipids 2: 37-40. Ryland, L.B. and M.W. Tamele. 1970. Thiols. In The Analytical Chemistry of Sulfur and Its Compounds. J.H. Karchmer, (Ed.) Wiley-Interscience, New York. Part I. pp. 465-519. Stevens, R.K. 1967. A rapid specific method for the gas chromatographic determination of organophosphate pesticides in cold pressed citrus o i l s . J. Ass. Off. Anal. Chem. 50: 1236-1242. - 141 -GENERAL SUMMARY AND CONCLUSIONS Information on s o i l l i p i d fractions i s both meagre and fragmentary. Of these l i p i d fractions the sulfolipid in s o i l has been ignored. The primary reason for this retarded development of sulfolipid research i s that suitable analytical methods and procedures are not available. The primary focus of this investigation, then, was to consider some aspects on the distribution of l i p i d sulfur in soils. A significant proportion of the work was devoted to a study of the characterization of l i p i d sulfur using gas-liquid, thin-layer and s i l i c i c acid column chromatography. The third chapter was an attempt to determine the.contents of l i p i d sulfur in thirty-seven soils and to identify relationships of the distribution of l i p i d sulfur with other s o i l factors. The distribution of the l i p i d sulfur was examined for a l l soils, organic and mineral samples, and soils of a kind grouped according to vegetation, drainage and horizon type. Lipid sulfur was found in a l l soils examined and the amount was very variable. There was a significant difference in contents of l i p i d sulfur between organic and mineral samples, whereas there did not seem to be a clear-cut distribution of the l i p i d sulfur between the corresponding mineral horizons of forest and grassland soils. However, i t is evident that l i p i d sulfur contents in mineral horizons are lower than those in organic horizons of the same forest soils and the l i p i d sulfur i s concentrated in organic so i l s . The l i p i d sulfur accounted for small parts of both the total sulfur and total l i p i d . Lipid sulfur contents as percent of total sulfur and as parts - 142 -per million of total l i p i d did not show a significant difference between organic and mineral samples. However, the distribution of l i p i d sulfur in the five different groups was significantly different depending on the expressions of the l i p i d sulfur contents. Fairly close relationships of the l i p i d sulfur were found with total sulfur, Hi-reducible sulfur and organic carbon. On the other hand, the relative l i p i d sulfur content was significantly correlated only with total l i p i d when the l i p i d sulfur content was expressed as % of total: sulfur, and with total sulfur when expressed as ppm of total l i p i d . The l i p i d sulfur content of mineral soils was not significantly correlated with any other s o i l factors whether expressed as ppm of s o i l , as % of total sulfur or as ppm of total l i p i d . On the other hand, the l i p i d sulfur contents of organic samples were significantly correlated with total sulfur and Hl-reducible sulfur and only with total sulfur when expressed as ppm of total l i p i d , and only with total l i p i d when expressed as % of total sulfur. These relationships were not consistent for a l l samples and for organic and mineral samples when samples were considered as five groups. The stepwise elimination regression analysis showed that the distribution of l i p i d sulfur in soils can be best explained by two s o i l factors, total l i p i d and total sulfur contents when the l i p i d sulfur was expressed as ppm of s o i l . When the l i p i d sulfur was expressed as % of total sulfur, the distribution of the l i p i d sulfur can be best explained by organic carbon and total l i p i d contents. However, the l i p i d sulfur can be best predicted by organic carbon and total sulfur contents when the l i p i d sulfur was expressed as ppm of total l i p i d . It is therefore imperative to use a suitable expression for the study of l i p i d sulfur distribution, so that the s o i l factors can' be chosen accordingly. - 143 -Fractionation of s o i l total l i p i d s was accomplished by using a s i l i c i c acid column chromatography. The distribution patterns of the three classes of lipids on the s i l i c i c acid column were similar for a l l eight soils studied regardless of s o i l types. Glycolipids were the dominant class of s o i l l i p i d s and they accounted for more than half of the total l i p i d s . The neutral lipi d s were around one-third of the total l i p i d s , polar lipids were only 4% of the total being the smallest component of the s o i l l i p i d s . The uniformity of the distribution in each fraction of the column chromatography suggests that the l i p i d s in these soils were similar in type and origin or that their distributions were affected by and made more uniform through interaction of microorganisms with other s o i l environmental factors. On the contrary, the s o i l l i p i d sulfur had no such a consistent similarity in the distribution pattern between corresponding classes for eight s o i l s , differing from s o i l to s o i l . Furthermore, i t was found that significant amounts of sulfur were, in a l l cases, recovered in these three classes. This finding clearly suggests that s o i l l i p i d sulfur i s present in a variety of forms. The ratios of the l i p i d sulfur to l i p i d as ppm of the l i p i d in each corresponding fraction and the ratios for whole soils have shown that the ratios for polar l i p i d fractions of a l l soils, except one, were always higher than those for whole soils and the ratios for neutral and glycolipid classes had lower values than the values for whole soils, except for neutral l i p i d fractions of two Humisol samples. This finding indicates that the neutral l i p i d fraction of these two Humisols could be the most useful fraction for further investigation to characterize the s o i l l i p i d sulfur, since this fraction, particularly - 144 -for two Humisols, contained the highest amounts of the l i p i d sulfur and yet the contents of the l i p i d sulfur in this fraction as ppm of l i p i d are higher than those in whole soils. Thin-layer and gas-liquid chromatographic behavior of the corresponding four fractions prepared from two Humisol samples using s i l i c i c acid column chromatography as described in the previous chapter were reported in the last chapter. Although the column chromatographic behavior of total li p i d s and l i p i d sulfur of two soils studied were similar to each other, thin-layer chromatographic behavior df the corresponding four fractions of two soils were not similar. These findings indicate that the individual component of one general l i p i d class fractionated from one s o i l differs from the same l i p i d class of the other s o i l even though the column chromatographic distribution of total lipids was similar for both. It was shown that the multiple development technique with the current developing solvent system could be used as a preparative TLC without applying the second column chromatography. It was also found that the sulfur-containing contaminants in the solvents used for l i p i d extraction and for elution from column chromatography masked gas-liquid chromatographic monitoring of sulfur-containing compounds in l i p i d extracts and fractions of column chromatography. However, the results provided by GLC equipped with a sulfur detector suggest that the GLC could be used in monitoring of the l i p i d sulfur by developing a sulfur base line for the solvent blank. This report has been of an exploratory nature, however some practical implications could be drawn from the information that was presented. Important suggestions were made with respect to methodology - 145 -and some of the information presented appears important in evaluating the suitability of a sample for the i n i t i a t i o n of the study on the sulfur-containing constituents of l i p i d in s o i l in particular. Several potentially useful aspects for future research activities are evident from this work, which would illuminate the characteristics of s o i l l i p i d sulfur fractions. Some of these aspects would include work on methods for quantitative and qualitative evaluation of s o i l l i p i d sulfur and chromatographic fractionation of them and considering interactions of the sulfur fraction with other l i p i d constituents in s o i l . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094753/manifest

Comment

Related Items