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

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THE D I S T R I B U T I O N OF L I P I D SULFUR  IN  S O I L S OF B R I T I S H COLUMBIA  BY  YEH MOON|CHAE  B. S c . , 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. S c . , W a s h i n g t o n 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 I N P A R T I A L FULFILLMENT  OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  xn  THE FACULTY OF GRADUATE STUDIES (Department  We a c c e p t t h i s  of S o i l  Science)  t h e s i s as conforming  required  to the  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA November 0  1979  Y e h Moon C h a e , 1979  In  presenting  an  advanced  the I  Library  further  for  his  of  this  written  shall  agree  thesis  in  at  University  the  make  that  it  thesis  purposes  for  partial' fulfilment  freely  permission may  representatives.  be  It  for  gain  of  British  Columbia  for  extensive  granted  of  University  British  by  the  is understood  financial  2075 Wesbrook Place Vancouver, Canada V6T 1W5  of  available  permission.  Department The  degree  scholarly  by  this  shall  the  requirements  Columbia, reference  copying Head  that  not  of  of  copying  be a l l o w e d  agree  and  of my  I  this  that  study. thesis  Department or  for  or  publication  without  my  - i i-  ABSTRACT  The not  fully  possible  significance of s o i l  lipids  t o man's e n v i r o n m e n t i s  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 roles  i n influencing  soil  structure,  infiltration  characteristics, phytotoxicity,  particulargy  o f P a n d S.  the f i r s t  and n u t r i e n t  T h e l i p i d s may a l s o  The  reported  cycling  a c t as a s i n k f o r The p r e s e n t  study  investigation of sulpholipids  distribution of s o i l  lipid  suggest  h y d r o p h o b i c i t y and  p o l y n u c l e a r h y d r o c a r b o n s a n d some p e s t i c i d e s . constitutes  that  s u l f u r was  i n soil.  studied  particularly  i n r e l a t i o n to the factors which influence  the content  of  s u l f u r under d i f f e r e n t s o i l environments.  Further  the l i p i d  were c a r r i e d out t o f r a c t i o n a t e of  The soils  in had  lipids  column chromatography and t o c h a r a c t e r i z e  t h i n - l a y e r and g a s - l i q u i d  the  total  and l i p i d soil  s u l f u r b y means  lipid  sulfur, using  chromatography.  i n v e s t i g a t i o n of the d i s t r i b u t i o n of l i p i d  showed t h a t  lipid  sulfur i n  s u l f u r was f o u n d i n a l l s o i l s e x a m i n e d b u t  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  organic horizons than i n mineral horizons, higher l i p i d  studies  s u l f u r than f r e e l y drained  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 lipid  s u l f u r accounted f o r a small  total  lipids.  The l i p i d  soils.  drained  The h i g h e s t  drained organic s o i l s .  percentage of t o t a l  phosphorus contents.  correlated  only with  s u l f u r a n d 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  lipid  s u l f u r and o f  The l i p i d  total  soils  The  s u l f u r c o n t e n t s were on a v e r a g e n e a r l y  times higher than the l i p i d c o n t e n t was s i g n i f i c a n t l y  and p o o r l y  and  three  sulfur  Hl-reducible  factors  examined.  -  The  d i s t r i b u t i o n of l i p i d  the  two s o i l  factors,  when a s u i t a b l e the  sulfur i n s o i l  i.e., total lipid  c o n t e n t , when t h e l i p i d m i l l i o n of s o i l .  can be b e s t e x p l a i n e d by content and t o t a l s o i l  sulfur  s u l f u r content was expressed as p a r t p e r  Therefore, s o i l  factors  e x p r e s s i o n f o r the l i p i d  d i s t r i b u t i o n of the l i p i d The  i i i -  fractionation  p o l a r l i p i d s , on a s i l i c i c  s u l f u r content was used f o r  sulfur i n s o i l .  of s o i l  into three general classes,  total lipids,  i . e . , neutral acid  p a t t e r n of the t h r e e c l a s s e s  can be chosen a c c o r d i n g l y  f o r selected  soils,  l i p i d s , g l y c o l i p i d s and  column has shown t h a t  the d i s t r i b u t i o n  of l i p i d s was s i m i l a r f o r a l l e i g h t  s o i l s studied regardless of s o i l  type.  The u n i f o r m i t y of the  d i s t r i b u t i o n p a t t e r n s suggests that  the l i p i d s i n these s o i l s were  s i m i l a r i n type 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  by,  and made more u n i f o r m through, i n t e r a c t i o n  other s o i l had  environmental f a c t o r s .  no such a c o n s i s t e n t  d i f f e r i n g from s o i l was t h a t  The s o i l  affected  of microorganisms w i t h  lipid  s u l f u r , however,  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 p a t t e r n ,  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  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, r e c o v e r e d  i n each g e n e r a l c l a s s , i n c o n t r a s t t o the f i n d i n g  t h a t most o f the  t o t a l l i p i d s were r e c o v e r e d i n b o t h n e u t r a l  and g l y c o l i p i d  classes,  exclusively.  This finding  s u l f u r i s present i n a v a r i e t y  c l e a r l y suggests t h a t  classes,  was conducted to c h a r a c t e r i z e  soil  lipid  of forms.  O b s e r v a t i o n of the t h i n - l a y e r b e h a v i o r of g e n e r a l l i p i d  lipid  soil  and g a s - l i q u i d  fractionated lipid  sulfur.  chromatographic  from two s o i l  samples,  T h i n - l a y e r chromato-  - iv -  graphic  b e h a v i o r of the c o r r e s p o n d i n g l i p i d  were not  s i m i l a r to each o t h e r ,  b e h a v i o r of the l i p i d other. of one  c l a s s e s of the two  a l t h o u g h column chromatographic  c l a s s e s of the two  s o i l s were s i m i l a r to each  These d i s s i m i l a r i t i e s i n d i c a t e t h a t the general  lipid  b e h a v i o r of g e n e r a l  i n d i v i d u a l component  c l a s s f r a c t i o n a t e d from one  that of the o t h e r s o i l .  soils  soil differs  from  O b s e r v a t i o n on the g a s - l i q u i d chromatographic  lipid  c l a s s e s suggested t h a t GLC  c o u l d be used i n  m o n i t o r i n g the s u l f u r - c o n t a i n i n g compounds i n l i p i d  e x t r a c t s of  by choosing a s u i t a b l e column, and  of h i g h  organic  Although the attempt t o s e p a r a t e and containing  lipid  components by  phase of i n v e s t i g a t i o n f a i l e d of i n d i v i d u a l l i p i d and  solvents  characterize  purity.  individual sulfur-  chromatographic methods i n t h i s l a s t to s i g n i f i c a n t l y advance our  constituents,  knowledge  p a r t l y because of t e c h n i c a l problems  p a r t l y because of l a c k of time, i t served to re-emphasize  c o m p l e x i t y b o t h of s o i l  l i p i d s i n general,  containing  in particular.  constituents  soil  and  the  of t h e i r s u l f u r -  I t a l s o i n d i c a t e d some of  more p r o m i s i n g l i n e s of i n v e s t i g a t i o n f o r f u t u r e  studies.  the  - v -  TABLE OF CONTENTS  Page  ABSTRACT  i  TABLE OF CONTENTS  i v  L I S T OF TABLES  viii  L I S T OF FIGURES  x  ACKNOWLEDGEMENTS  x i i  CHAPTER 1.  INTRODUCTION  1  CHAPTER 2.  REVIEW OF L I T E R A T U R E  4  1.  SOIL L I P I D S  4  1.1  Introduction  4  1.2  Methods o f E x t r a c t i o n  5  1.3  Content o f S o i l L i p i d s  8  1.4  Significance of Lipids i n Soils  1.5  Persistence of L i p i d s i n Soils  1.6  Chemistry of S o i l L i p i d s  15  1.6.1  Waxes  15  1.6.2  Acids  17  1.6.3  Hydrocarbons  21  1.6.4  Fats  25  1.6.5  Phospholipids  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 .  1.6.7  Carotenoids  1.6.8  Ketones  29  1.6.9  Miscellaneous  29  and  . . .11 . . . 13'  .27  Chlorophylls.28  - v i -  2.  NATURALLY OCCURRING S U L F O L I P I D S  30  2.1  Introduction  30  2.2  Mammalian S u l f o l i p i d s  32  2.3  Plant Sulfolipids  35  2.4  Microbial  37  2.5  Analytical  2.6  Isolation  2.7  Soil  Sulfolipids.  M e t h o d s o f S u l f o l i p i d s . . 41 of S u l f o l i p i d s  44  Sulfolipids  .47  REFERENCES CHAPTER 3.  48  THE D I S T R I B U T I O N OF SULFUR I N L I P I D  EXTRACTS  OF S O I L S  , . 60  INTRODUCTION  60  METHODS AND MATERIALS  61  Soils  .61  Extraction Analytical  63 Methods  64  RESULTS AND D I S C U S S I O N . O r g a n i c C a r b o n and T o t a l P h o s p h o r u s  65  Sulfur  69  Lipid The  CHAPTER 4.  65  and L i p i d  Phosphorus  Distribution  of the Lipid  72 Sulfur i n S o i l s ^  CONCLUSION .  86  REFERENCES  89  THE COLUMN CHROMATOGRAPHIC FRACTIONATION OF TOTAL L I P I D S AND L I P I D SULFUR I N SOME SELECTED B R I T I S H COLUMBIAN S O I L S 92 INTRODUCTION . .  92  METHODS AND MATERIALS.  93  - v i i-  RESULTS AND D I S C U S S I O N  CHAPTER 5.  95  CONCLUSION  102  REFERENCES  104  OBSERVATIONS ON THE GAS-LIQUID AND THIN-LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS I N TWO SELECTED S O I L S  105  INTRODUCTION.  105  METHODS AND MATERIALS  106  Soils  106  A n a l y t i c a l Methods  J06  Column C h r o m a t o g r a p h y  108  T h i n - L a y e r Chromatography  109  G a s - L i q u i d Chromatography  HO  RESULTS AND D I S C U S S I O N  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  - viii  -  L I S T OF T A B L E S Table  Page  CHAPTER 3.  THE D I S T R I B U T I O N OF SULFUR I N L I P I D EXTRACTS OF S O I L S  3.1  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 basis)  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 p h o s p h o r u s c o n t e n t s a n d pH v a l u e s among s o i l g r o u p s  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  Relationships of t o t a l sulfur with the 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 g r o u p s  70  The d i s t r i b u t i o n o f l i p i d phosphorus i n s o i l s  74  3.2  3.3  3.4  3.5  3.6  3.7  3.8  3.9  3.10  and l i p i d  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 soils  80  sulfur i n  Correlation coefficients f o r 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  sulfur 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 a n d L i p i d S / L i p i d v e r s u s some s o i l properties  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 b e t w e e n l i p i d s u l f u r a s ppm o f s o i l , a s % o f t o t a l s u l f u r , a n d a s 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 o l u m b i a n s o i l s  85  - i x -  Table  Page  CHAPTER 4.  THE COLUMN CHROMATOGRAPHIC FRACTIONATION OF TOTAL L I P I D S AND L I P I D SULFUR I N SOME SELECTED B R I T I S H COLUMBIAN SOILS  4.1  Some c h e m i c a l a n a l y s e s o f s o i l  4.2  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  4.3  Association with l i p i d l i p i d s u l f u r and t o t a l and l i p i d s u l f u r  samples . . .  94  98  classes of s o i l recovery of l i p i d .101  CHAPTER 5.  OBSERVATIONS ON THE G A S - L I Q U I D AND THIN-LAYER CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND L I P I D SULFUR FRACTIONS I N 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  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 from S i l i c i c a c i d column  113  5.2  - x -  L I S T OF  FIGURES  Figure  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 I N TWO SELECTED S O I L S  5.1  T w o - d i m e n s i o n a l m a p p i n g 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 extract of s o i l 1 . . .  114  T w o - d i m e n s i o n a l m a p p i n g TLC o f F r a c t i o n s 1, 2, 3 a n d 4 e l u t e d f r o m S i l i c i c a c i d column of t o t a l l i p i d extract of s o i l 2  115  M u l t i p l e d e v e l o p m e n t 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 of s o i l 1 '  117  M u l t i p l e d e v e l o p m e n t TLC o f F r a c t i o n s 1, 2, 3 a n d 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 of s o i l 2  118  M u l t i p l e d e v e l o p m e n t TLC o f F r a c t i o n s A, B, C, D, E a n d 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 the f i r s t S i l i c i c a c i d column of s o i l 1 • •  119  One-dimensional mini-TLC of the F r a c t i o n s A - F e l u t e d from S i l i c i c a c i d columns o f s o i l 1  120  GLC o f c o n c e n t r a t e d r e a g e n t chloroform  122  5.2  5.3  5.4  5.5  5.6  5.7  5.8  5.9  5.10  GLC o f t o t a l soil 1  lipid  extracted  GLC o f F r a c t i o n 1 o f l i p i d from s o i l 1 GLC  grade  from 123 extracted  of concentrated e x t r a c t i o n blank  124 126  - x i -  5.11  GLC  5.12  GLC o f F r a c t i o n 1 o f t o t a l e x t r a c t e d from s o i l 2  lipid  GLC  extracted  5.13  of t o t a l l i p i d  extracted  of F r a c t i o n 1 of l i p i d  from s o i l  soil  2  127  128  1  130  5.14  GLC  of F r a c t i o n A from s o i l  1  131  5.15  GLC  of F r a c t i o n B from s o i l  1  132  5.16  GLC  of F r a c t i o n C from s o i l  1  133  5.17  GLC  of F r a c t i o n D from s o i l  1  134  5.18  GLC  of 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 from s o i l  1  136  - x i i-  ACKNOWLEDGEMENT S  My s i n c e r e a p p r e c i a t i o n i s extended  t o Dr. L.E. Lowe,  p r o f e s s o r , Department of S o i l S c i e n c e , f o r h i s encouragement and v a l u a b l e guidance a t a l l s t a g e s o f t h e study and p r e p a r a t i o n o f this thesis.  Suggestions by Dr. C A . Rowles, Dr. A.A. Bomke,  Dr. Mary Barnes and Dr. J . F . R i c h a r d s a r e a l s o g r a t e f u l l y acknowled A s p e c i a l thanks t o my w i f e f o r her c o n s t a n t p a t i e n c e and  encouragement throughout  t h e p e r i o d of my study.  CHAPTER 1  INTRODUCTION  P a s t r e s e a r c h on s o i l l a r g e l y w i t h the nature i n recent years,  organic matter  has been concerned  and o r i g i n o f t h e humic s u b s t a n c e 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 s u c h a s t h e a m i n o a c i d a n d fractions.  I n f o r m a t i o n on s o i l  fragmentary. from s o i l  lipid  fractions  carbohydrate  i s b o t h meager and  T h e y h a v e 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  chemists,  difficulty  although,  no d o u b t p a r t l y b e c a u s e o f t h e a l m o s t  insuperable  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 o f components  r e c e n t l y , but a l s o because these  lipids  from t h e p o i n t o f view of agronomists.  seemed  of l i t t l e  However,  until  significance  t h e p e r f e c t i o n o f 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 o f l i p i d s has been an important penetrating suggesting and  investigation.  f a c t o r i n opening t h i s f i e l d  t o more  Furthermore, there a r e i n d i c a t i o n s  possible roles influencing soil  structure, hydrophobicity  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  particularly  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 a s 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 a n d 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 been c o m p l e t e l y phospholipids  ignored.  components, t h e s u l f o l i p i 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  of a g r i c u l t u r a l  soils.  i n s o i l has on t h e  P h o s p h o l i p i d s have been  found  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 s o u g h t 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  than  - 2 -  phospholipids,  i t now a p p e a r s  development of s u l f o l i p i d  they a r e u b i q u i t o u s .  The r e t a r d e d  r e s e a r c h h a s 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 assayed  by t h e molybdenum b l u e method. The  primary focus of t h i s  investigation,  c o n s i d e r some a s p e c t s o n t h e d i s t r i b u t i o n o f l i p i d More s p e c i f i c a l l y  s u l f u r under d i f f e r e n t  F u r t h e r s t u d y was c a r r i e d  out t o determine  useful i n monitoring the l i p i d  a silicic  a c i d column.  sulfur  soil  thin-layer  and s i l i c i c ' a c i d  solvents exhibited  i n characterizing  chromatograph equipped second  from  sulfur using gas-  column chromatographies.  significant  development o f a " s u l f u r base l i n e "  The  chromatography  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 w o r k was  liquid,  a major problem  influence  i n each.fraction eluted  to a study of the c h a r a c t e r i z a t i o n of l i p i d  commercial  i n soils.  environments.  i fgas-liquid  devoted  all  sulfur  some o f t h e f a c t o r s w e r e c o n s i d e r e d w h i c h  the content of t h e l i p i d  is  t h e n , was t o  sulfur  Because  impurities, the  f o r t h e s o l v e n t blanks proved  soil  sulfur  t o be  l i p i d s u s i n g a gas  with a sulfur detector.  chapter has reviewed  naturally occurring sulfolipids.  a r t i c l e s on s o i l  The subsequent  l i p i d s and  chapters are i n the  form 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 in  scientific journals,  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  consideration of the topic outlined. d i s t r i b u t i o n of l i p i d closely  sulfur  i n soils.  The t h i r d  The c o n t e n t o f l i p i d  examined i n r e l a t i o n t o o t h e r s o i l  factors  carbon 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 , phosphorus c o n t e n t s .  c h a p t e r examines t h e sulfur  was  s u c h a s pH, o r g a n i c  and t o t a l and  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  lipid  attempted  - 3 -  from the a n a l y s e s t h a t were done, p r i m a r i l y to g a i n  information  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 f u r t h e r r e l a t i o n to the sulfur in  i s o l a t i o n and  of the  in  lipid  soils. The  f r a c t i o n a t i o n of l i p i d  eight selected  soil  lipid  in s o i l  adopted.  i n the f o u r t h c h a p t e r .  s u l f u r which had The  not  previously  i s o l a t i n g and  f i f t h chapter d e s c r i b e s characterizing  two  selected  soil  the  effectiveness  samples.  a study t h a t  individual lipid The  In  the been used  d i s t r i b u t i o n of t o t a l l i p i d s  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 The  a c i d column of  a c i d column chromatography f o r  f r a c t i o n a t i o n of the s t u d i e s , was  s u l f u r on a s i l i c i c  samples i s d e s c r i b e d  this investigation a s i l i c i c  lipid  structure-determination  studies  on  soil  and  environments.  examines methods of  s u l f u r components i n  r e s u l t s of t h i s study have demonstrated  of a combination of g a s - l i q u i d , t h i n - l a y e r and  chromatographies i n i s o l a t i n g one  particular sulfur-containing  column lipid  component. In the l a s t c h a p t e r , a l l t o p i c s o u t l i n e d f o u r and  f i v e are  on the b a s i s sections.  summarized and  of the  i n chapters  an attempt i s made to draw  e x p e r i m e n t a l r e s u l t s o b t a i n e d from the  threes  conclusions relevant  - 4 -  CHAPTER 2  REVIEW OF LITERATURE  1.  SOIL LIPIDS  1.1.  Introduction  The  importance  o f s o i l o r g a n i c matter i n modern s o i l s c i e n c e  has l o n g been r e c o g n i z e d .  P a s t r e s e a r c h has been concerned  largely  w i t h .humic substances and has p a i d l e s s a t t e n t i o n t o other components of are  s o i l organic f r a c t i o n s .  These components of s o i l o r g a n i c matter  v a r i o u s l y known i n s o i l s c i e n c e as waxes and r e s i n s , bitumens, or  simply as o r g a n i c substances e x t r a c t e d w i t h an a l c o h o l - b e n z e n e m i x t u r e . A c c o r d i n g t o s e p a r a t i o n c o n d i t i o n s from s o i l and t o many p r o p e r t i e s , 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 b i o chemistry.  The a l c o h o l - b e n z e n e f r a c t i o n may c o n t a i n some s p e c i f i c  humic substances, e.g., hymatomelanic a c i d , and some r e s i n o i d  substances  and f r e e amino a c i d s . S o i l s undoubtedly  c o n t a i n a wide v a r i e t y of l i p i d s r a n g i n g  from t h e r a t h e r s t a b l e p a r a f f i n i c hydrocarbons degradation products.  Lipids are distributed  t o ephemeral c h l o r o p h y l l extensively  the s o i l s o f t h e world, r a n g i n g from the h i g h l y weathered of  the humid t r o p i c s t o the weekly developed  In  normal a e r o b i c s o i l s ,  throughout laterites  tundras of t h e a r c t i c  zone.  t h e l i p i d s p r o b a b l y e x i s t l a r g e l y as remnants  of m i c r o b i a l t i s s u e ; low and v a r i a b l e q u a n t i t i e s of these c o n s t i t u e n t s  - 5 -  may  be a s s o c i a t e d w i t h undecomposed  living  p l a n t r e s i d u e s and t h e b o d i e s o f  and dead m i c r o f l o r a l o r o t h e r In  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  ( 1 9 0 7 - 1 9 1 1 ) , i n t h e i r now substances  organisms.  from s o i l s ,  Shorey  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  obtained s e v e r a l l i p i d preparations which  they were a b l e a t l e a s t p a r t l y  to characterize.  S i n c e t h e n t h e most  extensive s t u d i e s of the l i p i d s  of m i n e r a l s o i l s have been  r e p o r t e d by M e i n s h e i n and Kenny  ( 1 9 5 7 ) a n d Wang e t a l . ( 1 9 6 9 a n d  The l i p i d s , b y Howard  o r , more s p e c i f i c a l l y ,  Morrison  1.2  (1969).  (1969), B r a i d s and M i l l e r  (1975), and F r i d l a n d  1971).  reviewed  Extensive  l i p i d s w e r e p u b l i s h e d by S t e v e n s o n  (1966),  (1976).  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 of  t h e waxes o f p e a t have been  and Hamer ( 1 9 6 0 ) a n d W o l l r a b a n d S t r e b l  r e v i e w s of t h e s u b j e c t o f s o i l  those  factors.  M u c h o f t h e l i p i d s may  from  soil  be l i n k e d  i s c o m p l i c a t e d b y a number i n some f o r m o f  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 generally insoluble i n organic solvents. in  their  bially  s t u d y on l i p i d s  Wagner a n d M u z o r e w a  of m i c r o b i a l o r i g i n , pointed out that micro-  s y n t h e s i z e d p r o d u c t s o f a l i p i d n a t u r e i n s o i l may  incorporated  into soil  (1977),  humus w i t h o u t u n d e r g o i n g  become  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 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  significant materials.  -  It  6  i s o f t e n p o s s i b l e to l i b e r a t e protein-bound l i p i d  use  of  The  a d d i t i o n of ether  and  such solvent mixtures are  and  plant  ethanol.  However, f o r most l i p i d s , e t h a n o l  and  Muzorewa,  1977).  Bick,  The a f f e c t e d by  1967;  i r o n s a l t s and  is required  occur i n s o i l  t h e y c a n n o t be  s a l t s are poorly  and  i s o l a t e d by  s o l u b l e . i n organic  iron. i n the  s o i l s as  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  solvents.  the y i e l d  and  further  Part  of  such  the l i p i d s ,  form of aluminum  An  acid  pretreatment  Pretreatment with shown by H a n c e  of l i p i d  or  because  through 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  and  phosphate  Wang e t a l . ( 1 9 6 9 ) a l s o showed t h a t c l a y - b o u n d extracted  animal  well  Wagner  simple.extraction  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  t r i g l y c e r i d e was  ethanol,  inorganic material  t o e x t r a c t them ( F r i d l a n d , 1 9 7 6 ) .  extraction.  of  l i p i d s i s l i k e l y t o be  of. aluminum o r  f o r e x a m p l e , may  solvent.  e f f e c t i v e n e s s of  R e w a l d , 1944)  l a r g e amounts of  cations  by  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  soil  the presence of and  i s a poor  F u s t e c ^ M a t h o n e t a l . , 1977;  s o l u b i l i t y of  clay minerals  fatty acids  the  or benzene improves the  t i s s u e s ( B l o o r , 1915;  (Morrison  as  -  during  unsaturated acid  pretreatment.  drying lipid  of  The  extractability  the  soil,  s o l u b i l i t y by  f a t t y a c i d on  out  l i p i d s may  p r o d u c i n g changes i n the constituents  clay minerals  b e t w e e n f a t t y a c i d and (1963) p o i n t e d  soil  be  a f f e c t e d by  since i t i s generally considered  These changes i n the the  of  that'drying  acid  reduces  constituents.  p r o b a b l y enhance the a d s o r p t i o n  and  clay mineral  that the  fatty  the  l e s s e n the t o be  s o l u b i l i t y of  liability  broken. soil  of  H a n c e and  l i p i d s was  the  of  bonding  Anderson  greater  in  - 7 -  f r e s h than i n d r i e d s o i l s , but i f the l a t t e r were p r e t r e a t e d  with  a m i x t u r e o f h y d r o f l u o r i c and h y d r o c h l o r i c a c i d s the d i 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  a factor complicating  e x t r a c t s may a l s o be  the e x t r a c t i o n 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 n o t a l l be water s o l u b l e and many l i p i d s a r e not s o l u b l e i n a l l f a t s o l v e n t s . p a r t i c u l a r l y acetone and e t h a n o l , organic  Some l i p i d  can e x t r a c t i n o r g a n i c or n o n - l i p i d  substances, and t h i s p o s s i b i l i t y should  i n v e s t i g a t i o n s of s o i l l i p i d s .  solvents,  be c o n s i d e r e d i n  The water-washing procedure a l s o 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 t o 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 l o s s of l i p i d  of n o n - l i p i d substances i n t h e l i p i d Both t h e y i e l d and chemical by o r g a n i c  extract  and r e t e n t i o n  ( N a z i r and Rouser, 1966).  n a t u r e of the m a t e r i a l  extracted  s o l v e n t s from s o i l s a r e i n f l u e n c e d by the n a t u r e of the  s o l v e n t and t h e c o n d i t i o n s of e x t r a c t i o n .  More m a t e r i a l i s e x t r a c t e d  by s t r o n g l y p o l a r than by weakly p o l a r s o l v e n t s and the g r e a t e s t amount by a m i x t u r e of t h e two.  The e x t r a c t e d m a t e r i a l c o n s i s t s of a complex  m i x t u r e o f compounds o f f a i r l y h i g h m o l e c u l a r Bick  weight.  M o r r i s o n and  (1967) a r b i t r a r i l y separated, these i n t o a s p h a l t , r e s i n and wax  f r 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 p o l a r and non-polar solvents. Very r e c e n t l y S c i a c o v e l i e t al_. (1977) e x t r a c t e d lipid  different  f r a c t i o n s from a brown s o i l of I t a l y u s i n g weak o r g a n i c  with d i f f e r e n t p o l a r i t i e s .  solvents  The e x t r a c t i o n sequence was e t h y l e t h e r ,  benzene, acetone, dioxane, t e t r a h y d r o f u r a n ,  ethyl alcohol,  - 8 -  dimethylformamide,  p y r i d i n e , d i m e t h y l s u l f o x i d e and formamide.  By  i n f r a - r e d and para-magnetic resonance s p e c t r o m e t r i e s they found f r a c t i o n s e x t r a c t e d w i t h e t h y l e t h e r , benzene and acetone were c h a r a c t e r i z e d by a preponderance of a l i p h a t i c s t r u c t u r e s ; t h e f r a c t i o n s w i t h dioxane, t e t r a h y d r o f u r a n and e t h y l a l c o h o l were c h a r a c t e r i z e d by a h i g h c o n t e n t o f oxygenated  compounds and by t h e  presence o f hydroquinone u n i t s or hydroquinone p o l y m e r i c c h a i n s ; and t h e f r a c t i o n s w i t h dimethylformamide  and d i m e t h y l s u l f o x i d e were  c h a r a c t e r i z e d by t h e presence o f p a r t i c u l a r s k e l e t o n s , p r o b a b l y based upon h y d r o x y l a t e d naphtho- o r anthraquinone u n i t s . There a r e c l e a r l y many p o s s i b l e combinations of s o l v e n t s , and t h e i r s e l e c t i o n w i l l depend on t h e type of m a t e r i a l and the n a t u r e of the predominant  lipids.  However, t h e most e f f e c t i v e s o l v e n t f o r s o i l s  cannot be p r e d i c t e d because the n a t u r e 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 o f s o l v e n t s and combinations of them seems essential.  1.3  Content of S o i l  The l i p i d  Lipids  content o f s o i l o r g a n i c matter i s o n l y a s m a l l and  v a r i a b l e p a r t o f t h e o r g a n i c matter.  No p r e c i s e v a l u e can be estimated  s i n c e no s t a n d a r d or g e n e r a l l y accepted method of d e t e r m i n a t i o n i s available.  Much i n f o r m a t i o n i n t h e l i t e r a t u r e on t h e amount of l i p i d  e x t r a c t e d from s o i l s by o r g a n i c s o l v e n t s cannot be c o o r d i n a t e d because o f t h e use of d i f f e r e n t s o l v e n t s and c o n d i t i o n s .  However,  the method o f proximate a n a l y s i s proposed by Waksman and Stevens  (1930),  - 9 -  a l t h o u g h . t h e method i s n e i t h e r complete nor s e l e c t i v e f o r l i p i d s , p r o v i d e s the b e s t comparative range of  values a v a i l a b l e f o r a f a i r l y  wide  soils. The range f o r the l i p i d  and h i s coworkers (1930 and  c o n t e n t of humus found by Waksman  1935), w i t h few e x c e p t i o n s , f e l l w i t h i n  the range of 1 to 6% of the t o t a l o r g a n i c matter.  Similar  results  were o b t a i n e d by Shewan (1938) f o r some s o i l s from S c o t l a n d . range f o r the l i p i d agriculturally  This  c o n t e n t i s p r o b a b l y c h a r a c t e r i s t i c of most  important  s o i l s of the w o r l d .  For the m i n e r a l  soils  the h i g h e s t v a l u e s , up to 16% of the humus, o c c u r r e d i n the p o d z o l i c soils.  In the case of the o r g a n i c s o i l s the highmoor peats c o n t a i n e d  l a r g e r amounts than the lowmoor and  sedimentary  peats  (Stevenson,  1966). The h i g h e s t v a l u e r e p o r t e d f o r the l i p i d humus appears found  t o be t h a t of P i e t t r e  ( r e f e r e n c e i n Stevenson,  t h a t n e a r l y one h a l f of the o r g a n i c matter  p l a n t a t i o n s o i l s i n B r a z i l was  by Minsen ( r e f e r e n c e i n Stevenson, (1966),  soil 1966)  who  i n some c o f f e e  i n the form of f a t t y or waxy m a t e r i a l .  N e a r l y o n e - t h i r d ' of the o r g a n i c matter  Stevenson  content of  i n some p o l l e n peats  1966)  was  r e c o v e r e d as  examined  lipid.  on the b a s i s of r e s u l t s o b t a i n e d by  methods, i n d i c a t e d t h a t the v a r i a t i o n noted  i n the l i p i d  such  content of  humus can be e x p l a i n e d by d i f f e r e n c e s i n v e g e t a t i o n , pH or a combination  of these two  factors.  t y p i c a l l y a s s o c i a t e d w i t h low pH,  The h i g h l i p i d and  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 .  contents are  t h i s i n t u r n suggests The  a  f a c t t h a t the amount of  r e c o v e r e d from peats d e r i v e d from Phragmites and  Sphagnum was  lipid  lower  - 10 -  than from peats d e r i v e d from c o t t o n g r a s s , heather and S c i r p u s e x p l a i n s the v a r i a t i o n  i n the l i p i d  c o n t e n t s due t o d i f f e r e n c e s  in vegetation. Fustec-Mathon e t . a l . (1975 and 1977) the l e v e l of bitumens i n two and  soil  of bitumens than those of hydromorphic  levels  c h e m i c a l compounds.  microbial  They concluded t h a t the h i g h  o f bitumens i n these sandy p o d z o l s a r o s e from the low m i c r o b i a l  a c t i v i t i e s due  to the i n c r e a s e i n the a c i d i t y of the  environment.  They a l s o concluded t h a t the a c i d i t y of the environment significant role  had a more  i n the p r o d u c t i o n of the bitumens than hydromorphy.  Fridlarid  (1976), i n h i s r e v i e w a r t i c l e , p o i n t e d out  t h e r e 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  c o n t e n t of s o i l  o r g a n i c matter  that  extracted with  a l c o h o l - b e n z e n e m i x t u r e s and pH or m i c r o b i a l a c t i v i t y . lipid  soils.  slower t r a n s f o r m a t i o n and  of p l a n t d e b r i s and a more s i g n i f i c a n t  p r o d u c t i o n of new  on  that podzols contained  With r e s p e c t to the p o d z o l s , they observed decomposition  study  types under v e r y s i m i l a r v e g e t a t i o n  from the same sandy r e g i o n , observed  c l e a r l y higher l e v e l s  i n their  The maximum  i s c o n f i n e d to s o i l s w i t h  b i o l o g i c a l a c t i v i t y , i . e . , to hydromorphic  and v e r y a c i d i c  low  s o i l s , and  the minimum t o steppe s o i l s w i t h h i g h e r b i o l o g i c a l a c t i v i t y and forest pH.  s o i l s on c a l c a r e o u s p a r e n t m a t e r i a l ,  He a l s o noted  to  i . e . , s o i l s with a n e u t r a l  t h a t the content of l i p i d s i n c r e a s e s , as a  rule,  w i t h an i n c r e a s e i n the t o t a l carbon c o n t e n t , except  i n some s o i l s , and  t h a t the d i s t r i b u t i o n  the same i n most  s o i l groups. (the  The  of l i p i d s i n s o i l h o r i z o n s was  lipid  relative lipid  content as percentage of s o i l  content)  o r g a n i c matter  i n c r e a s e s s l i g h t l y w i t h depth w h i l e the  - 11 -  lipid  content as percentage  of s o i l weight  (the a b s o l u t e l i p i d  content) decreases s h a r p l y because of the g e n e r a l decrease i n carbon content.  The  s l i g h t i n c r e a s e i n the r e l a t i v e l i p i d  content w i t h  depth i s p r o b a b l y a s s o c i a t e d w i t h the decrease i n m i c r o b i a l on them, r e s u l t i n g relative lipid  i n their preservation.  action  However, sometimes the  content a l s o d e c r e a s e s down the p r o f i l e , a p p a r e n t l y  because the p r e s e r v a t i o n of the l i p i d s cannot be compensated by decrease i n t h e i r supply t o deeper  horizons.  the  It i s also uncertain  whether l i p i d s as they a r e , can m i g r a t e down the p r o f i l e because they a r e i n s o l u b l e i n water per d e f i n i t i o n , a l t h o u g h l i p i d s of low waters o l u b i l i t y a r e expected as a bound l i p i d  t o m i g r a t e w i t h s o i l humic and  fraction  ( S c h n i t z e r , 1975).  Fridland  f u l v i c acids (1976) a l s o  found t h a t 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 in  the  horizon  of the z o n a l s o i l  same curve of the Ch:Cf r a t i o s , r a t i o have a minimal  lipid  s e r i e s i s a m i r r o r image of the  i . e . , s o i l s w i t h the h i g h e s t Ch:Cf  content.  T h i s shows t h a t l i p i d s  the e f f e c t of the same f a c t o r s as humic and f u l v i c  1.4  experience  acids.  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  the p o s s i b l e causes of s o i l n a t u r e may  content  waterproof  i n s o i l has been c o n s i d e r e d as one of  'fatigue'.  Waxy substances of  lipid  s o i l p a r t i c l e s to prevent movement of n u t r i e n t s  from m i n e r a l s u r f a c e s i n t o s o l u t i o n f o r p l a n t uptake.  They  may  s i m i l a r l y r e t a r d m i n e r a l i z a t i o n of o r g a n i c n i t r o g e n (Wagner and Muzorewa, 1977).  Low m o l e c u l a r weight  components may  behave as o r g a n i c  - 12 -  n u t r i e n t s , and a r e a l s o l i k e l y growth.  to produce harmful  e f f e c t s on p l a n t  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 agents f o r s o i l m i e r o f l o r a l populations. e t a l . (1975) p o i n t e d  out t h a t l i p i d s  microorganisms of s o i l .  Fustec-Mathion  as a whole a r e i n h i b i t o r s f o r  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 , o n pH o f s o i l o r i g i n of m i c r o f l o r a .  The d e p r e s s i v e  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 groups of l i p i d s  and on t h e  a c t i o n a l s o d e p e n d s t o some  and t h e n a t u r e  of f u n c t i o n a l  ( F u s t e c - M a t h o n e t a l . , 1977) . ;  There 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 long-chain  compounds c a n r e s u l t  substances, of  soil  lipid  possibly polysaccharide  crumbs  gum,  components c o u l d e x e r t s t a b i l i z i n g  l o o s e l y held aggregates  e f f e c t on s o i l  of  which increase the s t a b i l i t y  ( F e h l and Lange, 1965; M a r t i n e t a l . , 1959).  cementing m i n e r a l p a r t i c l e s to  i n increased m i c r o b i a l production  f o r c e s upon s o i l  together,, and.imparting (Wagner and M u z o r e w a ,  Numerous  a g g r e g a t e s by  water r e p e l l e n c y  1977).  An  indirect  f e r t i l i t y i s thus p o s s i b l e .  A t t e m p t s t h a t h a d b e e n made t o r e l a t e t h e p r o d u c t i v i t y o f soils  to the presence of l i p i d s or t h e i r  revealed  transformation  t h a t a n i n c r e a s e i n t h e amount o f l i p i d  productivity.  D e t a i l e d information concerning  s o i l has been g i v e n  i n a review  by S t e v e n s o n  in soil  products, reduces  soil  the r o l e of l i p i d s i n (1966).  - 13 -  1.5  P e r s l s t a n c e 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 systems i n and on the s o i l . in s o i l ,  of b i o l o g i c a l  In the decomposition of these r e s i d u e s  c a r b o h y d r a t e s and p r o t e i n s undergo h y d r o l y s i s to y i e l d water  s o l u b l e p r o d u c t s o f low m o l e c u l a r weight,  that are u l t i m a t e l y  d e s t r o y e d i n b i o c h e m i c a l and c h e m i c a l p r o c e s s e s .  In c o n t r a s t , many  l i p i d s which a r e a l s o s u b j e c t t o some changes f o l l o w i n g from b i o l o g i c a l systems,  elimination  a r e e i t h e r p r e s e r v e d i n t a c t or converted  into  t r a n s f o r m a t i o n p r o d u c t s t h a t a r e s t a b l e and tend to be p r e s e r v e d . l i g n i n , among the b i o l o g i c a l p r o d u c t s , behaves more or l e s s as do l i p i d s w i t h r e s p e c t to p r e s e r v a t i o n (Stevenson, 1966;  Breger,  Only the  1966).  T h e r e f o r e , one might  expect t o f i n d l i p i d s i n sea water d e 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 s l o w l y i n s o i l s and t h e i r decomposition i s r e t a r d e d m a i n l y by the a n a e r o b i c n a t u r e of g l e y e d or s a t u r a t e d s o i l s and  acidic  soils. Very l i t t l e decompose i n s o i l s . resistant  i s known of the r a t e a t which i n d i v i d u a l  lipids  I t seems, however, t h a t those compounds most  t o m i c r o b i a l decomposition,  such as h i g h e r a l c o h o l s and  a l k a n e s , s h o u l d p e r s i s t f o r l o n g e r p e r i o d s of time than those t h a t a r e a t t a c k e d r e a d i l y by microorganisms,  such as the g l y c e r i d e s ,  p h o s p h o l i p i d s and u n s a t u r a t e d f a t t y a c i d s . are p a r t i c u l a r l y r e s i s t a n t  Waxes of h i g h e r p l a n t s  to decomposition and  survive  unchanged over l o n g g e o l o g i c a l p e r i o d s (Stevenson,  essentially  1966).  - 14 -  A study by F u s t e c - M a t h i o n e t a l . (1977) of t h e . a c i d and n e u t r a l f r a c t i o n s o f the bitumens e x t r a c t e d from s u r f a c e v e g e t a t i o n and v a r i o u s h o r i z o n s o f s o i l s ,  showed t h a t t h e a c i d f r a c t i o n s and  the hydrocarbons underwent l e s s t r a n s f o r m a t i o n and l e s s i n A l h o r i z o n s o f the p o d z o l s s o i l horizons.  than i n the c o r r e s p o n d i n g  degradation hydromorphic  The n e u t r a l f r a c t i o n s a r e f o r the most p a r t  s l i g h t l y transformed,  only  but a r e m o r e r a p i d l y degraded, i n s o i l s .  Jones (1970) a l s o has shown-that the p l a n t d e r i v e d alkanes  in soil  appeared t o be r e s i s t a n t t o m i c r o b i a l a l t e r a t i o n under the e x p e r i m e n t a l conditions. From the r e s u l t s o f a l a b o r a t o r y i n c u b a t i o n study (1943) concluded  t h a t s t e r o l s were decomposed r a p i d l y i n s o i l ,  i n one experiment i n which c h o l e s t e r o l was added t o a m u l l 60% was decomposed w i t h i n a year; a e r a t i o n and h i g h water content  than w o o d l a n d . s o i l s  anaerobic  soil  although only  He a l s o p o i n t e d out t h a t l a c k of  were f a c t o r s which i n h i b i t e d  Gorham and Sanger (1967) r e p o r t e d  carotenoids.  Turfitt  decomposition.  t h a t l a k e muds were v e r y much r i c h e r  i n pigments of c h l o r o p h y l l d e r i v a t i v e s and  They a t t r i b u t e d the r i c h e r pigments i n l a k e muds to t h e  condition provided.  Except f o r the paper by P i e t t r e  ( r e f e r e n c e i n Stevenson, 1966), no r e p o r t has been p u b l i s h e d 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  productive a g r i c u l t u r a l s o i l ,  even when l a r g e annual increments of  r e s i d u e s and manures have been a p p l i e d . apparently  Most c u l t i v a t e d  c o n t a i n s u f f i c i e n t numbers of the proper  organisms t o d e s t r o y residues.  decomposable l i p i d s i n  the l i p i d m a t e r i a l contained  soils  k i n d s of m i c r o -  i n p l a n t and animal  - 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 c o n t a i n waxes, f a t t y hydrocarbons, g l y c e r i d e s , p h o s p h o l i p i d s , carotenoids,  c h l o r o p h y l l s and  methods to s e p a r a t e and mixtures.  In r e c e n t  others.  steroids,  acids,  terpenoids,  E a r l y workers l a c k e d  efficient  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  years,  use-has t e e n made of modern a n a l y t i c a l  methods f o r c h a r a c t e r i z i n g l i p i d f o l l o w i n g survey of l i p i d s  constituents  identified  in soil.  in soils,  In  o n l y a few  the examples  can be c i t e d where the newer methods have been a p p l i e d to the of s o i l l i p i d s because our  study  knowledge of t h i s m a t e r i a l i s h i g h l y  fragmentary.  1.6.1  Waxes  A v a i l a b l e evidence i n d i c a t e s t h a t m i x t u r e s of waxes comprise a l a r g e p a r t of the l i p i d s of s o i l s . however, d i f f i c u l t  to s e p a r a t e ,  and  I n d i v i d u a l components are, evidence of c o m p o s i t i o n i s u s u a l l y  based on i d e n t i f i c a t i o n of d e g r a d a t i o n p r o d u c t s (see r e v i e w by Stevenson, 1966). In the study conducted by M e i n s c h e i n and f r a c t i o n s were r e c o v e r e d  by  successive  Kenny (1957) f o u r  e l u t i o n w i t h 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 e x t r a c t s . benzene c o n s i s t e d the wax  l a r g e l y of waxes.  The m a t e r i a l r e c o v e r e d The  types of a c i d s and  e s t e r s were determined by c o n v e r t i n g  the e s t e r s to  with alcohols i n saturated  - 16 -  hydrocarbons by h i g h p r e s s u r e h y d r o g e n a t i o n ; the hydrocarbons were f u r t h e r f r a c t i o n a t e d by chromatography Even carbon numbered  and a n a l y z e d by mass s p e c t r o m e t r y .  (C-even) waxes were p r e s e n t i n c o n s i d e r a b l y  h i g h e r amounts than t h e i r odd carbon numbered wax  ranged from  (C-odd) homologs.  to C^^ and the C-even waxes were formed  C-even normal a l i p h a t i c a c i d s and normal primary a l i p h a t i c  The  from alcohols.  The waxes i n urea non:-adduct- samples were c h i e f l y e s t e r s of c y c l i c a l c o h o l s and normal a l i p h a t i c a c i d s .  The wax  f r a c t i o n examined  accounted f o r about 5% of the benzene-methanol B u t l e r et_ a l . a wax  which was  (1964) o b t a i n e d from an A u s t r a l i a n green  a m i x t u r e o f hydrocarbons and e s t e r s of normal  a c i d s w i t h normal primary a l c o h o l s . chromatography fractions.  C„.  2H  i t was  By s a p o n i f i c a t i o n and  s e p a r a t e d i n t o hydrocarbon, a c i d and  The a c i d s ranged from G-^  predominating.  extracts.  t  o  ^30'  w  i  t  fatty  column alcohol  the even numbers  n  The a c i d s p r e s e n t i n g r e a t e s t amounts were  (22%) and C „ , (21%). lb  soil  (13%),  The a l c o h o l s gave a range of compounds  s i m i l a r t o t h a t of the a c i d s . Himes and B l o o m f i e l d an o r g a n i c mat  (1967) i s o l a t e d a m i x t u r e of waxes from  i n an o r c h a r d 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 . an examination o f the wax  by IR, NMR  and mass spectrometry they  concluded t h a t the g e n e r a l s t r u c t u r e was o b t a i n e d f o r x and y i n d i c a t e d n-C^g  CH (CH ) COO(CH )^GH . 3  t h a t the a c i d  2  x  2  3  components were  and n-C^g, and the primary a l c o h o l s were n - C ^ ,  n  ~^28  A l c o h o l s and a c i d s of t h e s e c h a i n length-have been r e p o r t e d c o n s t i t u e n t s of p l a n t waxes.  From  Values  n-C^,  a n c  *  n -  ^30"  as  However, the carbon numbers of the a c i d s  - 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. R e c e n t l y M o r r i s o n and B i c k (1967) e x t r a c t e d the  wax  f r a c t i o n of a garden s o i l and a peat by e x t r a c t i n g them s u c c e s s i v e l y w i t h a m i x t u r e of benzene-ethanol, . l i g h t petroleum, and a m i x t u r e of isopropanol-ethanol. the  By s a p o n i f i c a t i o n and column chromatography  of  methyl e s t e r s , they o b t a i n e d a f r a c t i o n c o n s i s t i n g of l o n g - c h a i n  saturated f a t t y acids.  G a s - l i q u i d chromatography  acids  p r e s e n t and t h a t about 80% were C-even;  about  n -  C^g  t  o  -'34  n_<  w  e  r  e  60% of these a c i d s were n-C„., n-C„.,, n - C  0 0  showed t h a t  and n - C  ori  .  the  The  u n s a p o n i f i a b l e f r a c t i o n c o n t a i n e d normal primary a l c o h o l s from n-C. „ to.n-C,„.  S i m i l a r r e s u l t s were o b t a i n e d w i t h wax  from p e a t .  They a l s o o b t a i n e d , from IR s p e c t r a , i n d i c a t i o n s of the presence of hydroxy a c i d s i n the s a p o n i f i a b l e f r a c t i o n of the n e u t r a l l i p i d s the  same s o u r c e s as l a c t o n e s .  from  The same a u t h o r s a l s o found i n the  e a r l i e r study (1966) t h a t the r e s i n and a s p h a l t - f r e e wax  accounted f o r  0.6%  i n the peat.  1.6.2  of the o r g a n i c matter i n the garden s o i l s and 1.59%  Acids  Crude s o i l l i p i d p r e c i s e n a t u r e of which  e x t r a c t s c o n t a i n much a c i d i c m a t e r i a l ,  i s s t i l l obscure.  the  O r g a n i c a c i d s a r e of  v a r i o u s t y p e s ; more than a dozen f r e e f a t t y a c i d s both u n s a t u r a t e d and h y d r o x y l a t e d , and low m o l e c u l a r weight o r g a n i c a c i d s , i n c l u d i n g f o r m i c , a c e t i c , o x a l i c and b u t y r i c , have been i s o l a t e d from l i p i d  extracts  of  1966).  both m i n e r a l and peaty s o i l s  (see review by Stevenson,  .- 18 -  I t was  a l s o r e p o r t e d t h a t o r g a n i c a c i d s of v a r i o u s types occur i n  s m a l l q u a n t i t i e s i n the r h i z o s p h e r e of p l a n t r o o t s ( R o v i r a , and  i n raw p o d z o l humus ( S a l l a n s , et a l . ,  (1965) observed  S c h r e i n e r and  of  Leo  communities; u n d e r l y i n g l a y e r s of b l a c k mud  p r o g r e s s i v e l y d e p l e t e d i n the u n s a t u r a t e d  a peat.  Parker and  t h a t t h e r e were f a t t y a c i d s c o n t a i n i n g 12 to 20  atoms i n a l g a l mat  paraffinic acid  1937).  1962)  Shorey  became  acids.  (1910) i s o l a t e d  (C^g) from a s i l t  carbon  two o r g a n i c a c i d s ,  loam and l i g n o c e r i c a c i d  (C24)  ^  In the l i g h t of p r e s e n t knowledge i t seems.probable t h a t  r o m  both  these p r e p a r a t i o n s were m i x t u r e s , m a i n l y of u n s a t u r a t e d l o n g - c h a i n  fatty acids. (&2±)  acid  Another  was.also  hydroxy  f a t t y a c i d which they c a l l e d a g r o c e r i e  identified  from a N o r t h Dakota chernozemic  A c o u p l e of y e a r s e a r l i e r ,  the same a u t h o r s  9,10-dihydroxystearic acid  (C-^g) i n 27 d i f f e r e n t  mono-hydroxystearic  a c i d probably  the p o s i t i o n of the hydroxy presence o f both o l e i c matter acid  (O^g)  types of s o i l s and  i n peat were demonstrated  n  The  (C^g) a c i d s i n s o i l o r g a n i c  and an a p p a r e n t l y u n s a t u r a t e d a c i d which i s c a l l e d  (C )  a  (G-^g) of which  not r i g o r o u s l y e s t a b l i s h e d .  elaidie  soil.  identified  o£-hydroxystearic a c i d  group was and  (1908) a l s o  type  by K h a i n s k i i  humoceric  ( r e f . i n Morrison,  iy 1969)  and Aschan ( r e f . i n M o r r i s o n , 1969), r e s p e c t i v e l y . M o r r i s o n and B i c k (1966), i n a study of the decomposition  wax  from a garden  and hydroxy  acids.  s o i l and from a peat, o b t a i n e d l o n g - c h a i n f a t t y a c i d s The  same authors  l i p i d p r e p a r a t i o n from a garden  (1967), l a t e r o b t a i n e d a p u r i f i e d  s o i l which c o n t a i n e d about  20% of f r e e  a c i d s , 55% of which c o n s i s t e d of a m i x t u r e of f a t t y a c i d s n-C^rj n - C , w i t h 80% of even carbon number. Q /  of  The  even-numbered  acids,  t  o  - 19  n-C^^  to n - C ^ j  obtained  together  evidence from IR  -  made up  75%  of the f r a c t i o n .  They a l s o  s p e c t r a of the presence of a  significant  f r e e hydroxy f a t t y a c i d 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  identified.  More r e c e n t l y Wang•et a l . (1969)  reported  t h a t the main  f r e e higher  f a t t y a c i d s i n the s o i l s they s t u d i e d were p a l m i t i c (C^.) ,  palmitoleic  (C..,) and io  oleic  (C, ) lo 0  acids.  i n v e s t i g a t e d by means of t h i n - l a y e r and the l i p i d  L a t e r Wang et a l . (1971) gas  c o n t e n t of s o i l s under v a r i o u s  liquid  chromatography,  crops i n d e t a i l .  The  predominant f r e e f a t t y a c i d s they found were m y r i s t i c (0-^)> p a l m i t i c (C. ,) , p a l m i t o l e i c (C,,), lb ±b (C^)  stearic ( C ) , oleic lo 1 Q  a c i d s , a l l even-carbon a c i d s .  a c i d of which IR s p e c t r a was  (C. „) and lo  They a l s o found d i o c t y l p h t h a l i c  i d e n t i c a l to t h a t of d i o c t y l p h t h a l a t e  found i n the documentation of m o l e c u l a r s p e c t r o s c o p y . Fustec-Mathon et a l . (1977) found t h a t f a t t y a c i d s obtained majority  o n  O J  (mono- and  di-)  0 /  0 0  0 0  (1972) r e p o r t e d  from,the humic a c i d p r e p a r a t i o n s  sediments r e v e a l e d and  Very r e c e n t l y  from a c i d sandy s o i l s had. n - C to n - C range w i t h the zu zo being C „ C , C „ , and C w i t h preponderance of C-even. ZU z4 zo Zo Povoledo _et al.  extracted  arachidic  t h e ^ p r e s e n c e of odd,  t h a t a n a l y s i s of of some Canadian  even, s a t u r a t e d ,  branched-chain f a t t y a c i d s as major c o n s t i t u e n t s .  lipids lake  unsaturated  The  purified  e s t e r i f i c a t i o n p r o d u c t s of the l i p i d s gave r i s e to some 40 gas  liquid  chromatographic peaks, 22 of which were i d e n t i f i e d as m e t h y l e s t e r s of a wide v a r i e t y of f a t t y a c i d s r a n g i n g C^g.  i n c h a i n l e n g t h between  They a l s o i n d i c a t e d t h a t t h e s e f i n d i n g s a r e  preponderance of l i p i d s d e r i v e d  a n c  ^  i n keeping w i t h a  from microorganisms r a t h e r  than from  - 20  t e r r e s t r i a l rooted extracted  plants.  -  S c h n i t z e r and Neyroud  f a t t y a c i d s from humic and  f u l v i c acid preparations  a B l a c k Chernozemic s o i l i n C e n t r a l A l b e r t a and P o d z o l i n P r i n c e Edward I s l a n d , r e s p e c t i v e l y . f a t t y a c i d s ranged from n-C.  r  ange.  from the two  r e s p e c t i v e l y of n - C , and lb  10  drained these  i n the C  .  -  IH  C-even preponderance.  The  humic.preparations contained  n-C  of  Carbon atoms of  0 0  These f a t t y a c i d s had  acids extracted  of a p o o r l y  to n - C w i t h most being jo  lz C22  (1975) a l s o  fatty  60 and  80%  a c i d s , which suggested a m i c r o b i a l  lo  origin. I t i s p r o b a b l e t h a t s a l t s of f a t t y a c i d s are present mineral acid  s o i l s of r e l a t i v e l y h i g h pH.  s a l t s a r e i n d i c a t e d by  mineral  a d d i t i o n a l m a t e r i a l has f a t t y a c i d s and  presence of these f a t t y  the f a c t t h a t pretreatment of s o i l s  a c i d s u s u a l l y r e s u l t s i n an  subsequently extracted'by  The  in  lipid  increase  s o l v e n t s , but  never been studied..  i n the amount of  with  material  the p r e c i s e n a t u r e o f Local concentrations  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,  f r e q u e n t l y found a t b u r i a l s i t e s of human or other  salts  this of  are  l a r g e animal  corpses.  These l o c a l a c c u m u l a t i o n s of l i p i d s have been g i v e n the name " a d i p o c i r e " and  have been d i s c u s s e d  i n some d e t a i l by Bergmann (1963).  L i p i d preparations  from s o i l s a r e l i a b l e to c o n t a i n  amounts of aromatic a c i d s such as b e n z o i c , acids.  Larger  amounts of p h e n o l i c  humic substances and may alkaline hydrolysis. a number of p h e n o l i c  p-hydroxybenzoie or  acids are chemically  small vanillic  associated  with  be l i b e r a t e d , at l e a s t i n p a r t , by a c i d or  Thus S c h n i t z e r and  Neyroud  a c i d s from the humic and  (1975) have r e l e a s e d  f u l v i c acid  preparations.  - 21 -  T h i s suggests t h a t i n the humic substances f a t t y a c i d s r e a c t w i t h p h e n o l i c OH groups to form e s t e r s .  1.6.3  Hydrocarbons  H i g h e r a l k a n e s a r e g e n e r a l l y p r e s e n t i n s o i l s but, compared w i t h waxes and a c i d s , i n r e l a t i v e l y s m a l l q u a n t i t i e s . p a r a f f i n i c hydrocarbons i n the C^^  to C ^  Normal  range have been found i n the  n o n s a p o n i f i a b l e f r a c t i o n of l i p i d e x t r a c t s of s o i l by S c h r e i n e r and h i s associates ^31'  ( r e f . i n Stevenson, 1966).  Normal a l k a n e s , n - h e n t r i a c o n t a n e ,  ( S c h r e i n e r and Shorey, 1911b) and n - p e n t a t r i a c o n t a n e , ^35'  n - t r i t r i a c o n t a n e , £33*  (Titow, 1932)  a n (  ^  have been i s o l a t e d from a N o r t h  C a r o l i n a o r g a n i c s o i l and from a R u s s i a n peat, r e s p e c t i v e l y , by u s i n g such c r i t e r i a  as m e l t i n g p o i n t and elementary a n a l y s i s .  However, t h e s e  e a r l y attempts t o i s o l a t e i n d i v i d u a l components must be s u s p e c t , f o r it  i s o n l y w i t h the development  of chromatographic methods t h a t  s e p a r a t i o n s have become r e a l l y Stevens et_ a l . range i n s e v e r a l s o i l  such  feasible.  (1956) have found normal a l k a n e s i n the t y p e s , among which n-nonacosane (C^g)  n - h e n t r i a c o n t a n e ( C ^ ) predominated.  I n an a l i p h a t i c  f r a c t i o n from a peat bitumen, G i l l H a n d  a n <  to *  hydrocarbon  and Howard (1968) have shown  t h a t the hydrocarbon f r a c t i o n c o n s i s t e d m a i n l y of the C-odd n - a l k a n e s , C^g of  (11%),  (40%), and  (34%), but i t c o n t a i n e d a l s o s m a l l amounts  a l l o t h e r n-alkanes from C^-j to  Butler et a l .  ^rom  a  n  Australian  soil,  (1964) o b t a i n e d a hydrocarbon f r a c t i o n which gave peaks  c o r r e s p o n d i n g to n-alkanes from  to  w i t h odd and  even-carbon  - 22 -  numbers i n a p p r o x i m a t e l y e q u a l amounts. i n t h i s A u s t r a l i a n s o i l was r a n g i n g from (1965).  to  The d i s t r i b u t i o n of n-alkanes  remarkably s i m i l a r to t h a t of n-alkanes  i n an a n c i e n t sediment r e p o r t e d by Oro et a l .  Furthermore the n-alkanes i n t h i s c h e r t a l s o showed no  p r e f e r e n c e of C-odd t o C-even carbon atoms. M o r r i s o n and B i c k (1967) a l s o o b t a i n e d n-alkane  fractions  from b o t h a garden s o i l and a peat which c o n s i s t e d l a r g e l y of a l k a n e s from n - C  and n-C„„ w i t h about 87% odd-numbered. iy 3j i n  i n the f r a c t i o n from the garden s o i l were n-C^ and n - C  3 3  (15%).  R e c e n t l y Jones  e x t r a c t from an upland moorland to  The main components  (21%), n - C ^  (31%)  (1970) r e p o r t e d t h a t the l i p i d s o i l c o n t a i n e d n-alkanes i n the  C „ , range demonstrating a d e f i n i t e preponderance of  odd-carbon  JO  numbered.  Wang et a l . (1971) r e p o r t e d t h a t p a r a f f i n i c  f r e q u e n t l y appeared  i n the s o i l  they s t u d i e d .  hydrocarbons  Very r e c e n t l y F u s t e c -  Mathon et a l . (1977) a l s o r e p o r t e d t h a t n-alkanes c o n t a i n e d i n a c i d sandy s o i l s were i n the range from b e i n g C^y,  ^29' ^31  a n  ^  to C^^ w i t h main components  w i t h preponderance of C-odd.  S c h n i t z e r and Neyroud  (1975) e x t r a c t e d a l k a n e s from  humic and f u l v i c p r e p a r a t i o n s of a B l a c k Chernozemic  s o i l and a p o o r l y  d r a i n e d P o d z o l , of which carbon numbers v a r i e d from C ^ m a j o r i t y b e i n g i n the s t r a i g h t - c h a i n C^g odd to even C r a t i o of 1.0. to  (1969).  to C^g,  the  to C^^ range, and w i t h an  The d i s t r i b u t i o n of n-alkanes and the odd  even C r a t i o were s i m i l a r to t h o s e of m i c r o b i a l  r e p o r t e d by Jones  two  hydrocarbons  - 23 -  P o l y n u c l e a r a r o m a t i c hydrocarbons have a l s o been d e t e c t e d 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 c o n c e n t r a t i o n estimated was  (IV)  perylene  (V)  15  ppm.  coronene  A l t h o u g h Cooper and L i n d s e y (1953) suggested t h a t the p r e s e n c e of chrysene i n s o i l s was  due t o atmospheric s e t t l i n g  from combustion gases, i t seems l i k e l y indigenous t o s o i l s ,  of dust p a r t i c l e s  t h a t t h i s hydrocarbon may  be  s i n c e the chrysene and o t h e r p o l y n u c l e a r aromatic  hydrocarbons such as 3,4-  and 1,2-benzpyrene,  f l u o r a n t h e n e ( I I ) , pyrene ( I I I ) ,  phenanthrene,  p e r y l e n e ( I V ) , anthanthrene,  anthracene, t r i p h e n y l a m i n e benzanthracene, b e n z f l u o r e n e , 1,12-  - 24 -  benzperylene and coronene  (V) were d e t e c t e d 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 i n d u s t r i e s  (Blumer,  1961).  C h e s h i r e , jit al. (1967) have a l s o i d e n t i f i e d many of t h e s e aromatic hydrocarbons as p r o d u c t s of t h e d i s t i l l a t i o n of humic a c i d s w i t h zinc dust. Swan (1965) a l s o i d e n t i f i e d dehydroabietene (VI) from a f o r e s t s o i l o f B e l l a C o o l a , B r i t i s h Columbia. indicated  Wang e t alL. (1971)  t h a t p a r a f f i n i c hydrocarbons f r e q u e n t l y appeared  i n Taiwan  s o i l s they s t u d i e d and i n some s o i l s , an unknown nonpolar compound and d i o c t y l p h t h a l a t e were a l s o 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 t h e t y p i c a l C-H s t r e t c h i n g f r e q u e n c i e s a t 2853-2962 cm \  C-H d e f o r m a t i o n f r e q u e n c i e s a t around 1450 cm  as w e l l as s k e l e t a l v i b r a t i o n a t 720 cm  (VI)  1  1  and 1380 cm  i n t h e IR spectrum.  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 S e r r a and F e l b e c k (1972) were a homologous s e r i e s of normal alkanes.  The bound l i p i d  f r a c t i o n s they examined were o b t a i n e d by  e x t r a c t i o n of t h e a c i d h y d r o l y s i s p r o d u c t of t h e o r g a n i c matter from  - 25 -  a muck s o i l by u s i n g chloroform-methanol  1.6.4  azeotrope.  Fats  From t h e a l k a l i n e e x t r a c t o f a s i l t  loam, S c h r e i n e r and  Shorey (1911a) d e t e c t e d g l y c e r o l and a.mixture of u n i d e n t i f i e d a c i d s i n the product  o b t a i n e d by s a p o n i f i c a t i o n of t h e s o i l  fatty  lipids.  S i n c e then t h e r e have been no r e p o r t s o f the d e t e c t i o n or d e t e r m i n a t i o n of  simple g l y c e r i d e s i n s o i l s u n t i l Wang e^t a l . (1969) o b t a i n e d  t r i g l y c e r i d e s from s o i l chromatography.  lipid  Although  g l y c e r o l moiety o f f a t s ,  extracts using S i l i c a  they d i d not mention the d e t e c t i o n of t h e they i d e n t i f i e d  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 . of  palmitic  (C..,), p a l m i t o l e i c lo  w i t h docosanoic pentacosanoic  1.6.5  the f a t t y a c i d s a f t e r  The s o i l t r i g l y c e r i d e s c o n s i s t e d  (CL,). and o l e i c lo  (C^)» 22-methyltrieosanoic  ( C , ) a c i d s together lo 0  (C^)  a n c  ^ 24-methyl-  (C^g) ° i d s as t h e i r predominant components. a  r e p o r t s on the presence reasonable  g e l .column  Although  o f f a t s i n s o i l s a r e s c a r c e , i t seems  to b e l i e v e t h a t s m a l l amounts a r e u s u a l l y p r e s e n t .  Phospholipids  C o n s i d e r a b l e a t t e n t i o n has been g i v e n to the o c c u r r e n c e of p h o s p h o l i p i d s i n s o i l because these compounds a r e p o t e n t i a l sources of phosphorus f o r p l a n t growth.  The presence  of phospholipids i n s o i l  has been i n f e r r e d from the s m a l l amounts of phosphorus e x t r a c t e d from s o i l with l i p i d solvents.  The amount of phosphorus thus e x t r a c t e d  - 26  i s s m a l l , u s u a l l y 1 to 7 ppm, and G o r i n g  (1953).  Lipid  -  but  34 ppm  has been r e p o r t e d by  phosphorus accounts f o r l e s s than 1% of  o r g a n i c phosphorus i n s o i l s .  Hance and Anderson (1963) o b t a i n e d  of l i p i d phosphorus f o r f i v e s o i l s r a n g i n g phosphorus per 100  g soil,  Black  from 0.31  to 0.70  the values  mg  e q u i v a l e n t to o n l y 0.6-0.9% of the  total  o r g a n i c phosphorus. Simoneaux and reported l i p i d represented  (1965), f o r n i n e s o i l  phosphorus c o n t e n t s  from 0.06  selected s o i l s .  Caldwell  The  to 1.02%  ranging  total lipid  lacustrine soil  to 0.089 ppm  lacustrine soil  (Domaar, 1970).  i n the t h i n b l a c k Bm  14.5  and  subsurface  P.  h o r i z o n of a  An L-H  h o r i z o n s had. n i l to 0.6  that phospholipid  s o i l s ranged from 0.6  horizon contained  ppm  l i p i d P.  I t was  from the r e s u l t s t h a t most of the t o t a l p h o s p h o l i p i d P may from the combined b a c t e r i a l and  f u n g a l biomass.  McKercher  (1971b) a l s o r e p o r t e d  that phosphatidyl  about 40%  of t o t a l p h o s p h o l i p i d P and  30%,  f o r Saskatchewan s o i l s .  This  of Chernozemic s o i l s  (1970a) r e p o r t e d  of 20 Saskatchewan s u r f a c e m i n e r a l  but  ppm.  i n the brown Ah h o r i z o n of a  contents  ppm  to 1.70  phosphate content  Kowalenko and McKercher  averaged 3.4  ppm  of the o r g a n i c phosphorus i n the  of Southern A l b e r t a ranged from 13 ppm  ppm  0.24  extracts,  30.5  to  ppm  P  suggested accumulate  Kowalenko and  phosphatidyl  choline  represented  ethanolamine about  - 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 in  p l a n t and  in  soil.  animal  tissues,  t r i t e r p e n o i d s are widely there i s l i t t l e  However, t h e r e i s l i t t l e  amount o f them i n s o i l . soil  -  is p -sitosterol,  The  and  Shorey  (1909  soils.  One  w h i c h had had  and  McLean et a l . ,  1911c) o b t a i n e d  two  identified  p r e p a r a t i o n from a s o i l  and  from  1958).  T h i s compound was  subsequently  C and  was  saponification..  The  compound but  in this  t o as " a g r o s t e r o l " .  " p h y t o s t e r o l " had  melting  obtained  from a peat by  a process  former,  a g r o s t e r o l and  the l a t t e r p h y t o s t e r o l  might have been a t r i t e r p e n o i d (Morrison,  and  different  known s u b t a n c e  referred  has  peats  contained a c r y s t a l l i n e  t o any  in  Schreiner  p r e p a r a t i o n s from  other p r e p a r a t i o n which they c a l l e d  and  ^? - s i t o s t e r o l ,  involving  respectively  1969). Turfitt  presence  and  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 the c h o l e s t e r o l group,  p o i n t 135  nil  ( B e r g m a n , 1963)  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  group. The  i n f o r m a t i o n on t h e n a t u r e  occur  a common s t e r o l o f h i g h e r p l a n t s , w h i c h  H o w a r d , 1968  and  doubt t h a t they  only steroid positively  been o b t a i n e d from a garden s o i l (Gilliland  distributed  ( 1 9 4 3 ) e x a m i n e d a number o f E n g l i s h s o i l s f o r  o f f r e e s t e r o l s and  t o 12 mg  per kg  p o o r l y a e r a t e d and of arable s o i l s . 1% o f t h e s o i l the presence  of s o i l , acidic  On  found  t h a t t h e r a n g e o f v a l u e s was  from  the 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  s o i l s and  the lower v a l u e s  I v e s and  O ' N e i l l ( 1 9 5 8 a and  of  characteristic  t h i s b a s i s f r e e s t e r o l c o u l d account  lipids.  the  f o r about  1958b) r e p o r t e d  i n sphagnum p e a t moss o f t h e s t e r o l s and  the  triterpenoids  - 28  such as  o(-amyrin,  -  t a r a x e r o l and  taraxerone.  Meinschein  and  Kenny  (1957), i n t h e i r study of benzene-methanol e x t r a c t s of m i n e r a l subsoils, detected  s t e r o l s and  penta- and  a r e thought to be t r i t e r p e n o i d s .  The  h e x a c y c l i c compounds which  known o c c u r r e n c e s  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 a r e summarized by Bergmann (1963)  and  Breger  1.6.7  (1966), r e s p e c t i v e l y .  Carotenoids  and C h l o r o p h y l l s  Carotenoids  and  c h l o r o p h y l l s are, i n general,  o x i d i z e d i n the presence of a i r .  readily  In t h i s r e g a r d , Hoyt (1971) p o i n t e d  out t h a t c h l o r o p h y l l s a r e l e s s s t a b l e i n s o i l environments than i n l a k e s . I t i s reasonable  to b e l i e v e t h a t c a r o t e n o i d s and  c h l o r o p h y l l s are  r e a d i l y degraded i n most s o i l s and a r e d e t e c t e d o n l y i n s m a l l quantities i n arable s o i l s . p u b l i s h e d on the o c c u r r e n c e  Indeed no r e p o r t seems to have been of c a r o t e n o i d s and  i n a g r i c u l t u r a l s o i l s , although of.chlorophyll  the presence of d e g r a d a t i o n  ( V a l l e n t y n e , 1957)  i n anaerobic  c a l c a r e o u s s u r f a c e woodland s o i l s years.  Gorham and  Sanger  d e r i v a t i v e s and  l a k e mud  (Gorham, 1959)  (1967) p o i n t e d out  much r i c h e r than woodland s o i l s  chlorophyll derivatives  in  non-  has been known f o r many  t h a t l a k e muds are v e r y  i n the c o n t e n t s  of c h l o r o p h y l l  c a r o t e n o i d s , c h i e f l y because of the  environment p r o v i d e d .  and  products  anaerobic  Povoledo et a l . (1972) n o t i c e d a c o l o r e d spot  i n the b i d i m e n s i o n a l TLC  of e t h y l ether e x t r a c t from a l a k e sediment  humic a c i d p r e p a r a t i o n of which c o l o r , n o n f l u o r e s c e n t Rf v a l u e s were c h a r a c t e r i s t i c of c a r o t e n o i d s .  f e a t u r e and  They a l s o r e v e a l e d  the  I  - 29 -  presence  i n l a c u s t r i n e humus of c h l o r o p h y l l d e r i v a t i v e s , n o t a b l y  pheophytin a.  1.6.8  Ketones  M o r r i s o n and B i c k (1966), from a garden s o i l and a peat, o b t a i n e d a f r a c t i o n c o n s i s t i n g o f methyl ketones  (n-alkan-2-ones)  w i t h c a r b o n - c h a i n l e n g t h r a n g i n g from C^^-C^^ and w i t h the oddcarbon numbered members p r e d o m i n a t i n g . the o c c u r r e n c e of methyl ketones  T h i s i s the f i r s t r e p o r t of  i n t h i s range as n a t u r a l p r o d u c t s ;  the lower members, up t o C ^ , a r e w e l l known as products of the a c t i o n o f m i c r o f u n g i on m i l k f a t t y a c i d s i n c e r t a i n Very r e c e n t l y S e r r a and F e l b e c k  cheeses.  (1972) a l s o suggested t h e  presence o f d i k e t o n e s i n the s o l u b l e f r a c t i o n i n  chloroform-methanol  azeotrope o f a c i d h y d r o l y s a t e s • o f t h e o r g a n i c matter by means of gas chromatography, mass spectrometry  from a muck  soil  and i n f r a - r e d  spectrometry.  1.6.9  Miscellaneous  Many other substances  of a l i p i d n a t u r e such as t o c o p h e r o l s  and p o r p h y r i n s from h i g h e r p l a n t s and p o l y n u c l e a r quinones origin are l i k e l y Chlorinated  to be p r e s e n t i n s o i l s , but o n l y i n v e r y s m a l l amounts.  i n s e c t i c i d e s and t h e i r d e g r a d a t i o n p r o d u c t s , a l t h o u g h not  l i p i d s i n the s t r i c t 1969) .  of f u n g a l  sense, would be a s s o c i a t e d w i t h them ( M o r r i s o n ,  - 30  -  2.  NATURALLY OCCURRING SULFOLIPIDS  2.1  Introduction;  The  s u l f o l i p i d s have been known s i n c e the c l a s s i c a l study of  Thudichum (1874) on the chemical  composition  o u t s i d e of b r a i n t i s s u e , however, was (1959) o n l y twenty years have been r e p o r t e d n e a r l y the e n t i r e  ago.  in  f i r s t r e p o r t e d by Benson et a l .  S i n c e t h i s f i r s t r e p o r t , the  sulfolipids  biosphere. to the nomenclature of Haines (1971  the term s u l f o l i p i d denotes any  sulfonolipid  Their occurrence  i n a v a r i e t y of l i v i n g systems, which i n c l u d e  According  is a sulfolipid  of b r a i n .  and  sulfur-containing l i p i d .  1973b),  The  i n which the s u l f u r occurs as a s u l f a t e e s t e r and  i s used i n r e f e r e n c e  the s u l f o n i c a c i d form.  a r e termed t h i o l i p i d s .  and  they be  the  to s u l f o l i p i d s which c o n t a i n s u l f u r  S u l f o l i p i d s w i t h s u l f u r i n the reduced form  A l t h o u g h no  l i p i d has  been r e p o r t e d  containing  the s u l f o x i d e or s u l f o n e s t a t e s of s u l f u r , he suggested t h a t lipid  sulfatide  sulfoxo-  s u l f o n e l i p i d can be used i n r e f e r e n c e to such compounds  should  identified. Mammalian s u l f a t i d e s i n c l u d e c e r e b r o s i d e  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), g a n g l i o s i d e sulfate  ( L e i k o l a et a l . , 1969)  1973).  Microbial sulfatides include g l y c o l i p i d  bacteria  (Kates j2t a l . , 1967  Tubercle  bacillis  sulfates  (Haines and  V a g e l o s , 1969;  and  glycolipid  and M a r s h a l l and  (Goren, 1970a and Block,  1962;  s u l f a t e s of h a l o p h i l i c •  Brown, 1968)  and  of  1970b), a l k y l s u l f a t e s and h a l o a l k y l  Mayer and  Haines e t a l . , 1969)  s u l f a t e ( I s h i z u k a , et a l . ,  Haines, 1967;  of Ochromonas d a n i c a ,  Elvoson and  and  - 31 -  p h o s p h a t i d y l g l y c e r o s u l f a t e of Halobactefium cutirubrum Kates, 1973).  Another  (Hancock and  c l a s s o f 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  d i s c o v e r e d by Benson et_ a l .  (1959) .  Reports of t h i o l i p i d s i n s m a l l  amounts i n y e a s t s and p l a n t s have appeared  (Chu et a l . ,  1968).  In a d d i t i o n t o these s u l f o l i p i d s t h e r e a r e many r e p o r t s of s u l f o l i p i d s which have not been f o l l o w e d up w i t h f u l l A sulfolipid  characterization.  s i m i l a r t o t h a t of the p l a n t s u l f o n o l i p i d has been  d e s c r i b e d i n the sea u r c h i n (Isono and Nagai, 1966). s e v e r a l r e p o r t s of s u l f o l i p i d s i n b a c t e r i a Kates e t a l . , et a l . , 1972)  1968;  1955).  Goren, 1971;  A sulfolipid  There  ( K a r l s s o n et a l . ,  Gangadharam e t a l . ,  found i n diatoms  1963;  Jack, 1964a and in insects  1964b).  ( G i r a l , 1941;  ( r e f e r e n c e i n Haines,  1971;  Roberts  (Kates and Tornabene,  does not correspond t o the p l a n t s u l f o n o l i p i d .  s e v e r a l r e p o r t s of fungal s u l f o l i p i d s  are  There a r e a l s o  ( C o l l i e r and Kennedy,  1963;  A v a r i e t y o f s u l f o l i p i d s have been r e p o r t e d G i r a l _et/_al., ' 1946)  1971).  and c h i c k e n eggs  However, i t i s p o s s i b l e t h a t  these  substances a r e not s u l f o l i p i d s i n the u s u a l sense, but s t e r o i d  sulfates  or s u l f a t e e s t e r s of o t h e r o r g a n i c m o l e c u l e s . S u l f o l i p i d s of d i v e r s e s t r u c t u r e d i f f e r t h e i r c h e m i s t r y and b i o c h e m i s t r y .  The  significantly in  f o r m a t i o n 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 b i o s y n t h e t i c r o u t e than t h a t of the s u l f o n o l i p i d s , and t h e i r m e t a b o l i c b e h a v i o r should be different.  radically  T h e i r e x t r a c t i o n , i s o l a t i o n and c h a r a c t e r i z a t i o n should  a c c o r d i n g l y f o l l o w up w i t h 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 sulfonic acid  ester,  i n plants  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 .  mammalian s u l f a t i d e s have b e e n c h a r a c t e r i z e d cerebroside sulfate  i n brain  (Blix,  sulfate,  (Leikola  (Ishizuka  s u l f a t e , from boar t e s t i s and  (VII)  i n 1874 (Thudichum).  was t h e f i r s t  1 9 2 5 when L a n d s t e i n e r a n d L e v e n e  (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 glycolipid.  sphingosine, cerebroside acid, of b r a i n  sulfatide.  s u l f a t i d e t o be  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  sulfate-containing  t h e substance as a  A f e w y e a r s l a t e r , B l i x (1933) f o u n d g a l a c t o s e and s u l f a t e  He s u g g e s t e d t h a t  the s u l f u r i c ester  I  HO /  I  NH V  \ o - C H  2  - C H  OH I I - C H - C H =  oso;  (VII)  This  position  g r o u p o n t h e c a r b o n - 6 o f t h e g a l a c t o s e m o i e t y was  CH2OH  -0  i n h i s preparation  t h e s u l f a t e g r o u p was o n t h e  g a l a c t o s e moiety, probably i n t h e carbon-6 p o s i t i o n . of  ganglioside  e t a l . , 1973; Hatanaka e t a l . , 1975).  Cerebroside sulfate discovered  (Martensson, 1966),  e t a l . , 1969) and a g l y c o l i p i d  sulfoglycerogalactolipid  spermatozoa  These a r e  1 9 3 3 ; Yamakawa e t a l . , 1 9 6 2 ) ,  l a c t o s y l ceramide s u l f a t e of kidney s u l f a t e i n hard t i s s u e s  t o date.  Four  a cerebroside  sulfate  CH-(CH2)  I 2  -CH  3  - 33 -  believed  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;  Jantzkewitz,  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 . ( 1 9 6 2 ) s u g g e s t e d t h a t t h e s u l f a t e g r o u p 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  confirmed by S t o f f y n and S t o f f y n  s t r u c t u r e has s i n c e  (1963).  The proposed  was f u r t h e r c o n f i r m e d f r o m a d i f f e r e n t p o i n t Yamakawa  been  structure  o f v i e w by Taketomi and  (1964). 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 a n d c h a r a c t e r i z e d b y M a r t e n s s o n (1963b and 1966) a n d b y B e n s o n (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 o m human k i d n e y .  . One o f them h a s t h e same c h e m i c a l c o m p o s i t i o n a s  brain cerebroside  sulfatides.  glucose-galactose  sulfate.  from bovine kidney  The other  This  i s probably an acyl-sphingosine-  second s u l f a t i d e appears t o be absent  ( K a r l s s o n 'et. a l . ,  1968),  although r a t brain  (Cumar e t a l . , 1 9 6 8 ) a p p e a r s t o c o n t a i n a n enzyme f o r i t s b i o s y n t h e s i s . Subsequently, Martensson  (1966) e s t a b l i s h e d  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  with  the p o s i t i o n of thesulfate t h e s u l f a t e group on t h e  CH 0H  CH 0H  2  2  NH OH I  I  0-CH -CH-CH-CH = CH-(CH ), 2  (VIII)  l a c t o s y l ceramide s u l f a t e  2  2  -  3-position of galactose. confirmed  34 -  T h e s t r u c t u r e o f t h e s u l f a t i d e was t h e n  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 .  (1968). I n a n i n v e s t i g a t i o n o n human e p i d e r m i s , (1967, i n H a i n e s , 1971)  Nieminen e t a l .  found a s u l f u r - c o n t a i n i n g l i p i d  w h i c h i n TLC m i g r a t e d s l i g h t l y a h e a d o f t h e c e r e b r o s i d e esters.  Since  then a l i p i d  isolated  f r o m human k i d n e y ,  bovine and horses'  fraction with  and from t h e w a l l s o f  hooves ( L e i k o l a e t a l . , 1969).  found t o c o n t a i n  ceramide, s i a l i c  and  s u l f a t e i n e q u i m o l a r amounts.  was  a ganglioside  component o f g a n g l i o s i d e s .  They c o n c l u d e d t h a t  The s u l f o l i p i d s r e p o r t e d  1962;  characterized  t o date  t h e new  b y Maruyama  characterized.  of sulfur-containing  glycolipid  ( B e n s o n e t a l . , 1 9 5 9 ; Yamakawa e t a l . ,  M a r t e n s s o n , 1966; K a t e s e t a l . , 1967; G o r e n , 1970b; Hancock and  Kates, 1973), I s h i z u k a ether  e t a l . (1973) r e p o r t e d  alkyl  lipid  o f t e s t i s a n d s p e r m a t o z o a o f b o a r t o w h i c h t h e name  ( I X ) was g i v e n . contained  glycolipid  the occurrence of a  novel  that  lipid  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 group, a l t h o u g h they were n o t w e l l In addition to s i x kinds  galactosamine,  acid i s a characteristic  ( 1 9 6 2 a a n d 1962b i n H a i n e s , 1 9 7 1 ) i n c h i c k e n  already  The s u l f o l i p i d  acid, galactose,  sulfate, f o rthe s i a l i c  sulfate  s i m i l a r b e h a v i o r has been  h a i r and n a i l s ,  was  fraction,  Subsequently  the seminolipid  s u l f a t e as t h e major sugar  "Seminolipid"  they found that guinea p i g t e s t i s  (Suzuki  the s u l f o l i p i d s reported  containing  e t a l . , 1973).  by K o r n b l a t t  group a l t h o u g h they were n o t f u l l y  I t i s also  also  likely  e t a l . (1972) b e l o n g i n t h i s  characterized  beyond t h e components  - 35 -  CH o I II HC-O-C -(CH ) -CH I H C-0-(CH ) -CH 2  2  2  (IX)  of  2  | 4  l s  3  3  a seminolipid  the g l y c o l i p i d found  to contain galactose, chimyl a l c o h o l , p a l m i t i c  a c i d and s u l f a t e .  2.3  Plant  The  Sulfolipids  term " p l a n t s u l f o l i p i d "  (X) was f i r s t  i n t r o d u c e d by Benson  and h i s coworkers (Benson e t a l . , 1959 and 1960; D a n i e l _et _ a l . , 1961; Lepage e t a l . , 1961; Miyano and.Benson, 1962a and 1962b). most widespread and b e s t c h a r a c t e r i z e d s u l f o l i p i d s u l f o n o l i p i d has been found  This  i n many green p l a n t s and i s not r e s t r i c t e d  to t h e h i g h e r p l a n t s and green a l g a e . (Benson and Shibuya,  t o date.  I t i s the  I t has been r e p o r t e d i n r e d a l g a e  1962 and Radunz, 1969), and b l u e - g r e e n  a l g a e and p u r p l e b a c t e r i a (Radunz, 1969). the p l a n t s u l f o n o l i p i d appear t o occur  Highest  a l g a e , brown  c o n c e n t r a t i o n s of  i n t h e marine r e d a l g a e  (Burwell,  - 36 -  CH S0 H 2  J r . , 1945)  3  a l t h o u g h i t has been found i n b a r l e y , c l o v e r , s p i n a c h ,  c h i v e and c o l e u s (Benson et a l . , 1959),  in alfalfa  (O'Brien et a l . ,  1964) , i n maize, runner beans and P a u l ' s s c a r l e t r o s e (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 a l _ . ,  M i y a c h i ^ t a l . , 1966). Nagai and Isono  (1965) i s o l a t e d  a sulfonolipid  from the  sperm and eggs of the sea u r c h i n , Pseudocentrotus d e p r e s s u s . structural  s t u d i e s suggest t h a t the sugar moiety i s a 6-deoxyhexose-6-  s u l f o n a t e such as s u l f o q u i n o v o s e of the p l a n t s u l f o n o l i p i d . 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  1966) , or t h a t of a mussel, H y p r i o p s i s s c h e g l l i the  Their  amurensis  (1967).  (Isono,  (Isono et a l . , 1967),  s u l f o n o l i p i d has been r e p o r t e d i n another sea u r c h i n ,  p u l c h e r r i m u s by Isono  Although  Hemicentrotus  - 37  -  Haines (1971) p o i n t e d  out  t h a t the  difficulty.in  e s t a b l i s h i n g the s t r u c t u r e 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 f a c t t h a t i t took seven p u b l i c a t i o n s Peat et a l . , 1960; Miyano and  D a n i e l et a l . , 1961;  Benson, 1962a and The  (Benson et a l . , 1959 Shibuya and  at a l l pH v a l u e s  i n aqueous s o l u t i o n s .  Microbial  For  these reasons, the  the d i s c o v e r y  (Nagai and  and  been r e p o r t e d  Isono, 1967).  Chamydomonas. ( M i y a c h i  of i t s absence i n Ochromonas d a n i c a i n c o r r e c t and  due  In 1959, providing  plants  his  i n various  eggs of the sea  I t has  i n a v a r i e t y of microbes, Euglena g l a c i l i s and  i n green  Scendesmus, • by Benson and  been found i n the sperm and  Isono, 1965  Ochromonas d a n i c a  i t would  extracts.  of the p l a n t s u l f o l i p i d  coworkers (1959) the s u l f o n o l i p i d has I t has  and  late  Sulfolipids  and microorganisms, C h l o r e l l a and  organisms.  of  L i k e the s u l f a t e e s t e r s , t h i s compound i s i o n i z e d  remain i n the i n t r a c t a b l e r e s i d u e of p l a n t  Since  has  i s among the most p o l a r  appearance of these s u l f o n o l i p i d s i s e a s i l y e x p l a i n e d  2.4  1961;  s u l f a t e on h y d r o l y s i s ,  i n mammalian t i s s u e s and  the p o l a r l i p i d s .  Benson,  1960;  1962b). to complete the s t r u c t u r a l p r o o f .  s u l f o n o l i p i d does not y i e l d  not been r e p o r t e d  and  the  a l s o been  (Davies et^ al., et a l . , 1966).  (Haines and  B l o c k , 1962)  urchin  reported 1965) A  report was  to growth c o n d i t i o n s  (Miyachi  et a l . , 1966)..  M i d d l e b r o o k jet ad.  reported  investigations  evidence t h a t the m a t e r i a l r e s p o n s i b l e  and  found  f o r the f i x a t i o n of  n e u t r a l r e d , a s t a i n which d i s t i n g u i s h e s 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 suggested that  this sulfolipid  l a t e r , M a y e r and sulfate ester by  the  Haines  and  not  as  the  H a i n e s and sulfolipid and  acid.  of  He  A b o u t one  sulfolipid  has  trehalose sulfate  authors decade  was  a  confirmed  characterized  the  (2,3,6,6'-tetraester  XI). Block  ( 1 9 6 2 ) and  The  a t e t r a ester  docosanediol d i s u l f a t e  of  (XII)  s u l f a t i d e comprises the  contain a polar  the  The  T h i s s u g g e s t i o n was  of Goren (1970b).  C h l o r e l l a pyrenoidosa.  alkyl  a sulfonic acid.  from p h y t o f l a g e l l a t e s ,  (XI)  as  a sulfonic  tetraester  2'-trehalose sulfate,  was  sulfolipid.  (1967) s u g g e s t e d t h a t  structural studies  sulfolipid  t y p e of  Haines  (1965) r e p o r t e d a  O c h r o m o n a s d a n i c a , 0.  new  sulfolipid  trehalose  by  o n l y group of the  later  mehamensis, characterized  sulfate  M a y e r and  g r o u p a t b o t h ends of  was  new  Haines  (1967).  This  l i p i d molecules  which  molecule.  - 39 -  osor I ( C H ) — CH — 3  CH 3  2  7  (XII)  (CH )| 2  2  —  CH OS0 ~ 2  3  docosanediol d i s u l f a t e  The presence o f 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 n o t i c e d by E l v o s o n and Vagelos  (1969).  first  L a t e r on, these c h l o r o a l k y l  s u l f a t i d e s were f u r t h e r c h a r a c t e r i z e d as a group of p o l y c h l o r o d e r i v a t i v e s of 1,14-docosanediol (C ^) 2  (^22' X I I I ) and 1 , 1 5 - t e t r a c o s a n e d i o l  i n which t h e c h l o r o - g r o u p s s u b s t i t u t e f o r v a r i o u s hydrogens  0S0 I C H - ( C H ) — C — CH - (CH ),, — I CI 3  3  2  (XIII)  7  2  CH 0S0 2  3  a chlorodocosanediol" d i s u l f a t e  on  - 40 -  the c h a i n , but never more than s i x ( E l v o s o n and V a g e l o s , 1969 1970;  Haines, 1973b; Haines  et a l . , 1969).  and  These s u l f o l i p i d s are  a l s o unique as the o n l y h a l o l i p i d s to be c h a r a c t e r i z e d  i n natural  fats. A group the absence  of b r o m o s u l f a t i d e s was  of c h l o r i d e i o n and i n the presence of bromide  i n Haines, 1971  and 1973a).  (references  They i n c l u d e a hexabromo- and a monobromo-  compound, b o t h o f which appear compounds.  found i n c e l l s c u l t u r e d i n  t o be o t h e r w i s e i d e n t i c a l to the c h l o r o -  E v i d e n c e f o r the o c c u r r e n c e of i o d o s u l f a t i d e was  and f l u o r i d e was  equivocal  found to be t o x i c to the c u l t u r e (Haines, 1973a).  Kates et _ a l .  (1967), i n t h e i r expanded.studies  H a l o b a c t e r i u m c u t i r u b r u m (Sehgal et a l . , 1962;  of l i p i d s of  Kates et a l . , 1965a and  1965b), c h a r a c t e r i z e d a s u l f o l i p i d as the s u l f a t e e s t e r 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 o c c u r r e n c e of t h i s s u l f o l i p i d  i n one of the extreme  0  HO CH  2  HO  (XIV)  diphytanyl diether glycosulfate  (C  2 0  H i)-0-CH 4  - 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 b y M a r s h a l l Brown  and  (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 methods as r e v i e w e d by H a i n e s  c a n be a c c o m p l i s h e d by a v a r i e t y o f  (1971).  The c h o i c e  depend upon t h e n a t u r e o f t h e s u l f o l i p i d  o f a method  t o be s t u d i e d ,  the b i o l o g i c a l  source, the occurrence of i n t e r f e r i n g substances i n the 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 In general,  liberated  preparation,  of the laboratory.  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 o f two t y p e s .  type involves  will  The  first  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  sulfur.  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 u p o n 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 lipid  of the  or a d e r i v a t i v e of i t . T h e m o s t common d e s t r u c t i v e m e t h o d f o r t h e a s s a y o f s u l f a t i d e s  is  h y d r o l y s i s , although the procedure i s probably the l e a s t  L e e s e t _ a l . (1959) s u g g e s t e d t h a t influence  on t h e h y d r o l y s i s  s u l f o n i c a c i d of the plant excludes the p o s s i b i l i t y  the phospholipids  of s u l f a t i d e s .  Unlike  exert  a  sulfate  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  reliable. protective ester, that  of 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  conditions. The r e d u c t i o n  method o f M a r t e n s s o n (1963a) a p p e a r s 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 that but  more cumbersome the reductive  than o t h e r methods. method c o u l d  I t w o u l d a l s o seem  possible  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 ,  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  to sulfonolipids.  -  Haines  42 -  (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  combustion procedure  (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 o f  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  analytical  procedure f o r s u l f o l i p i d s . The  azure-A c o l o r i m e t r i c  p r o c e d u r e o f Kean (1968) c a n be  used e f f e c t i v e l y on crude p r e p a r a t i o n s of s u l f a t i d e s s c r a p e d s p o t s f r o m TLC.  T h e o n l y known i n t e r f e r i n g  p r o c e d u r e was c a r d i o l i p i n . appears t o be s p e c i f i c of  The of  methylene b l u e method o f Jones  a l k y l sulfates  sulfatide sulfonic  a c i d s and  labelling.  such as that  sulfonolipid. (1945) f o r t h e e s t i m a t i o n  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  for alkyl  sulfonolipids.  most v e r s a t i l e , s e n s i t i v e  the physical  acid  (1963)  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  i n mammalian t i s s u e s  The of  and t h e s e a u r c h i n  as  substance i n t h i s  The a n t h r o n e method o f Weenink  f o r 6-deoxy h e x o s e s u l f o n i c  the plant sulfonolipid  as w e l l  and g e n e r a l l y  convenient  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 sS  I t s o n l y l i m i t a t i o n , f o r e x a m p l e , i s t h a t human b r a i n  35  lipids  35 are  n o t amenable t o S  of McCandless  Likewise the activation  the application  to  sulfolipids after  their separation v i a  most u s e f u l  chromatography.  method f o r i d e n t i f i c a t i o n and  f o u r major peaks c h a r a c t e r i s t i c  found i n s u l f a t i d e s .  (1963),  of densitometry to charred plates are a l l applicable  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 are  analysis  (1964), t h e c r e s y l v i o l e t assay o f Svennerholm  and  The  labelling.  The l a r g e s t  S-0 b o n d a t 1 2 1 0 - 1 2 6 0 cm  1  structural  spectrophotometry.  o f i o n i c and c o v a l e n t  sulfates  due t o a s y m m e t r i c v i b r a t i o n  i s useful  to characterize ionic  There .  of the  sulfate.  - 43  (as d i s t i n g u i s h e d  -  from c y c l i c or  dicovalent).  to the symmetric v i b r a t i o n s of the S-0 region.  A t h i r d band i n the  bond of s u l f a t e e s t e r s and sulfates. the  s u l f o n i c a c i d s has The  to the 0-S  760-840 cm•  (SO2)  s t r e t c h was plant  region.  (SO2)  was  s t r e t c h i n g mode was  s u l f o n o l i p i d has  u r c h i n s u l f o l i p i d has  not  to the  magnetic resonance (NMR)  C-0  The  from a s u l f o n i c  i n f r a r e d spectra  found to be  cm  An  been p u b l i s h e d  not  cm  (Haines, 1971). cm  1  and  1  .  the  The  S-0 the  w h i l e a spectrum of the  of o r g a n i c  sea  N a g a i , 1966).  i n v e s t i g a t i o n of the  expect t h a t s u l f a t e e s t e r s would y i e l d NMR  of  i n f r a r e d spectrum of  (Isono and  been an  spectra  1350  a s s i g n e d a t 1160  been p u b l i s h e d  date t h e r e has  1  v i b r a t i o n a l modes, i d e n t i f i e s  (as d i s t i n g u i s h e d  1  c o n s i s t e n t l y a t 900  To  i s due  been examined i n s e v e r a l l a b o r a t o r i e s  asymmetric s t r e t c h i n g of  symmetric  region  1  due  d i s t i n g u i s h e s primary from secondary  A f o u r t h band, due  i s i n the  bond i n the 1040-1080 cm  935-1040 cm  substance as a s u l f a t e e s t e r  a c i d ) and  A second band i s  sulfate esters.  nuclear One  would  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 a l c o h o l from.which the s u l f a t e e s t e r i s derived.  The  presence of e l e c t r o n e g a t i v e  the e l e c t r o n d e n s i t y  around the protons attached  and  produce a s m a l l d o w n f i e l d s h i f t .  the  study of h i g h e r r e s o l u t i o n NMR  very productive  and  s u l f a t e on a sugar The  very l i k e l y (or any  to the s u l f a t e d carbon  I t would, t h e r e f o r e ,  appear  s p e c t r a of t h i s problem would  that be  s o l v e the problem of l o c a t i n g a  poly-hydroxyl  compound).  mass s p e c t r a of s u l f a t e e s t e r s have not  to the i n a b i l i t y s u l f a t e esters  s u l f a t e group should reduce  been explored  to overcome the problem of v o l a t i l i t y .  The  only  t h a t would appear to be open to i n v e s t i g a t i o n are  the  due  - 44 -  the c y c l i c s u l f a t e s , which.with few e x c e p t i o n s reported  2. 6  t o occur  n a t u r a l l y (Haines,  1971).  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 b r a i n s u l f a t i d e has been t h e o b j e c t o f much  e f f o r t and many p u b l i c a t i o n s . s u l f a t i d e were best and  have.not been  R e l a t i v e l y l a r g e amounts o f crude  e x t r a c t e d and i s o l a t e d by the procedure of F o l c h  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 t o i s o l a t e a q u a n t i t y o f c e r e b r o s i d e s u l f a t e . If  s m a l l amounts of h i g h l y p u r i f i e d  s u l f a t i d e a r e r e q u i r e d the most  r a p i d procedure would undoubtedly be. i s o l a t i o n of the crude by  sulfatide  t h e F o l c h e x t r a c t i o n procedure f o l l o w e d by p r e p a r a t i v e t h i n l a y e r  chromatography (Wagner, e t a l . , 1964; Haines, 1971).  Cerebroside  s u l f a t e has a l s o been i s o l a t e d by zone e l e c t r o p h o r e s i s u s i n g buffer  borate  (Svennerholm and Svennerholm, 1963). Martensson (1963a) e x t r a c t e d  from l y o p h i l i z e d  the l a c t o s y l ceramide s u l f a t e  t i s s u e w i t h chloroform-methanol (2:1).  This  extract  was chromatographed on s i l i c i c a c i d columns, then DEAE c e l l u l o s e and the f r a c t i o n s c o n t a i n i n g s u l f a t i d e were separated l a y e r p l a t e s of S i l i c a G e l G. was n e c e s s a r y t o r e p e a t  I n order  on p r e p a r a t i v e  thin  t o o b t a i n pure s u l f o l i p i d i t  t h e p r e p a r a t i v e t h i n l a y e r chromatography  twice  on S i l i c a G e l 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 g a n g l i o s i d e s u l f a t e from the w a l l s of horses'  hooves u s i n g F o l c h ' e t a l . procedure (1957) and p r e -  p a r a t i v e t h i n l a y e r chromatography.  S e m i n o l i p i d was a l s o  isolated  - 45 -  from boar t e s t i s and spermatozoa guinea p i g t e s t i s procedure  ( I s h i z u k a : et al.,,. 1973)  (Suzuki'et ' a l . ,  1973)  and  from  by the F o l c h et a l .  (1957) and subsequently by s i l i c i c  a c i d and t h i n  layer  chromatography. Kates e t a l . (1967)  a c h i e v e d 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  isolating  the i o n i c  lipids  as an acetone i n s o l u b l e p e l l e t which was d i s s o l v e d i n a minimum volume of  c h l o r o f o r m and p r e c i p i t a t e d w i t h t e n volumes of methanol.  p r e c i p i t a t e was  s u b j e c t e d t o chromatography  This  on c o n s e c u t i v e columns of  S i l i c a u s i n g stepwise e l u t i o n s of chloroform-methanol  (4:1) i n t h e i r  system. M i d d l e b r o o k et al_.  (1959) e x t r a c t e d 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 w i t h hexane c o n t a i n i n g 0.05% procedure s i m i l a r t o t h a t of Jones  decylamine.  Using a  (1945), the authors were a b l e to  f o l l o w the m a t e r i a l d u r i n g t h e i s o l a t i o n , by i t s h e x a n e - s o l u b l e n e u t r a l red  complex i n a water-hexane system.  i s o l a t i o n has been p u b l i s h e d elsewhere  A more d e t a i l e d procedure f o r the (Gangadharam et a l . ,  1963;  Goren,  1969) . Alkyl sulfatides et  al.,  1969;  Haines and B l o c k , 1962)  ( E l v o s o n and V a g e l o s , 1969; 1970)  (Haines, 1965;  Mayer and Haines, 1967;  and.the c h l o r o a l k y l  Haines e t a l . ,  1969;  Mayer  sulfatides  E l v o s o n and V a g e l o s ,  were e x t r a c t e d from broken c e l l s of Ochromonas d a n i c a w i t h a  chloroform-methanol u s i n g subsequent  (2:1) m i x t u r e ; the crude s u l f a t i d e s were i s o l a t e d  e x t r a c t i o n w i t h petroleum e t h e r and b u t a n o l a f t e r  s a p o n i f i c a t i o n o f the chloroform-methanol e x t r a c t s of the broken  cells.  - 46  -  Seven procedures (Benson et a l . ; 1959; 1962)  have been used f o r the i s o l a t i o n of the p l a n t  For the i s o l a t i o n of a r e a s o n a b l e the procedure of O'Brien and The  Y a g i and  column and  sulfonolipid.  q u a n t i t y of r a t h e r pure m a t e r i a l  Benson (1964) i s the method of  procedure begins w i t h a crude l i p i d  a Florisil  (w/v)  f r a c t i o n s are eluted with  2,2-dimethylpropane to m a i n t a i n  on the column d u r i n g chromatography. then p l a c e d The  on a D E A E - c e l l u l o s e  The  on  chloroform-methanol The  solvents  anhydrous c o n d i t i o n s  sulfonolipid fraction i s  column which separates  procedures are v e r y w e l l d e s c r i b e d  p r e p a r e the s u p p o r t s and  choice.  e x t r a c t , which i s p l a c e d  s o l v e n t s which i n v o l v e stepwise i n c r e a s e s of p o l a r i t y . c o n t a i n 5%  Benson,  a pure p r o d u c t .  i n c l u d i n g the methods used to  columns.  A l t h o u g h no r e p o r t on the use  of F o l c h ' s  e x t r a c t i o n procedure  (1957) i s a v a i l a b l e , i t would appear t h a t t h i s procedure would be a good one  f o r o b t a i n i n g a c r u d e p r e p a r a t i o n of the s u l f o n o l i p i d which  might be f u r t h e r p u r i f i e d has  on DEAE c e l l u l o s e i f n e c e s s a r y .  a h i g h p r o b a b i l i t y of success  s u l f o n o l i p i d on s i l i c i c  i n view of the p o l a r n a t u r e of  a c i d inpregnated  paper (Mumma and  C l e a r l y s m a l l amounts of the s u l f o n o l i p i d can be preparative  T h i s procedure  isolated  the  Benson, 1961). by  t h i n l a y e r chromatography ( 0 ' B r i e n j i t a l . , 1964).  E l e c t r o p h o r e s i s at low pH appears to be u s e f u l f o r s m a l l s c a l e p r e p a r a t i v e work (Benson, 1963) . Nagai and  Isono  (1965) e x t r a c t e d  sea u r c h i n w i t h chloroform-methanol (1:1). were then chromatographed on a s i l i c i c 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  the s u l f o n o l i p i d of The  ether  a c i d column.  c o l l e c t e d and  the  insoluble l i p i d s An  anthrone  d r i e d i n vacuo.  The  - 47 -  r e s u l t i n g w h i t e powder was s o l u b l e i n c h l o r o f o r m c o n t a i n i n g a s m a l l amount o f methanol and w a t e r .  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  a c c o r d i n g t o t h e p r o c e d u r e of R a d i n e t a l . (1956) t o o b t a i n a phosphorus-free preparation.  2.7  Soil  Sulfolipids  A l t h o u g h no r e p o r t s on t h e o c c u r r e n c e of s o i l  sulfolipids  c o u l d be found, one might expect s u l f o l i p i d s , e x c r e t e d by l i v i n g o r r e t u r n e d i n d e c a y i n g organisms, would be found i n s o i l s .  This  e x p e c t a t i o n i s supported by H a i n e s (1964) who o b t a i n e d a p o s i t i v e Jones methylene b l u e t e s t ( J o n e s , 1945) from t h e n u t r i e n t medium o f sorghum and w h i t e 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 Y o n k e r s , New York. Lowe and DeLong (1961) found t h a t i n a few Quebec s o i l s , a  t h i r d o f t h e o r g a n i c s u l f u r was i n t h e form o f s u l f a t e s u l f u r . (1961) a l s o e s t i m a t e d t h e s u l f a t e e s t e r c o n t e n t i n A u s t r a l i a n to be about 52% o f t h e o r g a n i c s u l f u r .  Freney soils  Thus a p p r e c i a b l e 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 t h e widespread d i s t r i b u t i o n o f 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 p r o d u c t s o f organisms, of w h i c h 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 o f s u l f o l i p i d s i n s o i l s , i f any, would h e l p i n the of  b e t t e r u n d e r s t a n d i n g o f b o t h t h e s o i l s u l f u r c y c l e and t h e p o t e n t i a l t h e s o i l environment as a s o u r c e o f p l a n t a v a i l a b l e  sulfate.  - 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 Research. R. P a o l e t t i a n d D. K r i t c h e v s k y , ( E d s . ) A c a d e m i c P r e s s , New Y o r k , V o l . 1, pp. 3 8 7 - 3 9 4 .  Benson,  A.A. 1964. P l a n t membrane l i p i d s . 1 5 : 1-13.  Benson,  A.A. a n d I . 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Proceedings of a symposium. Braunschweig, 6—10 Sept. 1976. Wagner, H., L. Horhammer, and P. Waeff. 1964. Dunnschichtchromatographie von Phosphatiden und Glycolipiden. Biochem. Z e i t s c h r i f t 334: 175-184. Waksman, S.A. "and I.J. Hutching. 1935. Chemical nature of organic matter i n different s o i l types. S o i l S c i . 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. S o i l S c i . 30: 97-116. Wang, T.S.C., Y.C. Liang, and W.C. Shen. 1969. Method of extraction and analysis of higher f a t t y acids and t r i g l y c e r i d e s i n s o i l s . S o i l S c i . 107: 181-187. Wang, T.S.C, P.T. Hwang and C.Y. Chen. 1971. S o i l l i p i d s under various crops. S o i l S c i . 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 f o r the i s o l a t i o n of brain cerebroside, sulfatide and sphingomyelin. J. Chromatog. 18: 503-511. Wollrab, V. and M. S t r e i b l . 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 s u l f o l i p i d . V. L y s o s u l f o l i p i d 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 s u l f u r i c 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 i n non-calcareous s o i l s of humid regions usually occurs i n organic combination, r e l a t i v e l y l i t t l e i s 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 i n s o i l s .  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 i d e n t i f i e d i n s o i l hydrolysates.  In two Australian s o i l s , Freney et a l . (1972)  reported that.amino acid sulfur accounted f o r 21 and 30% of the t o t a l organic s u l f u r .  Approximately 60% of the amino acid sulfur was  cystine s u l f u r .  The remainder of the organic sulfur remains largely  unknown, except to the extent that a major portion i s thought to occur in ester sulfate form. Although the presence of s u l f o l i p i d s i n s o i l has not yet been reported, the presence of s u l f o l i p i d s i n a l l l i v i n g matter (Haines, 1971 and 1973) indicates that a continual input to the s o i l system i s l i k e l y , and complexes with clay, protein and carbohydrates, or their presence i n microbial tissue could contribute to an accumulation i n the s o i l .  However, i t i s 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 s u l f o l i p i d since these forms are l i k e l y 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 i n the l i p i d extract) i n s o i l at a l l and no other authors have related phospholipid ,P ( l i p i d P), contents with other s o i l c h a r a c t e r i s t i c s , except f o r the report of Kowalenko and McKercher (1971b) on organic P. The purpose of t h i s study was to determine the content of l i p i d sulfur i n a range of s o i l s of B r i t i s h Columbia and to r e l a t e these values to other s o i l c h a r a c t e r i s t i c s , p a r t i c u l a r l y 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 i n this study were from various places i n the Province of B r i t i s h Columbia.  Eighteen of  the samples, representing three d i f f e r e n t horizon types, were forest soils.  Nine of them were organic s o i l s and ten of them were surface  horizons of either v i r g i n or a g r i c u l t u r a l grassland s o i l s .  Origin and  gross chemical composition of the s o i l s are given i n Table 3.1. A l l samples were a i r dried at room temperature, a 2 mm sieve.  crushed, and passed  through  For the chemical analyses the samples were further  crushed and passed through a 100 mesh (0.149 mm)  sieve.  T a b l e 3.1  O r 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  Dominant V e g e t a t i o n (Location/Classification S i t k a spruce  ii  (Port Renfrew)  II  II  Western hemlock (Jordan R i v e r ) II II n Lodgepole  ti  (Manning Park) II  Western red cedar ( V i c t o r i a ) D o u g l a s - f i r (Diamond Head)  ii  II  Aspen ( B e a t t o n R i v e r ) Hemlock-Cedar (Mt. Seymour) II  II  Western r e d cedar ( V i c t o r i a ) Aspen (Beaton River) n II Oak (Upland Park) D o u g l a s - f i r (Stamp F a l l s Park) Maple (Stamp F a l l s Park) Subalpine grass (Mt. Kobau) Grass ( S t . F t . J o h n / S o l o d i c Black) II  II  II  " / E l u v . Black) " (Oxbow/Orthic Black) " (Cloverdale/Humic E l u v . G l e y s o l ) " ( D e l t a / S a l i n e Humic G l e y s o l ) " (Langley/Hutnic E l u v . G l e y s o l ) " ( P r e s t / O r t h i c Humic G l e y s o l ) " (Hazelwood/ " Grass/sedge (Kamloops/Mesisol) n II " (Lulu Island/Humisol) " (Metchosin/Humisol) " (Whipsaw Creek/Mesisol) " ( " /Humisol) ( " /Mesisol) ( " /Humisol) Subalpine grass (Church Mt.)  LI Horizon  %  F H F H F H F F H F F H Ah Ah Ah, Ah^ Ah Ah Ah Ah Ah Ah Ah Ap Ap Ah Ah Ah Om Om Oh Oh Om Oh Om Oh Om  85.4 81.7 80.7 82.5 71.9 52.5 61.7 69.9 83.3 55.9 47.1 71.4 14.4 27.0 9.48 23.1 14.3 16.7 10.2 11.4 19.7 13.6 4.53 11.4 6.26 20.7 9.03 10.8 55.8 75.5 77.7 51.4 80.8 37.3 65.7 38.0 29.8  x  1  Ln  PH H0  3.7 3.6 3.9 3.0 4.6 4.7 5.5 3.8 3.5 5.9 3.6 3.4 6.1 5.6 5.6 5.5 5.5 5.7 5.9 6.6 5.9 5.1 8.5 5.5 5.8 5.2 5.2 5.2 5.2 5.7 3.3 5.2 5.0 3.7 4.3 4.1 4.6  2  ( a n a l y s i s expressed  C  Lipid  %  %  57.5 54.8 55.7 57.0 50.5 33.8 42.9 53.6 63.5 36.7 31.7 49.6 7.74 16.2 4.27 15.9 7.51 8.00 5.28 5.70 11.8 7.33 1.92 5.47 3.24 12.0 4.15 4.78 39.6 57.5 51.4 36.6 55.3 28.1 47.4 31.7 17.8  2.57 2.22 4.01 4.24 5.33 5.24 3.92 2.93 3.01 1.61 1.77 2.22 0.333 0.610 0.115 0.368 0.350 0.294 0.289 0.146 0.357 0.250 0.061 0.239 0.237 0.268 0.155 0.278 3.40 2.59 2.01 3.19 3.32 1.71 4.95 1.43 1.30  on oven d r y b a s i s ) .  Total-S ppm 2328 2117 1750 2016 936 887 1015 1063 1405 1726 985 1354 375 704 162 1219 213 543 542 421 858 926 286 684 581 928 367 510 2330 18321 23053 7193 3172 30431 3716 14135 1122  HI-S ppm 533 461 469 579 200 187 264 207 281 337 219 364 144 217 74.9 394 75.2 193 252 208 374 291 142 361 219 564 155 259 772 5259 7707 1995 1597 22758 1695 8477 397  Lipid-S ppm 24.2 22.8 37.5 37.5 14.8 30.8 13.8 14.7 14.8 16.6 13.8 11.0 5.33 7.13 1.59 7.04 4.48 3.94 . 4.17 2.85 7.81 5.15 2.50 9.65 12.9 6.54 3.30 9.19 39.0 192.0 150.0 181.0 68.3 291.0 46.5 146.0 25.4  Total-P ppm 1099 815 1005 637 862 921 830 683 599 1624 364 402 709 1749 535 1642 809 3668 1612 1308 1190 1415 578 1324 1458 1592 1240 1515 1281 954 777 1849 583 1040 1817 1387 1680  Lipid-! ppm 25.5 16.4 44.4 15.9 22.3 22.6 21.5 17.3 13.5 29.3 10.3 8.15 2.77 11.3 4.34 5.81 3.25 8.84 3.96 4.76 5.95 4.27 1.45 4.49 2.83 4.13 2.95 4.13 21.9 25.3 10.1 7.10 26.1 12.9 10.9 10.2 15.9  - 63 -  Extraction  Extraction of s o i l l i p i d s was carried out by a modification of the method of Bligh and Dyer (1959) without any pretreatment.  The  Bligh and Dyer method was adapted i n this study, since the merit of the method as examined by Kowalenko and McKercher (1970) rests i n i t s ease of adaptability to bulk extraction for q u a l i t a t i v e work and quantitative as well.  The a i r dried s o i l s were suspended i n s u f f i c i e n t  water to make the water content of the suspension 80 + 1%.  One volume  of the suspension was homogenized with three volumes of methanolchloroform (2:1, v/v) i n a Waring blender for 2 minutes.  One volume  of chloroform was then added while blending, and blending continued f o r a further 30 seconds.'  F i n a l l y 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 s l i g h t 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 f o r 30 minutes to an hour. of the chloroform layer was then recorded.  The volume  After removing the alcoholic  layer by aspiration, the chloroform layer, containing t o t a l l i p i d s (Bligh and Dyer, 1959), was reduced to a known volume at 35-40°C i n vacuo. The extraction of t o t a l l i 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 i n the concentrated t o t a l l i p i d s was s u f f i c i e n t to permit satisfactory analysis.  The concentrated t o t a l l i p i d s contained i n the volumetric  - 64 -  f l a s k were stored i n a freezer and a portion of this solution was used f o r analysis.  A n a l y t i c a l Methods  S o i l pH was determined with a Radiometer Model PHM 62 standard pH mater using a soil:water r a t i o of 1:5. Total organic carbon content was determined by the WalkleyBlack wet-combustion method (Allison, 1965) .  Samples of 0.02-0.3 g  were used depending on the carbon content. Loss on i g n i t i o n  (LI) was. determined "by" i g n i t i n g i n 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 f i n i s h as described by Kowalenko and Lowe (1972). Sulfur content i n 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 i n a stream of nitrogen gas using the methods f o r t o t a l 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 i d e n t i c a l to that of t o t a l sulfur except for the temperature of the sand bath (260-280°C).  - 65 -  Phosphorus content i n l i p i d extracts (lipid-P) was determined by the method of Dick and Tabatabai (1977) for t o t a l phosphorus after an aliquot of the l i p i d extract was taken to dryness as described for the determination of l i p i d - S . The determination of l i p i d contents followed the procedure of Bligh and Dyer (1959). of  The procedure was as follows:  a portion  the l i p i d extract containing 100 to 200 mg l i p i d was transferred  into a tared f l a s k ; the extract was evaporated to dryness under an IR lamp at 40-50°C under a stream of nitrogen; after drying over phosphoric anhydride i n a vacuum -desiccator, the dried residue was weighed.  RESULTS AND DISCUSSION  Organic Carbon and Total Phosphorus  The organic carbon contents for the thirty-seven s o i l s  fell  i n the range 1.92 to 63.5% with a mean of 29.0%, and the t o t a l phosphorus content varied from 364 to 3,688 ppm with a mean of 1,177 ppm (Table 2). The mean value of the t o t a l phosphorus content was considerably higher  1  than the means found by Walker and- Adams (1959) for New Zealand s o i l s (488 ppm), Williams et a l . (1960) for Scottish s o i l s (1,080) and Neptune jit a l . (1975) for Iowa (542 ppm) and B r a z i l (381 ppm) s o i l s .  This was.  probably due to the r e l a t i v e l y large number of organic samples included.  - 66  -  When the s o i l s were grouped i n t o o r g a n i c and m i n e r a l as shown i n T a b l e 3.2  the mean o r g a n i c carbon contents  samples were, as expected,  i n the o r g a n i c s o i l s . vegetation, drainage  of o r g a n i c  s i g n i f i c a n t l y h i g h e r than those of  m i n e r a l s o i l s , but the t o t a l phosphorus contents When the s o i l s are grouped  and h o r i z o n type, the mean o r g a n i c carbon  contents  well  significantly  of w e l l d r a i n e d f o r e s t , w h i l e the mean t o t a l phosphorus  tended to be lowest  horizons.  i n the w e l l d r a i n e d f o r e s t  organic  The mean v a l u e s f o r the r e s t of f o u r groups were s i m i l a r . There was  the pH,  coefficient  a c l o s e r e l a t i o n s h i p between the o r g a n i c  as i l l u s t r a t e d by the h i g h l y s i g n i f i c a n t  and pH v a l u e s  ( T a b l e 3.3).  Other f a c t o r s such as s o i l t e x t u r e  and  p r a c t i c e were d o u b t l e s s a l s o  c o n t r i b u t e d to the v a r i a t i o n s w i t h i n groups.  phosphorus content was  no  (r = 0.259) between the t o t a l phosphorus  d i f f e r e n c e s i n a g r i c u l t u r a l and m a n u r i a l i n v o l v e d and  carbon  correlation  ( r = -0.687***) f o r a l l s o i l samples; t h e r e was  significant correlation  may  contents  than those of the m i n e r a l h o r i z o n s of 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 and  content  lower  according-to  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 a l s o appeared to be  and  the  tended to be  of samples of p o o r l y d r a i n e d grass-sedge o r g a n i c s o i l s and  higher  soils  The  s i g n i f i c a n t l y c o r r e l a t e d with organic  ( r = -0.371*), but o n l y a t the 5% l e v e l .  T h i s low  total  carbon  correlation  i n d i c a t e t h a t i n o r g a n i c phosphorus of s o i l can be a predominant  component of the t o t a l phosphorus  present.  T a b l e 3.2  t  R e l a t i o n s h i p between o r g a n i c carbon and t o t a l phosphorus contents and pH v a l u e s among s o i l  Number o f S o i l Group  Samples  PH (H 2 0) Mean (Range)  Organic C (%) Mean (Range)  gtoups.  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)  21  4.29 (3.03-5.85)  45.4  (17.8-63.5)  1010 (364-1849)  Mineral  16  5.80 (5.10-8.49)  7.58 (1.92-16.2)  139(3 (535-3668)  12  4.09 (3.03-5.85)  48.9  (31.7-63.5)  82b (364-1624)  4.57  40.6  (Ah and Ap)  Well Drained F o r e s t LH P o o r l y Drained G r a s s l a n d Om/Oh  9  (17.8-57.5)  126*3 (583-1849)  (5.53-6.11)  9.94(4.27-16.2)  15lS (535-3668)  6.39 (5.10-8.49)  6.41(1.92-11.8)  1221 (578-1612)  5.35 (5.15-5.76)  5.93(3.24-12.0)  1426 (1240-1592)  Well Drained F o r e s t Ah  6  5.67  Well Drained G r a s s l a n d Ah  5  P o o r l y Drained G r a s s l a n d Ah/Ap  5  (3.32-5.77)  Table 3.3  Correlation matrix of some chemical characteristics of s o i l samples.  pH  pH  C  L  TS  LS  TP  LP  LS/TS  LS/L  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***  LS  -0.272  0.372*  0.233  . 0.935***  TP  0.259  -0.371*  -0.267  LP  -0.367*  0.733***  LS/TS  -0.129  0.165  0.Q13  -0.091  LS/L  HI-S  0.873***  1.000  0.037  -0.045  -0.029  1.000  0.704***  0.114  0.044  0.178  -0.152  1.000  0.486**  -0.160  -0.153  0.050  -0.128  0.335*  -0.163  C = Organic Carbon;  L = Lipid;  TP = Total Phosphorus;  LP = L i p i d Phosphorus;  *** = Very Highly Significant (99.9%).  1.000  0.816***  TS = Total Sulfur;  0.842***  0.794***  0.017  HI-S = Hi-reducible Sulfur;  * = Significant (95%);  -0.135  1.000 -0.088  LS = Lipid Sulfur  ** = Highly Significant (99%)  1.000  - 69 -  Sulfur  The  total  s u l f u r contents  varied  ppm w i t h a mean o f 3,524 ppm ( T a b l e 3.4).  b e t w e e n 162 ppm a n d 30,431 Therange 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 a n d t h e mean v a l u e was s u b s t a n t i a l l y  higher  than  t h o s e r e p o r t e d b y Rehm a n d 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 p p m ) ,  Tabatabai  et a l .  a n d B r e m n e r (1972) f o r I o w a s o i l s  (1973) f o r S a s k a t c h e w a n s o i l s Brazil  (294 p p m ) , B e t t a n y  (284 p p m ) , N e p t u n e e t a l . (1975) f o r  (166 ppm) a n d I o w a (319 p p m ) , a n d W i l l i a m s  Pembrokenshire s o i l s s o i l s can vary over  (710 ppm).  Since thet o t a l  an extremely  (1975) f o r N o r t h  West  sulfur contents of  w i d e r a n g e f r o m 0.002 t o 3.5%  ( W h i t e h e a d , 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 considering the proportion ofhighly  organic  i s not unexpected,  samples i n c l u d e d i n t h e  study. As contents  shown i n t h e T a b l e  for well  3.4, t h e r a n g e s o f t h e t o t a l  sulfur  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 both p o o r l y and  well drained grassland mineral horizons aresimilar t othe values d e t e r m i n e d b y Rehm a n d 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 Minnesota  (131 t o 940 ppm w i t h a mean o f 510 p p m ) . The  than  total  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  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  i n Table  3.4.  forest  horizons  s o i l s a s shown  T h e t r e n d s a r e i n a g r e e m e n t w i t h t h e r e p o r t b y Lowe  (1964) a n d J o n e s e t a ] - .  (1972), a n d T a b a t a b a i  L e v e s q u e (1974), r e s p e c t i v e l y . i n the.mean t o t a l  a n d B r e m n e r (1972) a n d  T h e r e was n o s i g n i f i c a n t  s u l f u r contents  g r a s s l a n d m i n e r a l h o r i z o n samples.  between w e l l This r e s u l t  and p o o r l y  difference drained  i si ncontrast t o the  T a b l e 3.4  R e l a t i o n s h i p s of t o t a l s u l f u r w i t h the H i - r e d u c i b l e s u l f u r (C-S) f o r s o i l groups.  Total S S o i l Group  All  Samples  Organic  (LH, Om and Oh)  Mineral  (Ah and Ap)  Well Drained F o r e s t LH  (ppm)  Mean (Range)  HI-S  (ppm)  Mean (Range)  (HI-S) and carbon-bonded  HI-S (% of t o t a l Mean (Range)  S)  sulfur  C-S  (% of t o t a l Meari (Range)  3,524 (162-30,431)  1,587 (74.9-22,758)  36.5 (19.5-74.8)  63.5 (25.2-80.5)  5,765 (887-30,431)  2,609 (187-22,758)  31.7 (19.5-74.8)  68.3 (25.2-80.5)  582 (162-1,219)  245 (74.9-564)  42.7 (30.8-60.8)  57.3 (39.2-69.2)  1,465 (887-2,328)  345 (187-579)  23.1 (19.5-28.7)  76.9 (71.3-80.5)  11,497 (1,122-30,431)  5,629 (397-22,758)  43.2 (27.7-74.8)  56.8 (25.2-72.3)  Well Drained F o r e s t Ah  536 (162-1,219)  183 (74.9-394)  36.4 (30.8-38.4)  63.6 (53.8-69.2)  Well Drained Grassland Ah  607 (286-926)  253 (142-374)  44.1 (31.4-49.7)  55.9 (50.3-68.6)  P o o r l y Drained G r a s s l a n d Ah/Ap  614 (367-928)  312 (155-564)  48.9 (37.7-60.8)  51.1 (39.2-62.3)  P o o r l y Drained G r a s s l a n d Om/Oh  S)  - 71 -  r e s u l t s o f Lowe ( 1 9 6 9 ) , who f o u n d total  sulfur  i n Gleysolic profiles  However, i n each case content  significantly phosphorus  t o t a l S tends  to increase with organic  total  sulfur  contents  o f t h e s o i l s were found  c o r r e l a t e d w i t h organic carbon  carbon  carbon  ( r = 0.282),  t o be n o t total  ( r = 0 . 0 3 7 ) , o r pH ( r = - 0 . 2 9 9 ) , a s shown i n T a b l e  poor c o r r e l a t i o n w i t h organic carbon  of Bettany  and  t h a n i n Chernozems and P o d z o l s .  (Table 3.2). The  The  considerably higher levels of  e t a l . ( 1 9 7 3 ) who f o u n d  ( r = 0.91***).  importance  i s contrast to the result  a very high correlation with  The c l o s e r e l a t i o n s h i p  of the organic sulfur  c o r r e l a t i o n s found  3.3.  i n those  organic  i n d i c a t e s t h e dominance soils.  The p o o r  i n t h i s s t u d y w e r e p r i m a r i l y d u e t o much m o r e d i v e r s e  s a m p l e s , a n d p a r t i c u l a r l y d u e t o some s a m p l e s f r o m o r g a n i c s o i l s have extremely and  high sulfur  t o t a l phosphorus The  contents but r e l a t i v e l y  f r a c t i o n of s o i l s  inorganic sulfate plus a part of the organic sulfur (Freney,  1961).  includes the thought  The H l - r e d u c i b l e s u l f u r  r a n g e d f r o m 74.9 t o 22,758 ppm w i t h a mean o f 1,587 ppm The  carbon  contents.  Hl-reducible sulfur  of organic s u l f a t e  low organic  which  to consist contents  (Table 3.4).  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 w e r e h i g h l y c o r r e l a t e d ( r = 0.924***)  with the t o t a l s u l f u r contents  ( T a b l e 3.3) a n d c o n s e q u e n t l y  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  r e d u c i b l e s u l f u r c o n t e n t s were expressed sulfur, soils  ( T a b l e 3 . 4 ) . When t h e H l as a percentage  of the t o t a l  i t c a n b e s e e n 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  (31.7%) were c o n s i d e r a b l y lower  (42.7%).  gave a  T h e mean v a l u e o b t a i n e d  than those f o r m i n e r a l  from t h e samples o f w e l l  soils  drained  -  forest organic horizons  72  -  (23.1%) were s i g n i f i c a n t l y  f r o m 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 ( 4 4 . 1 % and  lower  ( 3 6 . 4 % ) m i n e r a l h o r i z o n s , and  soils  trends but  Similar  for virgin soils 64.1%  respectively,  sulfur  i n G r e y Wooded LH  c o u l d be a c c o u n t e d  r e p o r t e d f o r Saskatchewan s o i l s Wooded s o i l s ) b y B e t t a n y  from  organic  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  o f A l b e r t a b y Lowe ( 1 9 6 5 ) who  of the t o t a l  those  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 (43.2%).  than  and  found  t h a t 32.5%  C h e r n o z e r m i c Ah  f o r as H l - r e d u c i b l e s u l f u r . ( 5 0 % f o r C h e r n o z e r m i c and  ( 1 9 7 3 ) , and  for Brazil  and  The  36%  ( 5 1 . 1 % ) and  horizons, values  f o r Grey  Iowa  (54.5%),  s o i l s b y N e p t u n e _et a l . ( 1 9 7 5 ) a r e i n c l o s e r a g r e e m e n t t o t h o s e f o r w e l l and  poorly drained grassland The  procedure  sulfur  (C-S)  alkali  (DeLong and  soils.  f o r the d i r e c t d e t e r m i n a t i o n of  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 Lowe, 1 9 6 2 ) , h a s b e e n c r i t i c i z e d  (1970).  They s u g g e s t e d  resulted  f r o m t h e d i f f e r e n c e b e t w e e n t o t a l and  Consequently  carbon^bonded  by F r e n e y e t a l .  t h a t a b e t t e r estimate of carbon-bonded  t h i s a n a l y s i s was  carbon-bonded s u l f u r  of  not  o b t a i n e d by  s u l f u r a r e shown i n T a b l e  3.4.  performed.  Hl-reducible sulfur. The  mean v a l u e s  t h e d i f f e r e n c e as a p e r c e n t The  sulfur  for  of t o t a l  trends described f o r the H l -  r e d u c i b l e s u l f u r are reversed w i t h the s o i l s from organic h o r i z o n s 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 percentage s u l f u r as c a r b o n - b o n d e d  L i p i d and  Lipid  The  of  of  total  sulfur.  Phosphorus  lipid  contents  of s o i l s v a r i e d over a very wide  range,  ;  - 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 s o i l s ) were reported very recently by Siem jit a l . (1975) for North Vietnam s o i l s .  The authors pointed out that the l i p i d contents  of North Vietnam s o i l s were very low, about one-tenth that of the s o i l s of USSR.  Moreover, their values included the l i p i d contents of  subsurface s o i l s which, i n general, contain l i t t l e l i p i d except i n the case of humic podzols. Fridland (1976) reported that the contents of l i p i d s i n the Horizon of most s o i l s 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 s o i l s were much higher than those (0.271%) for mineral s o i l s (Table 3.5).  I t also was  seen that well drained forest mineral horizons  had s l i g h t l y higher mean l i p i d content than the corresponding poorly and well drained grassland mineral horizons. contrast to the values of Jones (1970) who  These values are i n  found much higher l i p i d  content i n mineral horizon (A.,) samples (5.80% at around 20 cm depth and 3.72%  at around 40 cm depth) than i n 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 i n 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 i n Table 3.5,  i t can be seen that the mean value obtained from  Table 3.5  S o i l Group  A l l Samples Organic (LH, Om and Oh) Mineral (Ah and Ap) Well Drained Forest LH  The distribution of l i p i d and l i p i d phosphorus i n s o i l s .  Lipid (%)  Lipid (% of OM)  Mean (Range)  Mean (Range)  Lipid P (ppm) Mean (Range)  Lipid P (% of total P) Mean (Range)  1.82 (0.061-5.33)  3.22 (1.30-8.99)  12.5 (1.45-44.4)  1.36 (0.194-4.48)  3.00 (1.30-5.33)  3.95 (2.27-8.99)  18.5 (7.10-44.4)  2.11 (0.334-4.48)  0.271(0.061-0.61)  2.27 (1.30-4.20)  3.26 (1.61-5.33)  4.01 (2.35-8.99)  20.6 (8.15-44.4) 15.6 (7.10-26.1)  4.70 (1.45-11.3)  0.363(0.194-0.811) 2.53 (1.81-4.42)  Poorly Drained Grassland Om/Oh  2.26 (1.30-4.95)  3.87 (2.27-6.06)  Well Drained Forest Ah  0.345(0.115-0.610)  2.07 (1.34-2.70)  6.05 (2.77-11.3)  0.474 (0.241-0.811)  Well Drained Grassland Ah  0.221 (0.061-0.357)  2.05 (1.49-3.18)  4.08 (1.45-5.95)  0.333 (0.246-0.500)  Poorly Drained Grassland Ah/Ap  0.235 (0.155-0.278)  2.71 (1.30-4.20)  3.71 (2.83-4.49)  1.56 (0.384-4.48)  0.261 (0.194-0.339)  - 75 -  the organic s o i l s (3.95%) was considerably higher than that for mineral s o i l s (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 s o i l s (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% were reported by Morrison and Bick (1967). Stevenson  for mineral s o i l s )  In a review a r t i c l e ,  (1966) also pointed out that the range for the l i p i d  content  of humus of most a g r i c u l t u r a l l y important s o i l s 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 s o i l s 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 i n Table 3.6.  These results indicate that high l i p i d  content w i l l be found in.the s o i l s with a higher organic carbon content and a lower pH value as pointed out by Stevenson (1976) i n their review a r t i c l e s .  (1966) and Fridland  Although i t i s uncertain whether the  high l i p i d content of organic matter i n a c i d i c s o i l s results from i n a b i l i t y of microorganisms  to decompose completely the l i p i d s occurring  in plant remains, or larger quantities of l i p i d s are synthesized by microorganisms  (Stevenson, 1966), the maximum l i p i d content i s believed  to be 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 (Fridland, 1976). The l i p i d content was highly correlated with organic carbon content (r = 0.806***) only for mineral s o i l s , when s o i l s were grouped into two groups, i . e . , organic and mineral groups.  Similarly when  Table  3.6  S o i l Group  All  Samples  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  PH  -0.528***  Organic C  0.827***  content and other s o i l  properties.  Total S  HI-S  0.118  0.044  Lipid F  0.704*** ON  Organic (LH, Om and Oh) M i n e r a l (Ah and Ap)  0.125 -0.395  0.344 0.806***  -0.388  -0.369  0.439  0.219  0.283 0.717**  W e l l Drained F o r e s t LH  0.127  0.071  -0.350  -0.213  P o o r l y Drained Grassland Om/Oh  0.346  0.536  -0.477  -0.465  0.119  W e l l Drained F o r e s t Ah  -0.025  0.820*  0.470  0.404  0.620  W e l l Drained G r a s s l a n d Ah  -0.811  0.860  0.802  0.943*  0.800  0.035  0.431  0.638  0.608  0.652  P o o r l y Drained G r a s s l a n d Ah/Ap  0.287  - 77 -  the samples were separated into f i v e groups (Table 3.6), a s i g n i f i c a n t 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 s o i l s were grouped either into two or f i v e groups.  These results were, not expected and  they deserve further investigation. The l i p i d contents were also not s i g n i f i c a n t l y correlated with either t o t a l or Hl-reducible sulfur contents except for HIreducible sulfur content of well drained grassland Ah samples (r = 0.943*).  These poor correlations are probably due to the organic  s o i l s which have very high t o t a l sulfur contents and consequently  very  high HI-reducible sulfur contents. L i p i d phosphorus includes those  phosphorus-containing  materials which are components of l i p i d s and consequently phospholipid.  represents  Hance and Anderson (1963b) provided evidence that the  phosphorus i n a l k a l i n e 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 i d e n t i f i e d 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 l i p i d s i n a s o i l extract. L i p i d phosphorus represents a small percentage of the t o t a l phosphorus i n s o i l s .  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) f o r s o i l s of Great B r i t a i n ranging from 3.1 to 7.0 ppm with a mean of 4.9 ppm; by Simoneaux and Caldwell (1965) for U.S.A. s o i l s ranging from 0.24 to 1.70 ppm; by Dormaar (1970) for Alberta s o i l s from 0.32 to 1.30 ppm with a mean of 7.32 ppm; by Kowalenko and McKercher (1971b) f o r Saskatchewan s o i l s from 0.6 to 14.5 ppm with a mean of 3.4 ppm. The l i p i d phosphorus i s s i g n i f i c a n t l y correlated with the organic carbon content (r = 0.733***) and l i p i d content (r = 0.704***) as shown i n Table 3.3 and Table 3.6  Therefore the trends w i l l be  similar to l i p i d content d i s t r i b u t i o n of s o i l groups (Table 3.5).  The  high c o r r e l a t i o n of the l i p i d phosphorus with the organic carbon content and the poor c o r r e l a t i o n with t o t a l phosphorus were i n agreement with the r e s u l t 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 s i g n i f i c a n t c o r r e l a t i o n between l i p i d phosphorus and l i p i d content when s o i l s were classed into f i v e d i f f e r e n t groups on the basis of vegetation and drainage condition. However, there was s i g n i f i c a n t c o r r e l a t i o n between them (r = 0.717**) for mineral s o i l s alone, excluding organic s o i l s .  The poor c o r r e l a t i o n for organic s o i l s i s  l i k e l y to be due to the poor c o r r e l a t i o n between l i p i d and organic carbon content, which i s d i f f i c u l t to interpret : as described previously.  - 79  The  Distribution  The with l i p i d  of the L i p i d S u l f u r i n S o i l s  lipid  i n c l u d e s any  sulfate  reduced  (1973) f o u n d t h a t s u l f u r  therefore include a  sulfur  The extremely  lipid  sulfur  0.3  and  fraction in  (Table 3.7).  T h e r e was  ( 1 9 5 9 ) m e t h o d and  of the t o t a l  of the t h i r t y - s e v e n samples t o 291  ppm  soils.  When r e l a t i n g  s l i g h t l y h i g h e r mean v a l u e  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 m a l l and  t h e r a n g e s and  However, i t c a n be  said  horizons  samples a r e lower  of f o r e s t  o r g a n i c h o r i z o n s , and Table contents lipid  the  than  the  3.7  expressed  sulfur  had ppm  that the l i p i d  the l i p i d  sulfur  sulfur  seem of  The  d e v i a t i o n s were  in well  i s concentrated  as p e r c e n t a g e o f t o t a l s u l f u r .  large.  i n mineral drained  forest  i n organic  a l s o shows t h e d i s t r i b u t i o n o f t h e l i p i d  c o n t e n t s m e a s u r e d i n ppm,  horizons  There d i d not  contents  those  lipid  lipid  s u l f u r between s o i l s  standard  than  of  corresponding  g r a s s l a n d s a m p l e s f o r t h e same m i n e r a l h o r i z o n s .  d i f f e r e n c e was  two  w i t h a mean o f 40.1  h o r i z o n s of more f r e e l y d r a i n e d g r a s s l a n d samples.  and  these  sulfur.  to h o r i z o n types, the p o o r l y d r a i n e d m i n e r a l  o f g r a s s l a n d had  forest  e x t r a c t s of  a s i g n i f i c a n t d i f f e r e n c e i n the contents  s u l f u r b e t w e e n o r g a n i c and m i n e r a l contents  sulfur.  soils.  i n chloroform  Dyer  0.6%  contents  w i d e r a n g e , f r o m 1.59  thiol  variety  i n o x i d i z e d ( H i - r e d u c i b l e ) and  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  sulfur  e s t e r and  (carbon-bonded) form were p r e s e n t  forms of s u l f u r r e p r e s e n t e d  an  may  s u l f u r which i s associated  ester, sulfonic  much i s known a b o u t t h i s l i p i d  Kowalenko  two  sulfur  components i n s o i l s and  o f f o r m s , s u c h as Not  -  sulfur  In contrast to  the p e r c e n t . l i p i d  soils.  sulfur  the  did  not  Table  3.7  The d i s t r i b u t i o n  Lipid S o i l Group  All  Samples  Organic (LH, Om and Oh)  of l i p i d  S (ppm)  Mean + SD  a  (Range)  sulfur  in soils.  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)  40.1 + 65.5 (1.59-291)  1.33 (0.556-3.47)  2,455 (278-17,018)  66.3 + 77.3 (11.0-291)  1.51 (0.651-3.47)  2,523 (278-17,018)  5.85 + 3.02(1.59-12.9)  1.10 (0.556-2.22)  2,364 (1,169-5,489)  W e l l Drained F o r e s t LH  21.0 + 9.50 (11.0-37.5)  1.51 (0.813-3.47)  P o o r l y Drained G r a s s l a n d Om/Oh  127 + 88.7 (25.4-291)  1.51 (0.651-2.52)  4,965 (939-17,018)  Mineral  (Ah and Ap)  692 (278-1,031)  W e l l Drained F o r e s t Ah  4.92 + 2.09(1.59-7.13)  1.14 (0.578-2.10)  1,448 (1,169-1,913)  W e l l Drained G r a s s l a n d Ah  4.50 + 2.13(2.50-7.81)  0.757 (0.556-0.910)  2,348 (1,443-4,098)  P o o r l y Drained Grassland Ah/Ap  8.32 + 3.60(3.30-12.9)  1.41 (0.704-2.22)  3,481 (2,129-5,489)  SD = Standard D e v i a t i o n  - 81 -  show a s i g n i f i c a n t difference between organic and mineral s o i l s . Furthermore, the mean values for well drained forest organic horizons and organic s o i l s were i d e n t i c a l .  The l i p i d sulfur contents of the well  drained forest and poorly drained grassland mineral horizons were s i g n i f i c a n t l y higher than the values for well drained grassland mineral horizons.  The low values f o r well drained grassland samples  were probably due to the higher valuers for t o t a l sulfur content but lower l i p i d sulfur contents. There were highly s i g n i f i c a n t correlations between l i p i d and t o t a l sulfur  (r = 0.935***) and Hl-reducible sulfur  sulfur  (r = 0.873***),  but the l i p i d sulfur correlated with organic carbon (0.327*) s i g n i f i c a n t l y only at the 5% l e v e l (Table 3.8). On the other hand, the r e l a t i v e  lipid  sulfur content was s i g n i f i c a n t l y 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 t o t a l sulfur, and also was s i g n i f i c a n t l y correlated only with t o t a l  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 s o i l s were not s i g n i f i c a n t l y correlated with any other s o i l factors whether expressed as ppm of s o i l , as % of t o t a l sulfur or as ppm of l i p i d ; contents  However, the l i p i d  sulfur  of organic s o i l s were highly s i g n i f i c a n t l y correlated with  t o t a l sulfur and Hl-reducible sulfur when expressed as ppm of s o i l , and only with t o t a l sulfur when expressed as ppm of l i p i d , but s i g n i f i c a n t l y only at the 5% l e v e l with l i p i d contents when expressed as % of t o t a l 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  T a b l e 3.8  Correlation coefficients for lipid  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 .  Correlation Coefficient; S o i l Group  All  pH  Samples  -0.272  Organic (LH, Om and Oh) Mineral  (Ah and Ap)  W e l l Drained F o r e s t LH  •  P o o r l y Drained G r a s s l a n d Om/Oh W e l l Drained F o r e s t Ah W e l l Drained G r a s s l a n d Ah P o o r l y Drained G r a s s l a n d Ah/Ap  Organic C  0.327*  L i p i d S u l f u r versus  Lipid  Total S  0.233  0.935***  0.873***  0.178  0.925***  0.871***  -0.252  HI-S  0.090  -0.232  -0.303  -0.339  0.180  0.382  0.468  0.458  0.468  0.585*  0.885**  0.836**  -0.243  0.204  0.438  -0.215  -0.010  -0.381  0.047 -0.624 0.808  Lipid P  0.085  0.543 -0.262  0.922**  0.870*  0.768  0.729  0.396  0.943*  0.888*  0.850  0.970**  0.744  0.554  0.176  -0.314  -0.029  0.040  Table 3.9  Correlation coefficients for Lipid S/Total S and Lipid S/Lipid versus some s o i l properties.  Correlation Coefficient; Lipid S as % of Total S Correlation Coefficient; Lipid S as ppm of l i p i d S o i l Group  pH  Organic C  Lipid  Total S  pH  Organic C  Lipid  Total S  -0.129  0.165  0.486**  -0.160  0.013  -0.091  -0.163  0.816**'  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**  -0.085  -0.359  0.862**  A l l Samples Organic (LH, Om and Oh)  Poorly Drained Grassland Om/Oh  0.531  -0.273  0.184  -0.743*  -0.314  Well Drained Forest Ah  0.163  -0.396  0.028  -0.651  0.157  0.253  -0.212  0.616  0.860  -0.524  -0.715  -0.498  0.937*  -0.497  0.262  -0.002  Well Drained Grassland Ah  0.546  0.076  0.045  -0.306  Poorly Drained Grassland Ah/Ap  0.695  -0.686  0.285  -0.309  - 84 -  samples are grouped into f i v e d i f f e r e n t horizon types. If a l l correlations had been s i g n i f i c a n t i t would have been inferred that a l l those factors show a l i n e a r mathematical relationship with the d i s t r i b u t i o n 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 i n s o i l i s 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 s i g n i f i c a n t c o r r e l a t i o n 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 t o t a l 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 i n an examination of such relationships. A stepwise elimination multiple regression analysis was employed to f i n d 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 i s summarized i n 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 t o t a l sulfur values i s shown i n the 2 equation representing the f i n a l stage and the R  value was 0.889.  2 Although the R  value was s l i g h t l y decreased, as much as 89% of  the v a r i a t i o n i n 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  s i g n i f i c a n t l y 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  Table 3.10  2 Regression equations and coefficient of determination (R ) for relationships between l i p i d sulfur as ppm of s o i l , as % of total sulfur, and as ppm of l i p i d and s o i l factors of samples of some B r i t i s h Columbian s o i l s .  100 x R  Regression Equation  Lipid S (ppm of s o i l )  -51.9 + 7.15 pH + 0.224 C + 7.72 L + 0.00753 TS + 0.00280 HI-S + 0.00820 TP - 0.354 LP  Lipid S (ppm of s o i l )  -0.191 + 4.931 L + 0.00890 TS  Lipid S (% of total S)  2.27 - 0.140 pll - 0.0352 C + 0.402 L - 0.0000205 HI-S - 0.000185 TP + 0.0233 LP  Lipid S (ppm of l i p i d )  = -58.5 + 453 pH - 24.9 C + 0.230 TS + 0.334 HI-S - 0.188TP - 9.90 LP  Lipid S-(ppm of l i p i d )  = 2459 - 50.5 C + 0.415 TS  80.7 77.8  F i r s t and second equations for l i p i d S represent the i n i t i a l and f i n a l stages, respectively of the stepwise elimination procedures;  LP = Lipid Phosphorus;  L = Lipid;  TS = Total Sulfur;  LS = Lipid Sulfur.  49.9 41.6  = 1.21 - 0.0203 C + 0.435 L  C = Organic Carbon;  90.4 88.9  Lipid S (% of total S)  HI-S = Hl-reducible Sulfur;  TP - Total Phosphorus;  (%)  oo  - 86 -  s u l f u r along with t o t a l  sulfur.  T a b l e 3.10 a l s o shows t h a t the R  2  of r e g r e s s i o n of the l i p i d  s u l f u r as % o f t o t a l s u l f u r v a l u e s on a l l s o i l  f a c t o r s combined was  2 only 0.49 and the R soil  v a l u e s o f r e g r e s s i o n of the l i p i d  s u l f u r on a l l  f a c t o r s combined and on o r g a n i c carbon and t o t a l s u l f u r were  0.807 and 0.778 r e s p e c t i v e l y , when t h e l i p i d ppm of l i p i d .  s u l f u r was expressed as  These r e s u l t s i n d i c a t e t h a t o r g a n i c carbon c o n t e n t s of  s o i l s c o u l d be one of t h e b e s t p r e d i c t o r s whether l i p i d were expressed as % of t o t a l s u l f u r or as ppm of l i p i d In  sulfur  contents  c o n t e n t s of s o i l s .  view o f t h e number of f a c t o r s i n c l u d e d and the l i m i t e d  number o f samples i n each s o i l group," i t might not be u s e f u l t o 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 t o t h e u n d e r s t a n d i n g o f the c h e m i c a l and b i o l o g i c a l t r a n s f o r m a t i o n s or o v e r a l l turnover of s u l f u r in  soils.  CONCLUSION  L i p i d s u l f u r was found very v a r i a b l e .  The v a l u e s v a r i e d from as low as 1.59 ppm found  Ah h o r i z o n o f a f o r e s t (291 ppm) found to  i n a l l s o i l s examined and the amount was  soil  i n a humisol  i n an  t o n e a r l y two hundred times the amount sample.  the h o r i z o n types, the l i p i d  When s o i l s a r e grouped a c c o r d i n g  s u l f u r c o n t e n t s were h i g h e r i n the  o r g a n i c h o r i z o n s than i n t h e 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  soil  samples had h i g h e r l i p i d  The  s u l f u r than f r e e l y d r a i n e d s o i l  samples.  - 87 -  lipid  s u l f u r was c o n c e n t r a t e d  i n poorly drained grassland  organic  soils. The l i p i d sulfur ranging  s u l f u r accounted f o r small percentage of t o t a l  f r o m 0.56% f o u n d i n a n A h h o r i z o n o f E l u v i a t e d  C h e r n o z e m t o 3.47% f o u n d for  i n an H h o r i z o n o f f o r e s t s o i l .  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 were s i m i l a r  to those  sulfur  expressed  expressed  Black  The  trends  as percentage of t o t a l  a s ppm o f s o i l  except 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  forest  soils.  organic  and  The l i p i d  w e l l drained  s u l f u r contents  f o r e s t o r g a n i c h o r i z o n s were  The l i p i d lipid  content  a forest  sulfur  ranging  f r o m 278 ppm f o u n d i n a F h o r i z o n o f  t o 17,018 ppm f o u n d i n a h u m i s o l .  s l i g h t l y higher grassland  identical.  s u l f u r a l s o accounted f o r a s m a l l part of the t o t a l  of s o i l ,  soil  of poorly drained grassland  lipid  sulfur  than m i n e r a l h o r i z o n s .  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  content  than  Organic  (around  h o r i z o n s had  The p o o r l y  seven times)  drained  lipid  the w e l l drained forest organic horizons.  For  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 had  n e a r l y t w i c e a s much l i p i d  horizons.  The l o w e r  lipid  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  s u l f u r contents  i n w e l l drained  lipid  contents  i n these  lipid  overall lipid  sulfur  examined.  c o n t e n t s were n e a r l y t h r e e times h i g h e r  phosphorus contents  of s o i l s .  lipid  samples.  L i p i d p h o s p h o r u s was a l s o f o u n d i n a l l s o i l s overall  mineral  forest  m i n e r a l h o r i z o n s a s ppm o f t o t a l c o n t e n t s was d u e t o t h e l o w e r sulfur but the higher  horizons  The  than the  I n organic horizons, the  lipid  sulfur  contents were a l s o around t h r e e times h i g h e r  than the  lipid  p h o s p h o r u s c o n t e n t s w h i l e . t h e v a l u e s w e r e a b o u t 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 i n organic horizons than i n mineral horizons.  The l i p i d  sulfur contents were around eight times higher than the l i p i d phosphorus i n poorly drained grassland organic horizons, but s l i g h t l y over twice as high i n 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 i n well drained forest organic and grassland Ah horizons but s l i g h t l y lower i n well drained forest Ah horizons. For o v e r a l l samples, the l i p i d sulfur was highly s i g n i f i c a n t l y correlated  only with t o t a l and Hl-reducible sulfur but also  with organic carbon s i g n i f i c a n t l y only at 5% l e v e l . as % of t o t a l sulfur was correlated  correlated  The l i p i d  sulfur  s i g n i f i c a n t l y only with l i p i d .  However, the l i p i d sulfur as ppm of l i p i d was s i g n i f i c a n t l y correlated only with t o t a l sulfur. 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 can be best explained . (89% of the v a r i a t i o n i n the l i p i d sulfur content) by two s o i l factors, t o t a l l i p i d content and t o t a l 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 t o t a l sulfur, the d i s t r i b u t i o n of the l i p i d  sulfur  can be explained by organic carbon and t o t a l l i p i d contents • but only 42% of the v a r i a t i o n i n the l i p i d sulfur content can be explained. On the other hand, as much as 78% of the v a r i a t i o n i n the l i p i d content can be explained by organic carbon and t o t a l sulfur when the l i p i d sulfur was expressed  as ppm of l i p i d .  I t i s therefore imperative  to use a suitable expression f o r the l i p i d sulfur d i s t r i b u t i o n , so that the s o i l factors can be chosen accordingly.  - 89 -  REFERENCES  A l l i s o n , L.E. 1965. Organic Carbon. In Methods of S o i l Analysis. C A . Black, (Ed.) Amer. Soc. Agron. Monograph No. 9. Madison, Wisconsin. Part I I , pp. 1367-1378. Anderson, G. and R.E. Malcolm. organic phosphates.  1974. The nature of a l k a l i - s o l u b l e s o i l J . S o i l S c i . 25: 282-297.  Bettany, J.R., J.W.B. Stewart, and E.H. Halsted. 1973. Sulfur fractions and carbon, nitrogen, and sulfur relationships i n grassland, forest, and associated t r a n s i t i o n a l s o i l s . S o i l S c i . Soc. Am. Proc. 37: 915-918. Bligh, E.G. and W.J. Dyer. 1959. A rapid method of t o t a l l i p i d extraction and p u r i f i c a t i o n . 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 . 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In Progress i n the Chemistry of Fats and Other L i p i d s . R.T. Holman, (Ed.) Pergamon Press, Oxford. V o l . XI, Part 3, pp. 297-345. Haines, T.H. 1973. 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 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. S o i l S c i . 96: 94-98. Hance, R.J. and G. Anderson. 1963b. I d e n t i f i c a t i o n of hydrolysis products of s o i l phospholipids. S o i l S c i . 96: 157-161. 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.  Jones, L.H.P., D.W. Cowling, and D.R. Lockyer. 1972. Plant available and extractable s u l f u r i n some s o i l s of England and Wales. S o i l S c i . 114: 104-114. Kowalenko, C.G. 1973. Mineralization of s o i l sulfur and i t s r e l a t i o n to s o i l carbon, nitrogen and phosphorus. Ph.D. Thesis, University of B r i t i s h Columbia. Kowalenko, C.G. and L.E. Lowe. 1972. Observations on the bismuth s u l f i d e colorimetric procedure for sulfate analysis i n s o i l . Comm. S o i l S c i . Plant Anal. 3: 79-86. Kowalenko, C.G. and R.B. McKercher. 1970. An examination of methods f o r extraction of s o i l phospholipids. S o i l B i o l . Biochem. 2: 269-273. Kowalenko, C.G. and R.B. McKercher. 1971a. Phospholipid components extracted from Saskatchewan s o i l s . Can. J . S o i l S c i . 51: 19-22. Kowalenko, C.G. and R.B. McKercher. 1971b. Phospholipid P content Saskatchewan s o i l s . S o i l B i o l . Biochem. 3: 243-247.  of  Levesque, M. 1974. Relationship of s u l f u r and selenium i n some Canadian s o i l p r o f i l e s . Can. J . S o i l S c i . 54: 333-335. Lowe, L.E.  1964. An approach to the study of the sulfur status of s o i l s and i t s application to selected Quebec s o i l s . Can. J . S o i l S c i . 44: 176-179.  Lowe, L.E.  1965. Sulphur f r a c t i o n s of selected Alberta s o i l p r o f i l e s of the Chernozemic and Podzolic orders. Can. J . S o i l S c i . 45: 297-303.  Lowe, L.E.  1969. Sulfur fractions of selected Alberta p r o f i l e s of the Gleysolic order. Can. J . S o i l S c i . 49: 375-381.  Morrison, R.I. and W. Bick. and determination 18: 351-355.  1967. The wax f r a c t i o n of s o i l s : Separation of some components. J . S c i . Fd. Agric.  - 91 -.  Neptune, A.M.L., M.A. Tabatabai, and J . J . Hanway. 1975. Sulfur fractions and carbon-nitrogen-phosphorus-sulfur relationships i n some B r a z i l i a n and Xowa s o i l s . S o i l S c i . Soc. Amer. Proc. 39: 51-55. 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. Int. Meet. Humic Substances, Nieuwersluis, Pudoc, Wageningen. pp.233-258. Rehm, G.W. and A.C. Caldwell. 1968. Sulfur supplying capacity of s o i l s and the relationship to s o i l type. S o i l S c i . 105: 355-361. Siem, N.T., D.S. Orlov, and Ya. M. Ammosova. 1975. A study of alcoholbenzene extracts of the p r i n c i p a l s o i l s of North Vietnam. Moscow University S o i l Science B u l l e t i n 30: 9-12. Simoneaux, B.J. and A.G. Caldwell. 1965. Phospholipids i n selected s o i l s . Agron. Abstr., Annual Mtg. Amer. Soc. Agron. p. 77. Stevenson, F.J. 1966. L i p i d s i n s o i l . 203-210.  J . Am. O i l Chemist's Soc. 43:  Tabatabai, M.A. and J.M. Bremner. 1970. An alkaline oxidation method for determination of t o t a l sulfur i n s o i l s . S o i l S c i . Soc. Am. Proc. 34: 62-65. Tabatabai, M.A. and J.M. Bremner. 1972. D i s t r i b u t i o n of t o t a l and available sulfur i n selected s o i l s and s o i l p r o f i l e s . Agron. J. 64: 40-44. Wagner, G.H. and E.I. Muzorewa. 1977. Lipids of microbial o r i g i n i n s o i l organic matter. In S o i l Organic Matter Studies. Proceedings of a Symposium, Braunschweig, 6-10 Sept. 1976. J o i n t l y Organized IAEA and FAO i n 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 t o t a l phosphorus. S o i l S c i . 87: 1-10. Whitehead, D.C. 1964. S o i l and p l a n t - n u t r i t i o n aspects of sulfur cycle. S o i l Fert. 27: 1-8. Williams, C. 1975. The d i s t r i b u t i o n of sulfur i n the s o i l s and herbage of North West Pembrokeshire. J . Agric. S c i . , Camb. 84: 445-452. Williams, C.H., E.G. Williams and N.M. Scott. 1960. Carbon, nitrogen, sulphur, and phosphorus i n some Scottish s o i l . J . S o i l S c i . 11: 334-346.  - 92 -  CHAPTER 4  THE  COLUMN CHROMATOGRAPHIC FRACTIONATION TOTAL L I P I D S AND  L I P I D SULFUR I N  OF  SOME  SELECTED B R I T I S H COLUMBIAN SOILS  INTRODUCTION  A previous  report  ( C h a p t e r 3)  understanding the d i s t r i b u t i o n of l i p i d d i f f e r e n c e i n the dominant v e g e t a t i o n , horizon  types.  I t was  that the total  soil  s u l f u r and  organic  was  s u l f u r i n r e l a t i o n to  s u l f u r was variable.  sulfur, comprised only most c o n c e n t r a t e d  importance  of  the  and  found i n a l l s o i l s I t was  also  a s m a l l p o r t i o n of  i n the p o o r l y  found soil  drained  grassland  soils. No  laboratories lipid  lipid  were very  the  drainage conditions,  shown t h a t l i p i d  examined a l t h o u g h t h e v a l u e s  established  and  reports  have ever been found i n the  that describe  lipid  any  l i t e r a t u r e from  s e r i o u s attempt to s e p a r a t e the  sulfur into individual lipid  classes with  other  soil  quantitative  recovery. The for  the  o b j e c t i v e of  t h i s s t u d y was  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  means o f  silicic  sulfur with  useful information  of  lipid  be  fundamental to understand the soils.  l i p i d s and  a c i d column chromatography.  t h i s approach would p r o v i d e  in  soil  to i n v e s t i g a t e the  each c l a s s of  lipids,  and  I t was  lipid  methods sulfur  a l s o hoped  about the  by  that  associations  t h i s a s s o c i a t i o n would  c h a r a c t e r i s t i c s of the  lipid  sulfur  - 93 -  METHODS AND MATERIALS  The eight s o i l samples used i n this study were selected from thirty-seven s o i l s reported previously (Chapter 3).  The s o i l s  represent eight p r o f i l e types including two forest organic horizons, three organic s o i l s and three surface mineral horizons of forest and grassland s o i l s .  The samples were chosen so as to give a range of  s o i l c h a r a c t e r i s t i c s representing s o i l groups and yet to have high r a t i o s of l i p i d sulfur to t o t a l l i p i d contents to minimize problems i n sulfur analysis. i n Table 4.1.  Some chemical analyses of s o i l samples are given  The sample preparation, extraction of l i p i d and  a n a l y t i c a l methods used i n t h i s 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, C a l i f o r n i a ) 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 f r i t t e d disc was used.  A bed, 8.0 cm high, was prepared  by pouring a s l u r r y of about 15 g of the s i l i c i c acid i n chloroform into the column, and the bed was prewashed with three column volumes of chloroform.  The solvent l e v e l 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 s o l i d was transferred along with the soluble  material; quantitative transfer was ensured by thorough r i n s i n g of a l l glasswares with chloroform.  I f any insoluble f i l m remained on the  Table 4.1  Soil Number  Some chemical analyses of s o i l samples.  Dominant Vegetation (Location/Classification)  Drainage  Horizon  pH in H 0  Organic C %  2  Total S ppm  Lipid S ppm  Lipid %  Western hemlock (Jordan River)  Free  3.9  55.7  1750  37.5  4.01  Western hemlock (Jordan River)  Free  3.0  57.0  2016  37.5  4.24  Subalpine grass (Church Mt.)  Imperfect  Om  4.6  17.8  1122  25.4  1.30  Western red cedar (Victoria)  Free  Ah  6.1  7.74  375  5.33  0.333  Grass (Ft.St. John/Solodic Black)  Free  Ah  6.6  5.70  421  2.85  0.146  Grass (Delta/Saline Humic Gleysol)  Imperfect  Ah  5.8  3.24  581  12.9  Grass-Sedge ' (Whipsaw Cr./Humisol)  Imperfect  Oh  3.7  28.1  30431  291.0  1.71  Grass-Sedge (Lulu Island/Humisol)  Imperfect  Oh  3.3  51.4  23053  150.0  2.01  0.237  - 95 -  sample container after vigorous shaking with chloroform, the f i l m  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 p r i o r 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 f r a c t i o n s ; one  f r a c t i o n of the g l y c o l i p i d s containing monoglycosyl diglycerides (Fraction 2) was mixture;  eluted with 90 ml of chloroform-acetone (1:1,  the other f r a c t i o n of g l y c o l i p i d s containing  diglycosides and others (Fraction 3) was  v/v)  diglyceride  eluted with 700 ml of acetone.  Polar l i p i 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 c o l l e c t e d . solvent was volume.  The  The  evaporated on the f l a s h evaporator to reduce to a small l i p i d was washed out of the f l a s k with the desired pure  solvent or solvent mixture and diluted to a known volume i n a .volumetric f l a s k with a glass-stopper. f r a c t i o n was  determined by weighing a small aliquot that was  afterwards, as described  RESULTS AND  The weight of the l i p i d i n each discarded  by Bligh and Dyer (1959).  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 e s s e n t i a l l y quantitative separation  into three groups; less polar  (so c a l l e d neutral) l i p i d s , g l y c o l i p i d s , and phosphatides (Rouser  - 96 -  et  a l , 1967a).  Vorbeck  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 e a c h o t h e r and o t h e r lipid  c l a s s e s by e l u t i o n w i t h chloroform-acetone (1:1, v/v) m i x t u r e  f o l l o w e d by acetone.  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 of cerebroside s u l f a t e , plant polyhexoses w i t h acetone.  sulfolipid,  and c e r a m i d 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 separation procedure employing  silicic  acid that i s  e s s e n t i a l l y t h e o r i g i n a l procedure o f Borgstrom  (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 ( 1 9 5 9 ) .  I t was shown b y R o u s e r  et  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  spinach leaf l i p i d  e x t r a c t s , i n which g l y c o l i p i d  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 minor  i s h i g h and  component.  I t was f o u n d 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 a c t i o n study that a f a i r l y  l a r g e amount o f l i p i d s  f r a c t i o n eluted w i t h acetone. t h i s study f o rf r a c t i o n a t i o n by Rouser  fraction-  appeared  i n the  T h e r e f o r e t h e p r o c e d u r e was a d a p t e d i n of s o i l  e t a l . (1967a), the l i p i d  lipids.  U s i n g t h e scheme d e v e l o p e d  composition of the four  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 a s f o l l o w s : F r a c t i o n 1. lipids  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  including sterols,  s t e r o l e s t e r s , mono-, d i - , a n d 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 F r a c t i o n 2.  acids.  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  mixture separates monoglycosyl d i g l y c e r i d e from d i g l y c o s y l which w i l l  polar  be e l u t e d w i t h a c e t o n e .  Cerebrosides are also  completely separated from s u l f a t i d e s w i t h t h i s somewhat l e s s p o l a r l i p i d s  diglyceride almost  solvent mixture.  are eluted along with  monoglycosyl  Other  - 98 Table 4.2  Lipid sulfur and l i p i d distributions i n fractions of S i l i c i c acid column chromatography. Content (% of total applied)  Soil Number  Fraction  Lipid S  1  1 2 3 4 Whole Soil  .  2  • 1 .' 2 3 4 Whole Soil  3  1 2 3  4  Whole Soil  Lipid  L i p i d S/Lipld (ppm)  24.2 37.3 5.47 18.3  33.4 57.6 8.13 3.52  677 606 628 4875 935  12.9 36,8 4.08 16.6  22.5 64.6 12.1 5.28  506 503 297 2768 884  17.1 30.0 4.80 36.90  34.8. 54.6 9.15 4.59  960 1074 1025 15709 1954  4  1 2 3 4 Whole Soil  21.6 35.1 7.30 26.6  42.3 48.9 7.63 4.08  815 1149 1529 10471 1601  5  1 2 3 4 Whole Soil  24.9 30.6 6.18 36.1  37.3 53.7 7.60 5.00  1457 1249 1777 15715 805  6  1 2 3 4 Whole Soil  36.2 45.2 5.54 7.31  44.3 47.3 6.33 4.14  4506 5291 4789 9692 5443  7  1 2 3 4 Whole Soil  52.9 32.4 5.26 3.61  38.8 53.6 8.13 3.98  23202 10287 11072 15393 17018  8  1 2 3 4 Whole Soil  47.2 14.1 20.4 10.0  36.3 51.2 10.5 3.24  9683 2047 14500 23034 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. are almost completely separated  Cerebrosides  from s u l f a t i d e s by e l u t i o n with t h i s  solvent mixture. Fraction 3.  Acetone elutes d i g l y c o s y l diglycerides and  s u l f o l i p i d with plant l i p i d extracts. With b a c t e r i a l l i p i d s both neutral and acidic glycosyl glycerides are eluted, although these are not present i n extracts from a l l microorganisms.  With l i p i d  extracts  of animal organs ( p a r t i c u l a r l y brain) s u l f a t i d e s and ceramide polyhexosides are eluted with acetone. number of uncharacterized with acetone.  With f e c a l l i p i d extracts a large  substances devoid of phosphorus are eluted  No more than traces of phosphorus are found i n acetone  eluates except with samples that contain a large amount of diphosphatidyl glycerol that i s eluted i n 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 g l y c o l i p i d s . Fecal l i p i d extracts contain a v a r i e t y of substances devoid of phosphorus that are eluted with methanol along with phosphatides. The d i s t r i b u t i o n of l i p i d sulfur and l i p i d i n fractions eluted from s i l i c i c acid column are shown i n Table 4.2. studied gave similar e l u t i o n patterns f o r the l i p i d s . amount of l i p i d was recovered 64.6%  44.3%  The highest  i n the Fraction 2, varying from 47.3 to  of the t o t a l l i p i d ; Fraction 4 contained  varying from 3.24 to 5.28%.  A l l eight s o i l s  the least l i p i d ,  The Fraction 1 contained  from 22.5 to  and the Fraction 3 from 6.33 to 12.1% of the t o t a l l i p i d s .  For  - 99 -  the l i p i d sulfur elution, however, no consistent  s i m i l a r i t y i n elution  pattern was observed between corresponding fractions of the eight  soils.  Thus the amount of l i p i d sulfur eluted i n the Fraction 2 was the highest f o r the s o i l s 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  i n the Fraction 4 f o r s o i l 3 and 5, and i n the Fraction 1 (52.9% and 47.2%, respectively)  f o r s o i l s 7 and 8.  In contrast  to the d i s t r i b u t i o n  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 f o r s o i l s 7 and 8, of which l i p i d sulfur were the least i n the Fraction 4. When the d i s t r i b u t i o n s of three general classes of l i p i d s (Rouser et a l . , 1967a) are considered, the Glycolipids  (Fractions 2 and  3 together) comprise more than half of the t o t a l 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 l i p i d s (Fraction 1) were around one-third from 22.5 to 44.3% with a mean of 36.2%. lipid  of the t o t a l ranging  The mean value f o r the polar  (Fraction 4) was only 4.23% with a range of 3.24 to 5.28% (see  Table 4.3). Although these d i s t r i b u t i o n s of l i p i d i n the fractions are inconsistent with the d i s t r i b u t i o n pattern of a b a c t e r i a l l i p i d f o r a l l fractions reported by Langworthy et a l . (1974), the highest percentage (53.6%) of the t o t a l l i p i d was i n the g l y c o l i p i d f r a c t i o n (acetone fraction).  Unfortunately i t i s not possible to compare t h i s result  with others so as to understand the s o i l l i p i d component i n 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 i n t h i s study.  - 100 -  Table A.3 also shows that r e l a t i v e l y higher proportions of s o i l l i p i d sulfur (from 34.5 to 51.0% of t o t a l l i p i d sulfur) were associated with g l y c o l i p i d component of the s o i l l i p i d s .  However, f o r  s o i l 3, as much as 36.9% of the t o t a l l i p i d sulfur was associated with polar l i p i d s (mostly phosphatides), while 52.9% and 47.2% of the t o t a l l i p i d sulfur was associated with neutral or less polar l i p i d s f o r s o i l s 7 and.8 respectively.  V i r t u a l l y 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 d i f f e r 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 f o r 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  i n s o i l s remains  unclear, u n t i l the compositions of l i p i d sulfur i n vegetation and microorganisms  on and i n s o i l s are characterized.  The l i p i d sulfur expressed as ppm of l i p i d i n each f r a c t i o n and i n whole s o i l s are also shown i n Table 4.2.  The value f o r  Fraction 4 of s o i l 7 was s l i g h t l y lower but very similar to that for the whole s o i l .  In contrast, the values f o r Fraction 4 of other  seven s o i l s were always higher than those f o r whole s o i l s , and remaining fractions had lower values than the values f o r whole s o i l s except f o r 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 t o t a l amounts applied were shown i n Table 4.3.  Although, from a l l s o i l s , the recoveries of l i p i d were  over 100%, the recoveries of the l i p i d sulfur were, i n a l l cases, below 100%.  It was noticed that i n most cases every eluate of  - 101 -  T a b l e 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 of s o i l l i p i d t o t a l r e c o v e r y of l i p i d and l i p i d s u l f u r .  % of t o t a l Soil Number  Lipid Class '  Lipid  LS G P TR  sulfur  and  applied  S  Lipid  24.2 42.8 18.3 85.3  33.4 65.7 3.52 102.7  12.9 40.9 16.6 70.4  22.5 76.7 5.28 104.5  LS G P TP  17.1 34.8 36.9 88.8  34.8 63.8 4.59 103.1  LS G P TR  21.6 42.4 26.6 90.6  42.3 56.5 4.08 102.9  LS G P TR  24.9 36.8 36.1 97.8  37.3 61.3 5.00 103.6  LS G P TR  36.2 51.0 7.31 94.6  44.3 53.6 4.14 102.1  LS G P TR  52.9 37.7 3.61 94.2  LS G P TR  '  •-  LS G P TR  LS = Less P o l a r ; TR = T o t a l Recovery.  47.2 . 34.5 10.0 91.7  G =  Glycolipid;  •;"<''*  !  38.8 61.7 3.98 104.5 36.3 61.7 3.24 101.2  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 significant of  silicic  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 a c i d come a l o n g  glass disk.  with  the e l u t i o n solvent  I t was a l s o n o t i c e d  through  fritted  t h a t , i n a l l c a s e s , t h e r e were  c o l o r remained a f t e r e l u t i o n of a l l four  f r a c t i o n s w h i c h was  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.  Therefore the higher  o f l i p i d w e r e p r o b a b l y due t o t h e s i l i c i c the  lower recoveries  of l i p i d  l i p i d m a t e r i a l by b e i n g along  presumably recoveries  f r o m c o l u m n and  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  a d s o r b e d on column and t h e s i l i c i c  with e l u t i n g solvent  concentration  acid eluted  visible  d i d not influence the l i p i d  acid  eluted  sulfur  on measurement.  CONCLUSION  The f r a c t i o n a t i o n o f s o i l c l a s s e s on t h e s i l i c i c patterns soils  of the three  studied  class of s o i l  soil the  lipids.  4%  general  of s o i l  types.  G l y c o l i p i d s were t h e dominant  and t h e y w e r e more t h a n h a l f o f t h e t o t a l .  of the t o t a l  The u n i f o r m i t y  column chromatography  lipids  into three  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  n e u t r a l l i p i d s were around o n e - t h i r d l i p i d s were o n l y  lipids  a c i d c o l u m n h a s shown t h a t t h e d i s t r i b u t i o n  regardless lipids  total  of the t o t a l  being  lipids  the smallest  and  The  polar  component o f t h e  of the d i s t r i b u t i o n i n each f r a c t i o n of  ( i . e . , each l i p i d  c l a s s ) suggests that the  i n t h e s e s o i l s w e r e 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  their  d i s t r i b u t i o n s w e r e a f f e c t e d b y a n d made m o r e 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  other  soil  environmental factors.  - 103 -  The s o i l l i p i d sulfur had no 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 between corresponding classes f o r eight studies,  d i f f e r i n g from s o i l to s o i l .  soils  Perhaps the most s i g n i f i c a n t  finding of the study was that s i g n i f i c a n t amounts of sulfur were, i n a l l cases, recovered i n the less polar (neutral), g l y c o l i p i d and polar l i p i d fractions, with these three fractions accounting on average f o r 29%, 42% and 19% of the l i p i d s u l f u r , respectively.  This finding  c l e a r l y suggests that s o i l l i p i d sulfur i s present i n a variety of forms. The r a t i o s of the l i p i d sulfur to l i p i d as ppm of the l i p i d i n each corresponding f r a c t i o n and the r a t i o s f o r whole s o i l s have shown that the values f o r Fraction 4 (polar l i p i d class) of the seven s o i l s except f o r s o i l 7 were always higher than those f o r whole s o i l s . The values f o r the remaining fractions  (1, 2 and 3) had lower values  than the values for whole s o i l s 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 f r a c t i o n f o r further investigation of the c h a r a c t e r i s t i c s of the s o i l l i p i d sulfur since t h i s f r a c t i o n p a r t i c u l a r l y f o r 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  i n t h i s f r a c t i o n as ppm of l i p i d are higher than those i n whole s o i l s .  - 104 -  REFERENCES  Borgstrom, B. 1952. Investigation on l i p i d separation methods. Separation'of phospholipids from neutral fat and f a t t y acids. Acta Physiol. Scand. 25: 101-110. Langworthy, T.A. 1974. Long-chain g l y c e r o l diether and polyol d i a l k y l glycerol t r i e t h e r l i p i d s of sulfolobus acidocaldarius. J . B a c t e r i o l . 119: 106-116. Rouser, G.,  G. Kritchevsky, and A. Yamamoto. 1967a. Column chromatographic and associated procedure for separation and determination of phosphatides and g l y c o l i p i d s . In L i p i d 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 g l y c o l i p i d s . L i p i d s 2: 37-40.  Smith, L.M.  and N.K. Freeman. 1959. Analysis of milk phospholipids by chromatography and infrared spectrophotometry. J . Dairy S c i . 42: 1450-1462.  Vorbeck, M.L. and G.V. M a r i n e t t i . 1965. Separation of glycosyl diglycerides from phosphatides using S i l i c i c acid column chromatography. J . L i p i d Res. 6: 3-6.  - 105 -  CHAPTER 5  OBSERVATIONS ON THE GAS L I Q U I D AND  THIN  CHROMATOGRAPHIC BEHAVIOR OF L I P I D AND SULFUR FRACTIONS I N TWO  SELECTED  LAYER LIPID  SOILS  INTRODUCTION  In a previous distribution patterns lipids,  no c o n s i s t e n t  lipid  ( C h a p t e r 4) i t h a s b e e n shown t h a t t h e  of the three  classes of l i p i d s  of s o i l  similarity  classes, differing  Fraction 1 (less polar  type.  The s o i l  from s o i l  lipid  to s o i l .  lipid  fractions.  In a d d i t i o n the r a t i o s of l i p i d  s u l f u r among e i g h t  s o i l s and  four i n this  than those i n whole  These r e s u l t s suggested t h a t F r a c t i o n 1 could  be a u s e f u l  s u l f u r i n these  high  lipid  Fraction 1 represents  amounts o f l i p i d  sulfur associated  the less polar  lipid  s u l f u r were p r e s e n t i n t h i s with  the less polar  lipid  c l a s s , and fraction,  c l a s s may  s u c h s u l f u r f o r m s 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 are  one  soils. Since  the  that  contained  sulfur to l i p i d  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  fairly  soils  I t was a l s o shown  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  organic  polar  s u l f u r , however,  c l a s s ) o f two H u m i s o l s a m p l e s  amounts o f l i p i d  for  less  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  the highest  soils.  (i.e.,  g l y c o l i p i d s and p o l a r l i p i d s ) w e r e s i m i l a r f o r e i g h t  studied, regardless had  report  g e n e r a l l y l o w enough t o work w i t h  include  points  g a s - l i q u i d chromatography  - 106  ( R y l a n d and  Tamele, 1970).  This chapter o f i s o l a t i n g and  describes a study  t h a t examines the  c h a r a c t e r i z i n g the l i p i d  the l e s s p o l a r l i p i d  gas-liquid  possibility  s u l f u r i n the f r a c t i o n s  c l a s s f r a c t i o n a t e d f r o m two  a p p l y i n g t h i n - l a y e r and  METHODS AND  -  of  s e l e c t e d Humisols  by  chromatography.  MATERIALS  Soils  The  two  s o i l s used i n t h i s  s o i l s r e p o r t e d i n C h a p t e r 4.  study were s e l e c t e d from the  T h e s e two  s o i l s are Humisols sampled  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  The  samples were chosen because of t h e i r h i g h c o n t e n t s  s u l f u r and  a l s o the widest  a s shown i n C h a p t e r 4, attempts  t o i s o l a t e and  W h i p s a w c r e e k was slightly different The  lipid  Analytical  horizons of organic  s u l f u r to l i p i d  lipid  in Fraction 1  c h a r a c t e r i z e i t s components.  The  Humisol  resampled f o r the bulk e x t r a c t i o n study, chemical  analyses  soils.  of t o t a l  ratios  from  so t h a t F r a c t i o n 1 c o u l d b e u s e d f o r f u r t h e r  and  reported previously.  are given i n Table  from  has  p r o p e r t i e s from p r e v i o u s l y reported  sample p r e p a r a t i o n s were those  some c h e m i c a l  eight  Origin  data. and  5.1.  Methods  The  methods used f o r t h e e x t r a c t i o n , measurement  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 organic carbon,  total  and  sulfur,  shown  total  Table  5.1  Son.e c h e m i c a l  analyses  %. o f dried Soil Number  Dominant  Vegetation  (Location/Classification)  1  Grass (Lulu  pH in  of  soil  samples.  oven  ppm o f  soil  2  C  dried  ;  Organic  H 0  over  Lipid  soil —  Total  Total  Lipid•  S  HI-S  S  3.3  51.4  2.01  23,100  7,710  150  4.0  30.6  0.836  29,000  17,400  156  Island/Humisol)  2  Grass (Whipsaw  Cr./Humisol)  - 108  Hl-reducible reported  sulfur, total  -  lipid  and  lipid  s u l f u r i n s o i l s were  those  previously.  Column Chromatography  Total (Fractions procedure  1,  lipid  extracts  2, 3 and  were f r a c t i o n a t e d  4) on a s i l i c i c  1 was  into six fractions  further  (Fractions  e t h e r , m e t h a n o l , and  ( 1 0 0 - 2 0 0 m e s h ) , was  A,  (75 m l )  petroleum  Fraction  petroleum v o l u m e was  I t was  The 1) was  was  (1962).  4.  acid  column  F) u s i n g p e t r o l e u m  t h e i r mixtures  according to  B i o - S i l A,  clear.  The  washed w i t h 2 volumes  silicic  acid  lipid  cm  eluted  concentrated  nearly  i n diameter  (50 m l )  from the f i r s t  was to  a  of methanol,  3  3 volumes silicic  to dryness  and  of  acid  column  t a k e n up  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 n o t more t h a n  (v/v) e t h e r i n petroleum methanol i n ether F).  ether, the  graded m a t e r i a l  3 v o l u m e s o f e t h e r and  750 pi).  of each eluant were taken:  (Fraction  E and  p a c k e d i n a c o l u m n 2.0  of acetone,  ether.  C, D,  the  g r a d e d i n m e t h a n o l a t 5 m i n u t e s i n t e r v a l s t o remove  s u s p e n d e d i n m e t h a n o l and h e i g h t o f 7 cm.  B,  Block  the f i n e s u n t i l the supernatant  50 m l  a c i d column a c c o r d i n g t o  p u r i f i e d on a s i l i c i c  c h l o r o f o r m , and  m o d i f i e d m e t h o d o f H a i n e s and  (i.e.,  fractions  o f R o u s e r e t a l . (1967a) as d e s c r i b e d i n C h a p t e r Fraction  volumes  into four  ether  The  following  petroleum  fractions eluted  ether  (Fraction B), ether  (Fraction D), methanol  (Fraction A), (Fraction  ( F r a c t i o n E ) , and  C),  in  (total with 50% 50%  chloroform  - 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 f l e x i b l e support of solvent-resistant polyethylene terephthalate with fluorescent indicator incorporated i n 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).  V i s u a l i z a t i o n of separated  components was carried out by exposing the developed sheets to iodine vapor i n a closed chamber.  Attempts at i d e n t i f i c a t i o n of i n d i v i d u a l  spots by v i s u a l i z a t i o n with various spray reagents f o r s p e c i f i c functional groups were unsuccessful. Two-dimensional mapping TLC of four fractions (Fractions 1 through 4) were performed by using the developing solvents; chloroformmethanol-water  (65:25:4, v/v/v) f o r the f i r s t solvent ( v e r t i c a l ) and  chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1) f o r the second solvent (horizontal) (see Figures 5.1 and 5.2).  For the  multiple development TLC of the four fractions and the s i x fractions on second column chromatography chloroform-methanol-water  ( i . e . , Fraction A through F);  (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 s i x f r a c t i o n s , the chromato-  graphic sheet was cut out into several 2x6 cm mini sheets and developed i n the mini developing tank.  Various combinations of petroleum ether,  - no  -  ether, methanol, chloroform, acetic acid and water were used as developing solvent systems, i n the hope that the best resolution of components could be achieved. six fractions were as follows:  The best solvent systems for the 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 s o l i d state electrometer, a s o l i d 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 f o r sulfur analysis. Two  gas l i q u i d 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 s t e e l column packed with 5% OV-1  i . d . Stainless  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 before use.  was conditioned overnight at 350°C  - Ill -  For  these studies, the c a r r i e r gas was nitrogen.  oxygen and a i r were used as the burner gas mixture.  Hydrogen,  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 i n l e t temperature were 195 C and 125 C, respectively. Nitrogen c a r r i e r gas, hydrogen, oxygen and a i r flow rates were held at 80, 120, 18 and 38 ml per minute, respectively. other hand, f o r the stainless s t e e l column with 5% OV-1,  On the  column  temperature was programmed from 100 to 200°C at the same rate as f o r the glass column.  Detector base temperature and i n l e t 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 c h a r a c t e r i s t i c s of the two s o i l samples used i n t h i s study are shown i n Table 5.1.  The samples were chosen to  examine further i f these s o i l s could give a d i f f e r e n t response or r e s u l t s on the TLC and GLC although they have similar l i p i d and l i p i d sulfur d i s t r i b u t i o n on s i l i c i c acid column chromatography.  Also as  described i n a previous report (Chapter 3), s o i l 1 (Lulu muck) was the i d e n t i c a l s o i l sample reported i n 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 d i s t r i b u t i o n 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 i n Table 5.2.  Thin-Layer Chromatography  For monitoring column chromatography  and to compare the t h i n -  layer chromatographic behavior of each column chromatographic f r a c t i o n of two s o i l s , two-dimensional mapping and one-dimensional multiple developing TLC were carried out. Several attempts to v i s u a l i z e spots on the basis of s p e c i f i c reactive groups f a i l e d due to i n s i g n i f i c a n t response of spray reagents on the s p e c i f i c spots.  Reagents tested included:  copper acetate-  Rhodamine B f o r l i p o p h i l i c a l k y l sulfonates, copper sulfate for s u l f u r containing glycosides, and resorcinol-ammonia f o r sulfonic acids (Kates, 1972).  Therefore, only TLC v i s u a l i z e d by exposing to iodine  vapor are shown i n Figures 5.1 through 5.6. To obtain o v e r a l l developing patterns two dimensional mapping TLC of the four fractions was carried out and the r e s u l t s are shown i n Figures 5.1 and 5.2 for s o i l 1 and 2, respectively.  As shown i n 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 d i s s i m i l a r i t i e s  indicate that the i n d i v i d u a l components of the one general l i p i d fractionated  class  from one s o i l are d i f f e r e n t from the same l i p i d class from  the other s o i l .  In other words, although the column chromatographic  d i s t r i b u t i o n of t o t a l l i p i d s i s similar for the two s o i l s , the nature of i n d i v i d u a l l i p i d components d i f f e r from one another.  - 113 -  Table 5.2  Lipid and l i p i d sulfur distributions in fractions from • S i l i c i c acid column.  Content (% of total applied)  Soil Number  Fraction  1  Lipid  Lipid S  36.3  47.2  2  51.4  14.1  3  10.5  20.4  A Recovery  3.24  10.0  101.2  91.7  1  40.6  55.8  2  53.9  27.8  3  9.68  4.70  4  3.42  2.51  Recovery  107.6  90.8  - 114 -  4  3  'i * / yj, 1  * - -*. *  D\ i;  '.' \* >' II  /  \ J  ^  ; r J > ( ( v/  '  -  i  ) '  •1  'i  i'  N  d  i. \  i  \ \ \  1  <•  '.1  >  ll  Vi *x *  ' ' 1  t  1  1 — %  Qy-'"""'"  -  1  -  _ l  ,  1 1 1 , I  - ' " " " " I * *  *.  <  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 t o t a l l i p i d extract of s o i l 1. F i r s t solvent (I, v e r t i c a l ) , chloroformmethanol-water (65:25:4); second solvent (II, horizontal), chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1). Detection by exposing i n the iodine vapor.  - 115 -  V  3  4  A  i w  D ^  i  O  2  1 •—•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 t o t a l l i p i d extract of s o i l 2. F i r s t solvent (I, v e r t i c a l ) , chloroformmethanol-water (65:25:4); second solvent (II, horizontal), chloroform-acetone-methanol-acetic acid-water (15:6:3:1:1). Detection by exposing i n the iodine vapor.  - 116 -  The differences i n the composition of the corresponding l i p i d class of two s o i l s were also shown i n the one-dimensional multiple development thin-layer chromatograms i n 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 i n the chromatograms suggest that the multiple developing It may  technique may  only be used for preparative  TLC.  also suggest that a second column chromatography must be applied  prior to the current  TLC.  Multiple development chromatograms of the f r a c t i o n s , from A to F, eluted from a second s i l i c i c acid column of Fraction 1 of 1 are shown i n Figure 5.5.  The chromatograms indicate that the  soil  lipid  components i n Fraction A are d i f f e r e n t from those i n 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 d i f f e r e n t components of l i p i d s  which were not shown i n 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, carried out i n the hope that the best developing obtained.  was  solvents could be  Various d i f f e r e n t solvent r a t i o s of d i f f e r e n t solvent  mixtures had been applied to each f r a c t i o n and the TLC developed with the best resolving eluents for each f r a c t i o n were shown i n Figure  5.6.  Except for Fraction B, a l l other fractions f a i l e d 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 t o t a l l i p i d extract of s o i l 1. F i r s t solvent, chloroform-methanol-water (65:25:4); second solvent, hexane-ether (4:1). Detection by exposing i n the iodine vapor.  - 118 -  Figure 5.4  •  i  «  .  1  2  3  4  Multiple development TLC of Fractions 1, 2, 3 and 4 eluted from S i l i c i c acid column, of t o t a l l i p i d extract of s o i l 2. F i r s t solvent, chloroformmethanol-water (65:25:4); second solvent, Hexaneether (4:1). Detection by exposing i n 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. F i r s t solvent, chloroform-methanol-water (65:25:4); second solvent, hexane-ether (4:1). Detection by exposing i n 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-etheracetic acid (40:10:1); B, pet. ether-ethermethanol-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-etheracetic acid (20:10:40:1).  - 121 -  Fraction B, C and F giving a similar pattern on the multiple development TLC, gave d i f f e r e n t behavior on one-dimensional TLC.  Gas-Liquid Chromatography  The major v i r t u e of the gas-liquid chromatographic analysis of sulfur using the Melpar flame photometric response s p e c i f i c i t y of the detector.  detector i s the high  It responds to sulfur-containing  compounds with great s e n s i t i v i t y , e.g., s e n s i t i v e 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 i n the sulfur analysis i s not proportional to concentration (Brody and Chaney, 1966).  The detector also i s so  i n s e n s i t i v e to extraneous material i n a raw extract of b i o l o g i c a l tissue that compounds other than sulfur have v i r t u a l l y no  photometric  response as long as the concentration of the non-sulfur contaminant i n the sample injected does not exceed 20 jig (Stevens, 1967).  As a  r e s u l t , the high s p e c i f i c i t y of the detector should require fewer sample preparation steps to determine the organosulfur content i n extracts of natural product  (Bowman and Beroza, 1966b).  The r e s u l t s of gas-liquid chromatography (GLC) using the Melpar flame photometric  detector are shown i n Figures 5.7  The resolution of GLC was poor when 3% OV-1  through  5.19.  and 10% SE-30 were used as  column packing materials for the t o t a l l i p i d s and f r a c t i o n s 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  TEMPERATURE Figure 5.7  180  200  (°C)  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 a i r flow rates held at 80, 120, 18 and 38 ml/min, respectively. Detector base and i n l e t temperatures held at 210 and 125°C, respectively. 5 p i of sample (ca. 95.2 ppm S) i n chloroform.  RESPONSE  Figure 5.8  GLC of t o t a l 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 i n Figure 5.7. 5 JLII of sample (ca. 37.3 ppm S) i n 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 i n Figure 5.7. 5 yl of sample (ca. 43.2 ppm S) i n chloroform.  - 125 -  5% OV-1 was used f o r s o i l 1 and 10% EGSS-X for s o i l 2.  Therefore two  d i f f e r e n t column packing materials were used for the l i p i d s from two different s o i l s .  Although GLC of t o t a l l i p i d s and a l l f r a c t i o n s from  the f i r s t s i l i c i c acid column of s o i l s 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 t o t a l l i p i d s and Fraction 1 of s o i l 1 and 2 and s i x fractions ( i . e . , Fractions A through F) are presented because the results of GLC were not s p e c i f i c f o r other fractions and were quite s i m i l a r to each other. GLC of t o t a l l i p i d and Fraction 1 of s o i l 1 on 5% OV-1 are shown i n Figures 5.8 and 5.9, respectively. EGSS-X are shown i n Figures 5.11 and 5.12. extracted  Those of s o i l 2 on 10% GLC of the t o t a l l i p i d  from s o i l 1 showed at least 5 d i s t i n c t i v e 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) i n Figure 5.9.  GLC of t o t a l 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 i n Figure 5.11) and 6 peaks (peak numbers: 1, 2, 3, 4, 8 and 9 i n 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 i n the l i p i d extracts and fractions of column chromatography, since there were several d i s t i n c t i v e peaks i n the chromatogram and certain peaks i n the GLC of t o t a l l i p i d s did show i n the GLC of Fraction 1.  However, t h i s 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 i n the chromatogram of 5% 0V-1 of the extraction-solvent  separation  blank (Figure 5.7) and 17 peaks i n the GLC  11  67  RESPONSE  f—1 • <y> •  _L 100  120  140  160  TEMPERATURE  Figure 5.10  180  (  •C)  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 a i r flow rates held at 80, 120, 18 and 38 ml/min, respectively. Detector base, i n l e t temperatures held at 195 and 125°C, respectively. 5 }il of sample (ca. 92.5 ppm S) i n chloroform.  RESPONSE  to  _L_  100  I 120  ;  I  I  140  160  TEMPERATURE Figure 5.11  I 180  (°C)  GLC of t o t a l 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 i n Figure 5.10. 5 p.1 of sample (ca. 18.7 ppm S) i n chloroform.  RESPONSE  TEMPERATURE  ure 5.12  (°C)  GLC of Fraction 1 of t o t a l 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 i n Figure 5.10. ^.jil of sample (ca. 12.8 ppm S) i n chloroform.  - 129 -  on the 10% EGSS-X of the same extraction-solvent blank (Figure 5.10). For s o i l 1, the peaks i n GLC of t o t a l l i p i d s (Figure 5.8) and i n that of Fraction 1 (Figure 5.9) were a l l i d e n t i c a l to the peaks of the corresponding numbers i n the GLC of the concentrated  extraction-  solvent blank (Figure 5.7). For s o i l 2, seven out of 10 peaks i n GLC of t o t a l l i p i d extract (Figure 5.11) and a l l 6 peaks i n GLC of Fraction 1 (Figure 5.12) were i d e n t i c a l to the peaks of the corresponding numbers i n the GLC of the concentrated  extraction-solvent blank (Figure 5.10).  The three d i s t i n c t i v e peaks (peaks d, e and f ) i n the GLC of t o t a l l i p i d extract (Figure 5.11) were not shown i n the GLC of the concentrated extraction-solvent blank i n Figure 5.10. Therefore those three peaks i n the GLC of t o t a l l i p i d extract of s o i l 2 could represent soil.  sulfur-containing compounds extracted from the  However i t remains uncertain whether these peaks represent a l l the  sulfur-containing l i p i d s i n the s o i l or simply represent s u l f u r containing organic compounds which could be extracted i n 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 i n 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 i n each f r a c t i o n requires large volumes of the fractions from which sulfur determination  can be made.  small portions of the f r a c t i o n s were concentrated  Therefore  under a stream of  nitrogen gas, at which concentration the best resolution of peaks could be obtained.  1  RESPONSE  io  1  IOO  Figure 5.13  120  140 160 TEMPERATURE  180  200  (  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 a i r flow rates held at 80, 120, 20 and 38 ml/min, respectively. Detector base and i n l e t 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 i n 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 i n 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 i n Figure 5.13.  RESPONSE  CO  1  i  IOO  Figure 5.17  I20  I40 I60 TEMPERATURE  I80 (°C)  200  GLC of Fraction D from s o i l 1. On 5% OV-1. Temperature program as shown. Instrument and other conditions as i n 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 i n Figure 5.13.  RESPONSE  lOO  Figure 5.19  120  140 160 TEMPERATURE  180 (°C)  200  GLC of Fraction F from s o i l 1. On 5% OV-l. Temperature program as shown. Instrument and other conditions as i n 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 s i x f r a c t i o n s , Fractions B (peaks a and b i n  Figure 5.15), C (peaks a and b i n Figure 5.16), E (peak a i n Figure 5.18), and F (peak a i n Figure 5.19) showed additional peaks which were not shown i n the GLC of Fraction 1.  I t i s also uncertain whether these  peaks represent sulfur-containing l i p i 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 e l u t i o n solvents employed f o r 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 s o i l s were not similar to each other, although column chromatographic behavior of t o t a l l i p i d s and l i p i d sulfur of two s o i l s were similar to each other.  These d i s s i m i l a r i t i e s may indicate that  the i n d i v i d u a l components of one general l i p i d class fractionated from one s o i l d i f f e r from the same l i p i d class of the other s o i l even though the column chromatographic d i s t r i b u t i o n of t o t a l l i p i d s was similar between two s o i l s .  - 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 i n each f r a c t i o n , i t indicates that the second s i l i c i c acid column chromatography d i d not help very much i n further f r a c t i o n a t i o n 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 i n the solvents used for l i p i d extraction and f o r e l u t i o n from column chromatography masked gas-liquid chromatographic monitoring  of sulfur-containing  compounds i n l i p i d extracts and f r a c t i o n s , i t was suggested that the GLC could be used i n monitoring  the sulfur-containing compounds i n l i p i d  extracts and fractions of s o i l , e s p e c i a l l y f o r s o i l 2 with 10% EGSS-X column f o r example.  However i n the absence of complete characterization  i t i s uncertain whether the monitored sulfur-containing compounds represent the sulfur-containing l i p i d s extracted from s o i l or simply represent sulfur-containing organic compounds extracted i n the current extraction-solvent system. It i s now apparent that future experiments should be c a r e f u l l y 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 s u l f u r containing l i p i d s i n s o i l , future experiments should be planned i n the l i g h t of the r e s u l t s observed i n t h i s study.  C l e a r l y solvents of higher  purity w i l l be needed to take advantage of GLC techniques  i n such studies.  - 139 -  In conclusion, t h i s l a s t phase of the investigation, involving attempts to separate and characterize  individual sulfur-  containing l i p i d components by chromatographic methods, f a i l e d to s i g n i f i c a n t l y 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 i n general, and of their sulfur-containing in p a r t i c u l a r .  I t also indicated  investigation for future  studies.  constituents  some of the more promising l i n e s of  - 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 i n insect tissue. J . Ass. Off. Anal. Chem. 49: 1046-1052. Bowman, M.C. and M. Beroza. 1966b. Pesticide Residue. Determination of Imidan and Imidoxon i n sweet corn by gas chromatographic detection. J . Ass. Off. Anal. Chem. 49: 1154-1157. Bremner, J.M. and W'.L. Banwart. 1974. Identifying v o l a t i l e S . compounds. Sulfur I n s t i t . J . Spring 1974, pp. 6-9. Brody, S.S. and J.E. Chaney. 1966. The application of a s p e c i f i c detector for phosphorus and f o r sulphur compounds-sensitive to subnanogram quantities. J . Gas Chromat. 4: 42-46. Haines, T.H. and R.J. Block. 1962. Sulfur metabolism i n algae. I. Synthesis of metabolically inert chloroform-soluble sulfate esters by two Chrysomonads and C h l o r e l l a pyrenodosa. J. Protozool. 9: 33-38. Kates, M.  1972. Techniques of lipidology. Isolation, analysis and i d e n t i f i c a t i o n 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 g l y c o l i p i d s . In L i p i d 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 g l y c o l i p i d s . Lipids 2: 37-40. Ryland, L.B. and M.W. Tamele. 1970. Thiols. In The A n a l y t i c a l Chemistry of Sulfur and Its Compounds. J.H. Karchmer, (Ed.) WileyInterscience, New York. Part I. pp. 465-519. Stevens, R.K. 1967. A rapid s p e c i f i c method for the gas chromatographic determination of organophosphate pesticides i n cold pressed c i t r u s 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. ignored.  Of these l i p i d fractions the s u l f o l i p i d i n s o i l has been  The primary reason for t h i s retarded development of s u l f o l i p i d  research i s that suitable a n a l y t i c a l methods and procedures are not available.  The primary focus of t h i s investigation,  then, was to  consider some aspects on 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 . A s i g n i f i c a n t 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 t h i r d chapter was an attempt  to determine  the.contents  of l i p i d sulfur i n thirty-seven s o i l s and to i d e n t i f y relationships of the d i s t r i b u t i o n of l i p i d sulfur with other s o i l factors.  The  d i s t r i b u t i o n of the l i p i d sulfur was examined for a l l s o i l s , organic and mineral samples, and s o i l s of a kind grouped according to vegetation, drainage and horizon type.  L i p i d sulfur was found i n a l l s o i l s examined  and the amount was very variable.  There was a s i g n i f i c a n t  difference  i n contents of l i p i d sulfur between organic and mineral samples, whereas there did not seem to be a clear-cut d i s t r i b u t i o n of the l i p i d  sulfur  between the corresponding mineral horizons of forest and grassland soils.  However, i t i s evident that l i p i d sulfur contents i n mineral  horizons are lower than those i n organic horizons of the same forest s o i l s and the l i p i d sulfur i s concentrated i n organic s o i l s .  The  lipid  sulfur accounted for small parts of both the t o t a l sulfur and t o t a l lipid.  L i p i d sulfur contents as percent of t o t a l sulfur and as parts  - 142 -  per m i l l i o n of t o t a l l i p i d did not show a s i g n i f i c a n t difference between organic and mineral samples.  However, the d i s t r i b u t i o n of l i p i d  sulfur  i n the f i v e d i f f e r e n t groups was s i g n i f i c a n t l y d i f f e r e n t depending on the expressions of the l i p i d sulfur contents.  F a i r l y close relationships  of the l i p i d sulfur were found with t o t a l sulfur, Hi-reducible sulfur and organic carbon.  On the other hand, the r e l a t i v e l i p i d sulfur content  was s i g n i f i c a n t l y correlated  only with t o t a l l i p i d when the l i p i d  sulfur  content was expressed as % of total: sulfur, and with t o t a l sulfur when expressed as ppm of t o t a l l i p i d .  The l i p i d sulfur content of mineral  s o i l s was not s i g n i f i c a n t l y correlated with any other s o i l  factors  whether expressed as ppm of s o i l , as % of t o t a l sulfur or as ppm of t o t a l lipid.  On the other hand, the l i p i d sulfur contents of organic samples  were s i g n i f i c a n t l y correlated with t o t a l sulfur and Hl-reducible sulfur and only with t o t a l sulfur when expressed as ppm of t o t a l l i p i d , and only with t o t a l l i p i d when expressed as % of t o t a l sulfur.  These relationships  were not consistent f o r a l l samples and f o r organic and mineral samples when samples were considered as f i v e groups.  The stepwise  elimination  regression analysis showed that 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 can be best explained by two s o i l factors, t o t a l l i p i d and t o t a l 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 t o t a l sulfur, the d i s t r i b u t i o n of the l i p i d sulfur can be best explained by organic carbon and t o t a l l i p i d contents.  However, the l i p i d sulfur can be best predicted by organic  carbon and t o t a l sulfur contents when the l i p i d sulfur was expressed as ppm of t o t a l l i p i d .  I t i s therefore imperative to use a suitable  expression for the study of l i p i d sulfur d i s t r i b u t i o n , so that the s o i l factors can' be chosen accordingly.  - 143  Fractionation  -  of s o i l t o t a l l i p i d s was  a s i l i c i c acid column chromatography.  The  accomplished by using  d i s t r i b u t i o n patterns of  the  three classes of l i p i d s on the s i l i c i c acid column were similar for a l l eight s o i l s studied regardless of s o i l types. dominant class of s o i l l i p i d s and the t o t a l l i p i d s .  Glycolipids were the  they accounted for more than half of  The neutral l i p i d s were around one-third of  the  t o t a l l i p i d s , polar l i p i d s were only 4% of the t o t a l being the component of the s o i l l i p i d s .  The  smallest  uniformity of the d i s t r i b u t i o n i n each  f r a c t i o n of the column chromatography suggests that the l i p i d s i n these s o i l s were similar i n type and affected  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  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  lipid  sulfur 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 between corresponding classes for eight s o i l s , d i f f e r i n g from s o i l soil.  Furthermore, i t was  found that s i g n i f i c a n t amounts of sulfur were,  i n a l l cases, recovered i n these three classes.  This finding  clearly  suggests that s o i l l i p i d sulfur i s present i n a variety of forms. r a t i o s of the l i p i d sulfur to l i p i d as ppm corresponding f r a c t i o n and  the r a t i o s for whole s o i l s have shown that  always higher than those for whole s o i l s and  were  the r a t i o s for neutral  and  lower values than the values for whole s o i l s ,  except for neutral l i p i d fractions of two Humisol samples. finding indicates  The  of the l i p i d i n each  the r a t i o s for polar l i p i d fractions of a l l s o i l s , except one,  g l y c o l i p i d classes had  to  This  that the neutral l i p i d f r a c t i o n of these two  could be the most useful f r a c t i o n for further investigation  Humisols  to  characterize the s o i l l i p i d sulfur, since t h i s f r a c t i o n , p a r t i c u l a r l y  - 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 i n this f r a c t i o n as ppm of l i p i d are higher than those i n whole s o i l s . 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 i n the previous chapter were reported i n the l a s t chapter.  Although the column  chromatographic  behavior of t o t a l l i p i d s and l i p i d sulfur of two s o i l s studied were similar to each other, thin-layer chromatographic  behavior df the  corresponding four fractions of two s o i l s were not similar.  These  findings indicate 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 the same l i p i d class of the other s o i l even though the column chromatographic t o t a l l i p i d s was similar for both.  d i s t r i b u t i o n of  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  i n the solvents used f o r l i p i d extraction and for elution from column chromatography masked gas-liquid chromatographic  monitoring of s u l f u r -  containing compounds i n l i p i d extracts and fractions of column chromatography.  However, the r e s u l t s provided by GLC equipped with a  sulfur detector suggest that the GLC could be used i n monitoring of the l i p i d sulfur by developing a sulfur base l i n e for the solvent blank. This report has been of an exploratory nature, however some p r a c t i c a l 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 i n evaluating the s u i t a b i l i t y of a sample f o r 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 i n s o i l i n p a r t i c u l a r .  Several  p o t e n t i a l l y useful aspects f o r future research a c t i v i t i e s are evident from t h i s work, which would illuminate the c h a r a c t e r i s t i c s of s o i l sulfur f r a c t i o n s . for quantitative  lipid  Some of these aspects would include work on methods and q u a l i t a t i v e evaluation of s o i l l i p i d sulfur and  chromatographic fractionation of them and considering interactions sulfur f r a c t i o n with other l i p i d constituents i n s o i l .  of the  

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