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J ourna l o f M i c rob i o log i c a l M e t hods 8 (1988) 131 - 137 131Elsevier

JMM 00259

E v a l u a t i o n o f a h ex a n e / i s o p ro p a n o l l i p i d s o l v en t

s y s t em fo r a n a l y s i s o f b a c ter i a l p h o s p h o l i p i d s a n d

a p p l i ca t i o n to ch l o ro fo rm -s o l u b l e Nu c l ep o re

polycarbonate) membranes wi th re ta ined bacter ia

James B Guckert and David C. WhiteD ep art m ent o f Biolo gical Science, Florida State Universi ty , Tallahassee, FL 32306 U SA )

(Received 31 August 1987) (Revised version received 28 December 1987) (Accepted 10 January 1988)

Summa r y

The recovery of phospholipid, ester-linked fatty acids and phospholipid phosphate from the

eubacterium Pseudomonas a t lant ica was shown to be equivalent for the modified Bligh and Dyer

(chloro form/methanol /wate r) procedure and a hexane/isopropanol (HIP) lipid extraction. In addition to

its previously suggested lower toxicity, the HIP solvent system allows lipid analyses to be routinely done

on chloroform-soluble materials such as Nuclepore polycarbonate membranes, useful for well-definedsize fractionation studies of bacterioplankton communities.

Key words: Bacterial lipid; Lipid analysis; Nuclepore filter

Introduct ion

The analysis of membrane phospholipids has become an important tool for the

quantitative description of the viable microbial biomass, community structure, and

nutrit ional status in complex, natural environments [1]. There are a diversity of

procedures utilized, however, for the extraction, isolation and purification of these

lipids. These procedures may not all be equivalent in recovery of the lipid classes o f

interest for microbial ecology research. New or modified methods need to be

evaluated in terms of quali ty and quantity of lipid recovered with respect to standard

analyses.

Correspondence to: James B Guckert, Department of Microbiology, Montana State University,

Bozeman, MT 59717, USA.

* Present address: Department of Microbiology, Montana State University, Bozeman, MT 59717, USA.** Present address: Institute for Applied Microbiology, University of Tennessee, 10515 Research Drive,

Suite 300, Knoxville, TN 37932-2567, USA.

0167-7012/88/ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)



132

T h e m o s t w i d e l y u s e d s o lv e n t s y s te m is c h l o r o f o r m : m e t h a n o l ( C M , 2 :1 , v / v ) w h i c h

is r e p o r t e d t o e x t r a c t l ip i d m o r e c o m p l e t e l y f r o m a n i m a l s , p l a n t s o r b a c t e r i a t h a n

m o s t o t h e r ' s i m p l e ' s o l v e n t s y s t e m s [ 2 ] . T h i s e x t r a c t i o n s y s t e m w a s i m p r o v e d b y

E . G . B l i g h a n d W . J . D y e r [3 ]. T h e ' B l i g h a n d D y e r ' ( B / D ) e x t r a c t io n u se s a

m o n o p h a s i c s o l u t i o n o f c h l o r o f o r m : m e t h a n o l : w a t e r ( 1 : 2 : 0 . 8 , v / v / v ) f o r t h e p r i m a r ye x t r a c t i o n . W a t e r h e l p s t h e e x t r a c t i o n o f t h e p o l a r l i p i d s a s it c a u s e s c e l lu l a r s w e l li n g

w h i c h f a c i l i t a t e s t h e p e r m e a b i l i t y o f t h e c e l l w a l l s f o r t h e s o l v e n t s [ 4 ] . A f t e r t h e

p r i m a r y e x t r a c t io n , c h l o r o f o r m a n d w a t e r a r e a d d e d t o a f i n a l r a ti o o f 2 :2 :1 .8

( c h l o r o f o r m : m e t h a n o l : w a t e r , v / v / v ) t o f o r m t w o p h a s e s . T h e s e r a ti o s r es u l t i n a l o w e r

p h a s e c l o se t o 1 0 0 c h l o r o f o r m (l ip i d - ri c h ) a n d t h e u p p e r p h a s e l a rg e l y o f m e t h a -

n o l / w a t e r c o n t a i n i n g t h e n o n - li p i d s . R e c o v e r y is r e p o r t e d t o b e 9 5 o f th e to t a l l ip i d

w i t h a s in g l e e x t r a c t i o n [ 3]. W h i t e e t a l . [5 ] s u b s t i t u t e d a 5 0 m M p h o s p h a t e b u f f e r

( 8. 7 g K z H P O 4 p e r l it er , a d j u s t e d w i t h 1 N H C 1 t o p H 7 . 4) f o r w a t e r i n t h e o r i g i n a l

p r o p o r t i o n s f o r e n v i r o n m e n t a l s a m p l e s to m i n i m i z e p H s h i ft s w h i c h c o u l d r e su l t i n

t r a n s e s t e r i f i c a t i o n a r t i f a c t s o c c u r r i n g d u r i n g t h e l i p i d e x t r a c t i o n p r o c e d u r e [ 2 ] . T h em o d i f i e d B / D ( m B / D ) w a s u s e d i n t h i s r e s e a r c h .

T h e m B / D e x t r a c t i o n d o e s h av e li m i t a ti o n s . A p r i n c i p a l c o n c e r n f o r li p id a n a l ys i s

i n m i c r o b i a l e c o l o g y is th e c h l o r o f o r m - s o l u b i l i t y o f m a n y c o m m o n m a t e ri a ls w h i c h

c o u l d c o n t r i b u t e i n t e r f e r e n c e t o t h e a n a l y si s. T h i s i n c lu d e s c e r t a i n p l a st ic s t h a t m a y

b e o f i n t e re s t a s s u b s t r a t a i n b i o f o u l i n g r e s e a r c h [6 ], a n d N u c l e p o r e f i lt e r s f o r

q u a n t i t a t i v e s i z e f r a c t i o n a t i o n s t u d i e s o f a q u a t i c m i c r o b i o t a [ 7 ] .

A n e x t r a c ti o n s y st em o f h e x a n e / i s o p r o p a n o l / w a t e r ( H I P ) w a s s u g ge s te d a s a lo w -

t o x i c i ty s u b s t i tu t e f o r th e B / D p r o c e d u r e [8 ]. T h e H I P w o u l d p e r m i t li p id a n a l y s is

o f m a t e r i a l r e t a i n e d o n N u c l e p o r e f i l t e r s s i n c e p o l y c a r b o n a t e i s s t a b l e i n t h e s e

s o l v e n ts [ N u c l e p o r e C a t a l o g , 1 98 4]. I n a d d i t i o n , i s o p r o p a n o l is a n i n h i b i t o r o f

p h o s p h o l i p a s e D a n d h a s b e e n u s e d i n p r e v i o u s w o r k t o k e e p p l a n t l i p i d s i n t h e i r

n a t i v e s t a te [ 2]. O t h e r a d v a n t a g e s o f H I P r e p o r t e d h a v e b e e n t h a t i t e x t r a c t s l es s

p i g m e n t a n d n o n - l i p i d s ( e s p e c ia l ly p r o t e o l i p i d s ) t h a n C M [8 ].

A c o m p a r i s o n o f H I P v s. m B / D w a s c a r r ie d o u t f o r r e c o v er y o f p h o s p h o l i p i d ,

e s t e r -l i n k e d f a t t y a ci d s ( P L F A ) o f a e u b a c t e r i a l c u l tu r e . P L F A , t h e p r i n c i p a l l ip i d s

o f e u b a c t e r i a [9 ], a r e u s e d a s b i o c h e m i c a l m a r k e r s o f th e v i a b le m i c r o b i o t a f o r

e n v i r o n m e n t a l s a m p l e s [ 1 ] a n d a r e t h e s p e c i f i c l i p i d s o f i n t e r e s t f o r m u c h o f c u r r e n t

m i c r o b i a l e c o l o g y li p id r e s e a r c h [ 10 ]. O n e p u r p o s e o f t h is c o m p a r i s o n w a s to v a l i d a t e

a l i p id e x t r a c t i o n s y s t em c o m p a t i b l e w i t h N u c l e p o r e f i lt e rs t o a l l o w l i pi d a n a l y s e s t o

b e d o n e o n b a c t e r i o p l a n k t o n c o m m u n i t i e s d e f i n e d b y s p e c if ic s i z e -f r a c ti o n a t io n .

T h e r e c o v e r y o f b a c t e r i a l l ip i d f r o m c e ll s re t a i n e d o n N u c l e p o r e f il te r s w as a l s o

i n c l u d e d i n t h e c o m p a r i s o n . A n a l y s i s o f re c o v e r e d l ip i d w a s b y m o l a r q u a n t i t ie s o f

p h o s p h o l i p i d p h o s p h a t e a n a l y s e d c o l o r i m e t r ic a l ly a n d t o t a l P L F A p e r g d r y w e i g ht

o f c e l l c u l t u r e q u a n t i f i e d b y g a s c h r o m a t o g r a p h y . T h e p r o f i l e s o f P L F A r e c o v e r e d

w e r e e v a l u a t e d f o r p o s s i b l e s e l e c t i v i t y o f t h e s o l v e n t s y s t e m s .

a t e r i a l s a n d e t h o d s

B a c t e r i a l c u l t u r e c o n d i t i o n s

P s e u d o m o n a s a t l a n t i c a H u m m ( A T C C 1 92 62 N C M B 3 01 ) w a s g r o w n f o r 18 h in

1 l it e r o f m e d i u m c o n t a i n i n g : 1 0 g g a la c t o s e , 5 g c a s a m i n o a c i d s , a n d s u f f i c i e n t





134

f r a c t i o n w a s s u b j e c t e d t o a m i l d a l k a l i n e m e t h a n o l i c t r a n s e s t e r i f i c a t i o n w h i c h

r e s u lt e d i n f a t ty a c id m e t h y l e s te r s F A M E ) a n d a q u e o u s - s o l u b l e g l y c e r o l p h o s p h a t e .

T h e F A M E w e r e q u a n t i f i e d b y c a p i ll a ry g a s c h r o m a t o g r a p h y G C ) w i t h o u t a n y

f u r t h e r p u r i f i c a t i o n . T h e G C c o n d i t i o n s a r e a s d e s c r i b e d [ 1 1] e x c e p t t h a t t h e

t e m p e r a t u r e p r o g r a m w a s 80 ° C - 2 1 0 ° C a t 3 ° / m i n f o ll o w e d b y a 1 0 m i n i s o t h e r m a lp r o g r a m . P e a k a r e a s w e r e q u a n t i f i e d u s i n g th e N e l s o n A n a l y t i c a l 3 50 0

C h r o m a t o g r a p h y S o f t w a r e p ac k a g e. T h e i n te r n a l s ta n d a r d o p t i o n w i th i n th e T a rg e t

C o m p o u n d s o f tw a r e p r o g r a m w a s e m p l o y e d re la ti v e t o 5 0 p m o l o f 1 9:0 a d d e d a s t h e

i n t e r n a l i n j e c t io n s t a n d a r d j u s t p r i o r t o G C a n a ly s is . I d e n t i f i c a t i o n o f t h e P.

at lant ica F A M E w a s b a s e d o n i d e n t i c a l r e t e n t i o n i n d ic e s w i t h p r e v i o u s l y a n a l y s e d P .

at lant ica l ip i d w h i c h w a s s t ru c t u r a l l y v e r i fi e d u s i n g G C / m a s s s p e c t r o m e t r y

G C / M S ) [11 ], a d im e t h y l d is u l fi d e D M D S ) d e r i v a ti z a ti o n w it h G C / M S [ 12 ] t o

d e t e r m i n e d o u b l e b o n d p o s i t i o n a n d g e o m e t r y , a n d F o u r i e r t r a n s f o r m i n f r a r e d

s p e c t r o s c o p y to v e r i f y t h e u n u s u a l t r a n s - g eo m e t r y o f s o m e o f P. a t lant ica s P L F A

[131.

P h o s p h o l i p i d p h o s p h a t e a n a ly s is

A l l l i p i d f r a c t i o n s f r o m t h e s i l i c i c a c i d c h r o m a t o g r a p h y w e r e a n a l y s e d w i t h

p e r c h l o ri c a c id d i g es ti o n s a n d c o l o r i m e t r i c f o r p h o s p h o l i p i d p h o s p h a t e P L P O 4 )

d e t e r m i n a t i o n a s d e s c r i b e d i n W h i t e e t a l . [ 5 ] .

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17 :0 17 :108c IN; I~T t1 8 ; 1 ~ 9 c

F i g. 1. C o m p a r i s o n o f t h e p h o s p h o l i p i d f a t ty a c i d (P L F A ) p r o f i le s r e c ov e r ed b y m o d i f i e d B l i g h a n d

D y e r ( m B / D ) e x t r a c t io n , h e x a n e / i s o p r o p a n o l ( H I P ) a n d H I P o f ce ll s r e t a i n e d o n N u c l e p o r e p o l y c a r-

b o n a t e m e m b r a n e f il te r s ( H I P / f i lt e r ) . T h e s y m b o l s i n d i c a te t h e a v e r a g e (n = 3 ) p e r c e n t o f t h e t o ta l m o l a rq u a n t i t y r e c o v e r e d f o r e a c h f a t t y a c i d . T h e e r r o r b a r s i n d i c a t e - -_ S D . W h e n e r r o r b a r s a r e n o t v i s i b le ,

t h e S D w a s e q u a l t o o r l e s s t h a n t h e w i d t h o f t h e s y m b o l .



135

Fa t t y a c i d n o m en c l a t u r e

F a t t y ac id s a r e d e s i g n a te d a s to t a l n u m b e r o f c a r b o n a t o m s : n u m b e r o f d o u b l e

b o n d s w i t h t h e p o s i t i o n o f t h e d o u b l e b o n d c l o se s t t o t h e a li p h a t i c ( o m e g a , w ) e n d

o f t h e m o l e c u l e in d i c a te d w i th t h e g e o m e t r y ' c ' f o r cis a n d ' t ' f o r t rans (e.g. 16:lw7t,

t rans-9 -hexadecenoa te .

S ta t i s t ical analysis

A l l a n a l y s e s w e r e d o n e o n t h e F l o r i d a S t a t e U n i v e r s i t y C y b e r 7 3 0 m a i n f r a m e

c o m p u t e r u t i l i z i n g p r o g r a m s a v a i l a b l e i n t h e s t a t i s t i c a l s o f t w a r e p a c k a g e S P S S .

L o g a r i t h m i c t r a n s f o r m a t i o n s o f d a t a w e r e d o n e t o m e e t t h e h o m o g e n e i t y o f e r r o r

v a r i a n c e a s s u m p t i o n s o f t h e a n a l y s is o f v a r i a n c e m o d e l u s e d [1 4].

R e s u l t s

C o m p a r i s o n o f m B / D a n d H I P l ip id e xt ra c ti on

A c o m p a r i s o n o f t h e P L F A p r o fi le s r e c ov e r e d b y m B / D , H I P a n d H I P f r o m f i lt er s

is s h o w n i n F i g . 1. T h e r e l a t i v e f a t t y a c id l e v e ls a r e e x p r e s s e d a s p e r c e n t o f t h e t o t a l

m o l a r q u a n t i t y r e c o v e r e d ( t o o l ) . T h e s y m b o l s i n d i c a t e t h e m e a n o f t h r e e r e p l ic a t e s

_+ 1 s t a n d a r d d e v i a t i o n ( S D ) . T h e r e w e r e n o s t a t is t i c a ll y s i g n i f i c a n t d i f f e r e n c e s

b e t w e e n t h e s e e x t r a c t io n m e t h o d s f o r a n y P L F A ( A N O V A o~ -- 0 .0 5 ).

T h e t o t a l m o l a r q u a n t i t y o f P L F A p e r g d r y w t. c e ll m a t e r ia l a n d t h e p h o s p h o l i p i d

p h o s p h a t e ( P L P O 4 ) p e r g d r y w t . f o r t h e t h r e e l i p i d t r e a t m e n t s a r e s h o w n i n

T a b l e 1. N o s i g n i f i c a n t d i f f e r e n c e s (o~ = 0 . 0 5 ) w e r e f o u n d f o r e i t h e r b i o c h e m i c a l

e s t i m a t e o f b a c t e r i a l b i o m a s s f o r t h e l ip i d e x t r a c ti o n m e t h o d s . T h e r a t i o s o fP L F A / P L P O 4 f o r t h e t h r e e t r e a t m e n t s a r e a ls o s h o w n in T a b le 1. T h e r e w e re n o

s i g n i f i c a n t d i f f e r e n c e s (e~ = 0 . 0 5 ) f o u n d f o r t h e s e d a t a , e i th e r .

i s c u s s i o n

T h e H I P p r o c e d u r e is a n e q u i v a l e n t s u b s ti t u te f o r t h e m B / D l ip i d e x t r a c t i o n fo r

b o t h t h e q u a n t i t y o f t o t a l P L F A a n d P L P O 4 ( T a bl e 1) a s w e ll a s th e P L F A p r o f i le s

( F ig . 1) r e c o v e r e d f r o m a b a c t e r ia l c u l tu r e , w h e t h e r h a r v e s t e d b y c e n t r i f u g a t i o n o r

N u c l e p o r e f i l t r a t i o n .

TABLE 1

COMPARISON OF PHOSPHOL1PID FATTY ACID PLFA) AND PHOSPHOL IPID PHOSPHA TE

PLPO4) RECOVERIES FROM HEX ANE: ISOP ROPA NOL HIP) FOR CELLS HARVESTED BY

CENTRIFUGAT ION OR NUCLEPORE MEMBRANE FILTRATION, COMPARED WITH THE

CORRE SPOND ING MODIFI ED BLIGH AND DYER roB/D) EXTRACT ION FOR P A TLA NTI CA

CELLS

HIP HIP/fi l ter mB/D

~mol/g dry weight cells)

PLFA 65.5 _+ 12.4 70.5 + 9.4 58.0 + 15.5

PL PO 4 26.5_+ 1.4 22.6_+ 1.5 26.1_+ 2.1

PLFA/PLPO 4 2.5 _+ 0.6 3.2 + 0.6 2.2-+ 0.8



136

P r e v i o u s e v a l u a ti o n s o f t h e H I P l ip i d e x t r a c t io n h a ve c o m p a r e d H I P v s. B / D

e x t r a c t i o n s o f b e e f l i p id [ 1 5 ] a n d H I P v s. C M e x t r a c t i o n s o f r a t b r a i n l ip i d [ 8 ]. I n

b o t h c a s e s, t h e H I P w a s f o u n d t o r e c o v e r l o w e r a m o u n t s o f p o l a r l i pi ds , w h e n

e v a l u a t e d b y w e i g h t o f l i p i d r e c o v e r e d . I t h a s b e e n s u g g e s t e d t h a t p r o t e o l i p i d s

e x t r a c t e d w i t h C M , b u t n o t H I P , a c c o u n t e d f o r t h is d i f f e re n c e [8 ]. W h e n t h e s o l v e n ts y s t e m s w e r e e v a l u a t e d a n a l y t i c a l l y b y g a s c h r o m a t o g r a p h y i n t h e p r e s e n t s t u d y , n o

d i f f e r e n c e i n t h e r e c o v e r y o f b a c t e r i a l d i a c yl p h o s p h o l i p i d s w a s f o u n d , h o w e v er , a

c o m p a n i o n s t u d y c o m p a r i n g t h e r e c o v e r y o f l ip i d c la s se s f r o m a u n i c e l l u l a r g r e e n

a l g a i n d i c a t e s t h a t H I P d o e s n o t r e c o v e r t h e g l y c o - a n d p o l a r l i p id s a s w e ll a s t h e

m B / D p r o c e d u r e f o r t h is e u k a r y o t i c m e m b r a n e s y s te m [16 ]. Tw o o f t h e p r e v io u s H I P

e v a l u a t i o n s [8 , 1 5] a s w e ll a s t h e p r e s e n t s t u d y ( F ig . 1) h a v e s h o w n t h a t t h e p r o f i l e o f

f a t t y a ci d s r e c o v e r e d b y t h e H I P is e q u i v a le n t t o t h e C M e x t r a c t io n p r o c e d u r e s .

T h e m B / D e f f i c ie n t l y r e m o v e s n o n - l ip i d s w h e n t h e p h a s e s a r e s e p a r a t e d [2 ].

P r o c e d u r e s h a v e b e e n s u g g e s t e d f o r a n a q u e o u s w a s h t o r e m o v e n o n - l i p id s i n th e

H I P e x t r a c t i o n [ 8]. N o n - l i p i d s w h i c h m i g h t b e r e c o v e r e d o n l y i n te r f e re w i t h n o n -

s p e c i f i c a n a l y s e s , s u c h a s g r a v i m e t r i c d e t e r m i n a t i o n s o f l i p id r e c o v e r y . T h e

p r o c e d u r e s u s e d i n t h e p r e s e n t s t u d y a r e s p e c i f i c f o r P L F A a n d P L P O 4 a n d a r e ,

t h e r e fo r e , u n a f f e c t e d b y n o n - l i p i d c o n t a m i n a t i o n . W h e n N a z S O 4 w a s u s e d t o s e p a -

r a te th e p r i m a r y H I P e x t r a c t i o n in t o t w o p h a s e s f o r n o n - l i p i d r e m o v a l as o r i g in a l l y

d e s c r i b e d [8 ], t h e t o t a l P L F A r e c o v e r y b y G C a n a l y s is d e c r e a s e d t o 5 6 o f t h e m B / D

( u n p u b l i s h e d d a t a ) . T h e s e re s u lt s s u g g e st e d t h a t n o p h a s e p a r t i t i o n i n g b e u s e d i n th e

H I P p r o c e d u r e w h e n s p e c i f i c a n a l y t i c a l e s t im a t e s o f l ip i d s m i n i m i z e t h e i n t e r f e re n c e s

o f n o n - l i p i d c o n t a m i n a n t s .

I n c o n c l u s i o n , t h e r e su l ts f r o m t h is e x a m i n a t i o n o f P atlantica l ip i d s i n d i c a t e s t h a t

t h e H I P e x t r a c t i o n o f H a r a a n d R a d i n [8 ] is e q u i v a l e n t to t h e m B / D f o r b a c t e r i a l

b i o m a s s d e t e r m i n a t i o n s o f e i th e r P L P O 4 o r t o t a l P L F A . T h e P L F A p r o fi le s fo r

b a c t e r i a l c o m m u n i t y s t r u c t u r e d e t e r m i n a t i o n s w e r e a l so f o u n d t o b e e q u i v a l e n t f o r

t h e t w o p r o c e d u r e s i n d i c a t i n g n o p r e f e r e n ti a l re c o v e r y o f b a c t e r i a l p h o s p h o l i p i d s . A

c o m p a n i o n s t u d y t o t h is p r e s e n t r e s e a r c h i n d i c a t e s t h a t t h i s a n a ly s i s w i ll o n l y b e v a li d

i n e n v i r o n m e n t s w i t h l o w m i c r o e u k a r y o t i c b i o m a s s d u e t o t h e l o w e r re c o v e r ie s o f

g l y c o - a n d p o l a r l i p id s f r o m t h e s e m e m b r a n e s y s t e m s b y H I P [1 6]. T h e u s e o f t h e

H I P e x t r a c t i o n i n m i c r o b i a l e c o l o g y l ip i d a n a ly s i s w il l p e r m i t t h e e x t r a c t i o n o f

b a c t e r i a l s a m p l e s o n c h l o r o f o r m - s o l u b l e c o m p o n e n t s s u c h a s p l a st ic s u s e d a s

a r t i f i c i a l s u b s t r a t a i n b i o f o u l i n g r e s e a r c h a s w e l l a s N u c l e p o r e p o l y c a r b o n a t e f i l t e r s

f o r s i z e f r a c t i o n a t i o n s t u d i e s o f a q u a t i c e n v i r o n m e n t s . W h e n t h e s e e n v i r o n m e n t s a r e

p r i m a r i l y b a c t e r i a l (e .g . g r o u n d w a t e r , s u b s u r f a c e w e l ls [ 1 7] a n d i n i ti a l m i c r o f o u l i n g

c o m m u n i t i e s [1 8]), th e u t i l i z a t io n o f l ip i d a n a ly s e s f o r b a c t e r ia l b i o m a s s , c o m m u n i t y

s t r u c t u r e a n d n u t r i t i o n a l s t a tu s [1] d o e s n o t h a v e t o b e r e s t r ic t e d to c h l o r o f o r m - s t a b l e

s u b s t r a t a o r f i l t e r s w i t h t h e u s e o f t h e H I P l i p i d e x t r a c t i o n p r o c e d u r e .

cknowledgements

T h i s r e s e a rc h w a s s u p p o r t e d b y c o l l a b o r a t i v e a g r e e m e n t C R - 8 1 3 7 2 5 f r o m t h e

R o b e r t S . K e r r E n v i r o n m e n t a l R e s e ar c h L a b o r a t o r y o f t h e U S E P A , g r a n t s

N 0 0 0 1 4 - 8 2 - C - 0 4 0 4 a n d N 0 0 0 1 4 - K - 0 0 5 6 f r o m t h e O f f i c e o f N a v a l R e s e a rc h , a n d a

S i g m a X i G r a n t - i n - A i d o f R e s e a r c h . T h e a u t h o r s a l s o a c k n o w l e d g e t h e g e n e r o u s g i f t

