analysis of free amino acids in microbially colonized sandstone by precolumn pitc

7/27/2019 Analysis of Free Amino Acids in Microbially Colonized Sandstone by Precolumn PITC

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A n a l y s i s o f F r e e Amino A c i d s i n M i c r o b i a l l y C o l o n i z e d S a n d s t o n e

b y P r e c o l u m n P h e n y l I s o t h i o c y a n a t e D e r i v a t i z a t i o n a n dH i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y

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I n s t i t u t f u r A l l g e m e i n e M i k r o b i o l o g i e , C h r i s t i a n - A l b r e c h t s - U n i v e r s i t a t ,D - 2 3 0 0 K i e l , F e d e r a l R e p u b l i c o f G e r m a n y

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A f a s t , s e n s i t i v e m e t h o d f o r e x t r a c t i o n a n d a n a l y s i s o f s o l u b l e f r e e a m i n o a c i d s f r o m m i c ro b i al l y c o l on i z ed

s a n d s t o n e i s d e s c r i b e d . A f t e r p r e c o l u m n p h e n y l i s o t h i o c y a n a t e ( P I T C ) d e r i v a t i z a t i o n , t h e ' P I T C - a m i n o a c i d s

w e r e i d e n t i f i e d a n d ' q u a n t i f i e d b y r e v e r s e d - p h a s e h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y . T h i s k i n d o f

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

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

m i c r o o r g a n i s m s i n e c o s y s t e m s ( 6 , 7 ) . I t i s w e l l d o c u m e n t e dt h a t a l g a e a n d o t h e r m i c r o o r g a n i s m s c a n e x c r e t e c o n s i d e r -a b l e a m o u n t s o f t h e s e c o m p o u n d s i n t o t h e e n v i r o n m e n td u r i n g g r o w t h i n c u l t u r e a n d i n s i t u ( 1 , 1 0 ) . O t h e r s o ur ce s o ft h e s e l o w - m o l e c u l a r - w e i g h t c o m p o u n d s a r e a u t o l y s i s o f c e l l sa n d e x o e n z y m a t i c d e g r a da t i o n o f c o m p l e x s u b s t r a t e s ( 1 2 ) .T h e l o w - m o l e c u l a r - w e i g h t f r a c t i o n o f d i s s ol v e d o r g an i c m a t -

t e r i s e a s i l y u s e d b y b a c t e r i a a n d a l g a e b e c a u s e t h e s es u b s t r a t e s a r e r e c o g n i z e d b y s e v e r a l u p t a k e s y s t em s a n d c a nb e a s s i m i l a t e d r e a d i l y ( 1 1 ) . B ac t e r i a i n c o rp o r at e a b o u t 8 0 t o90% o f a s s i m i l a t e d a m i n o a c i d c a r b o n p r e d o m i n a n t l y i n t op r o t e i n s ; 1 0 t o 20% i s r e s p i r e d ( 1 6 , 2 6 ) . I n t h e a b s e n c e o f f r e ea m i n o a c i d s , p r o t e i n s y n t h e s i s a c c o u n t s f o r 60% o f t h e

e n e r g y b u d g e t ( 1 8 ) . T h e r e f o r e , t h e i n c o r p o r a t i o n o f a m i n oa c i d s d i r e c t l y i n t o p r o t e i n s h o u l d r e s u l t i n s u b s t a n t i a l s a v i n g si n t h e e n e r g y c o s t o f g r o w t h . T h i s e ne rg y c o n s e r v a t i o n may

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

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

e n v i r o n m e n t s ( e . g . , s e e r e f e r e n c e s 3 a n d 1 7 ) . D i s s o l v e d f r e ea m i n o a c i d s p l a y a c r u c i a l r o l e i n a q u e o u s e n v i r o n m e n t s ( 6 ) .T h i s i s p r o b a b l y t r u e i n t e r r e s t r i a l s y s t e m s a s w e l l , b u te v i d e n c e t o s u p p o r t t h i s a s s u m p t i o n i s l a c k i n g . M i c r o b i a la c t i v i t y ( e . g . , e x c r e t i o n o f i n o r g a n i c a n d o r g a n i c a c i d s ) c a nc o n t r i b u t e t o b i o l o g i c a l w e a t h e r i n g p r o c e s s e s ( 1 6 a , 2 7 ) .M i c r o b i a l a c t i v i t y i s d e p e n d e n t o n n u t r i t i o n , a n d t h e r e f o r en u t r i t i o n a l c o n d i t i o n s ( c o n c e n t r a t i o n s a n d t y p e s o f s u b -s t r a t e s s u c h a s a m i n o a c i d s ) i n s t o n e s h o u l d b e i n v e s t i g a t e d .

T h e a n a l y t i c a l d e t e rm i na t i on o f a m i n o a c i d s i s u s u a l l yd o n e b y p r e c o l u m n d e r i v a t i z a t i o n w i t h o - p h t h a l d i a l d e h y d e( 1 4 , 1 5 ) . T h e d e r i v a t i v e s a r e s e p a r a t e d b y h i g h - p e r f o r m a n c e

l i q u i d c h r o m a t o g r a p h y ( H P L C ) a n d d e t e c t e d b y f l u o r e s -c e n c e . T h e s e n s i t i v i t y p r o v i d e d b y o - p h t h a l d i a l d e h y d e d e -r i v a t i z a t i o n a l l o w s d e t e c t i o n o f d i s s o l v e d f r e e a m i n o a c i d s i nt h e c o n c e n t r a t i o n s p r e s e n t i n t h e f r e e w a t e r c o l u m n ( 3 0 t o

7 2 0 nM) ( 1 3 , 1 5 ) . D i s a d v a n t a g e s o f t h e o - p h t h a l d i a l d e h y d em e t h o d a r e t h a t t h e d e r i v a t i v e s a r e u n s t a b l e a n d t h a t s e c -

o n d a r y a mi n es d o n o t d e r i v a t i z e . T h u s , r e a c t i o n t i m e s d u r i n gd e r i v a t i z a t i o n m u s t b e k e p t c o n s t a n t a n d t h e a n a l y s i s must

* C o r r e s p o n d i n g a u t h o r .

b e d o n e w i t h o u t d e l a y . N o r m a l l y t h i s h a s b e e n g u a r a n t e e d

b y f u l l a u t o m a t i z a t i o n o f t h e a n a l y t i c a l s y s t e m .We u s e d a p h e n y l i s o t h i o c y a n a t e ( P I T C ) p r e c o l u m n d e -

r i v a t i z a t i o n p r oc e d u re b a s e d o n t h e m e t h o d d e s c r i b e d b yH e i n r i c k s o n a n d M e r e d i t h ( 9 ) . T h e d e r i v a t i z e d a m i n o a c i d sa r e d e t e c t e d b y UV a b s o r b a n c e a t 2 4 5 n m . As l i t t l e a s ' 1 0

p m o l o f a m i n o a c i d c a n b e d e t e c t e d ; t h i s s e n s i t i v i t y i s

s u f f i c i e n t f o r i n v e s t i g a t i o n s i n o l i g o t r o p h i c m e d i a o r c o l o -n i z e d r o c k . T h e P I T C d e r i v a t i z a t i o n p r o c e d u r e h a s t w o

a d v a n t a g e s o v e r o - p h t h a l d i a l d e h y d e : s e c o n d a r y a m i n e s s u c h

a s p r o l i n e c a n b e d e t e c t e d a n d t h e d er i v at i z ed a m i n o a c i d sa r e s t a b l e f o r w e e k s when s t o r e d f r o z e n a t pH 6 . 8 ( 9 ) .We u s e d t h e P I T C m e t h o d o n t w o t y p e s o f m i c r o b i a l l y

c o l o n i z e d s a n d s t o n e . T h e f i r s t w a s a w e a t h e r e d ' c r u s t f r o mt h e S c h l e s w i g c o u r t h o u s e ( F e d e r a l R e p u b l i c o f G e r m a n y )( s a m p l e L i [ 1 6 a ] ) , a n d t h e s e c o n d w a s A n t a r c t i c B e a c o n

s a n d s t o n ec o n t a i n i n g

a

c r y p t o e n d o l i t h i cm i c r o b i a l commu-

n i t y ( 4 ) ( s a m p l e 8 4 5 / 2 3 9 f r o m t h e McMurdo D r y V a l l e y s o f

S o ut h ' V i c t o ri a L a n d ) . T h e c o u r t h o u s e s a m p l e was p r o c -e s s e d w i t h i n 2 h o f s a m p l e c o l l e c t i o n ; t h e A n t a r c t i c s a m p l eh a d b e e n s t o r e d a t - 2 7 ° C s i n c e i t s s a m p l i n g d a t e ( 1 9 8 5 ) .

One t o 2 g o f c o l o n i z e d s t o n e was c r u s h e d i n a m o r t a r . T h es o l u b l e f r e e a m i n o a c i d s ( S F A A ) w e r e e x t r a c t e d w i t h 0 . 1 5 MN a C l f r o m 1 0 0 t o 2 0 0 mg o f c r u s h e d s t o n e ( 1 : 1 [ w t / v o l ] ) a t4 ° C f o r 3 0 m i n o n a r o t a r y s h a k e r . N o s i g n i f i c a n t i n c r e a s e i nt h e a m i n o a c i d c o n c e n t r a t i o n was s e e n w i t h a l o n g e r e x t r a c -

t i o n t i m e ( 6 0 m i n ) . T h e s a m p l e s w e r e c e n t r i f u g e d , a n d 1 0 0 , u lo f s u p e r n a t a n t was d r i e d a n d u s ed f o r d e r i v a t i z a t i o n . A l ld r y i n g s t e p s w e r e d o n e i n a S p e e d Vac c o n c e n t r a t o r ( S a -v a n t ) . H y d r o x y p r o l i n e ( H P r ) was u s e d a s t h e i n t e r n a l s t a n -

d a r d ( 2 . 5 , u l o f a 1 0 mM s o l u t i o n ) . P r i o r t o P I T C d e r i v a t i z a -t i o n , t h e d r i e d s a m p l e s w e r e r e s u s p e n d e d i n 1 0 , u l o f r e d r y i n gs o l u t i o n ( e t h a n o l - w a t e r - t r i e t h y l a m i n e , 2 : 2 : 1 [ v o l / v o l / v o l ] )a n d r e d r i e d . P I T C d e r i v a t i z a t i o n was a c c o m p l i s h e d b y t h e

a d d i t i o n o f 2 0 , u l o f c o u p l i n g s o l u t i o n ( e t h a n o l - w a t e r - t r i e t h -y l a m i n e - P I T C , 7 : 1 : 1 : 1 [ v o l / v o l / v o l / v o l ] ) , f o l l o w e d b y i n c u b a -t i o ' n f o r 2 0 m i n a t room t e m p e r a t u r e . A f t e r a f i n a l d r y i n g s t e pi n w h i c h a l l PITC was r e m o v e d , t h e s a m p l e s were r e s u s -

p e n d e d i n 2 0 0 , u l o f 1 2 . 5 mM p h o s p h a t e b u f f e r ( p H 6 . 4 ) ,v o r t e x e d , a n d c e n t r i f u g e d f o r 2 m i n a t h i g h s p e e d . A 5 - p J la l i q u o t o f t h e s u p e r n a t a n t wa s u s e d f o r HPLC a n a l y s i s .

A n a l y s e s w e r e p e r f o r m e d w i t h a P h a r m a c i a - L K B HPLCs y s t e m c o n s i s t i n g o f a m o d e l 2 1 5 6 s o l v e n t c o n d i t i o n e r , ' a

m o d e l 2 2 4 8 HPLC p u m p , a m o d e l 2 1 5 7 a u t o s a m p l e r , a n d a

8 7 9



A P P L . E N V I R O N . M I C R O B I O L .

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0.0 5.0 1 0 , 0 15.0 20.0 25,0 30.0 35.0 40.0

4

1~~~~~~~~110~~~~~~~2

0 .0 5 .0 1 0 , 0 1 5 , 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0

Retention Time ( m i n )

FIG. 1. Comparison of the dissolved free amino acid content of

two colonized sandstones. PITC-amino acids were separated on a

SpherisorbODS I 5 - , u m column by HPLC. (A) Amino acid calibra-

tion standard AA-S-18 (Sigma), 250 pmol of each amino acid. (B)

Samples. 1, Asp; 2 , Glu; 3, Hpr (IS); 4, Ser; 5, Gly; 6, unknown; 7,

unknown; 8, Thr; 9 , Ala; 10, Pro; 11, His; 12, reagent peak; 13, Arg;

14, Tyr; 15, Val; 16, Met; 17, Cys; 18, Ile; 19 , Leu; 20, Phe; 21,

NH3/Try; 22, Lys.

model 2141 variable wavelength monitor. The column was a

Spherisorb octyldecylsilane I I C18 (5 , u m , 4.6 by 250 mm)

(Bischoff, Leonberg, Federal Republic of Germany) pre-

cededby a guardcolumn (5 p u . m , 4.6 by 20 mm). Thecolumns

were held at 3 6 ° C . The binary gradient was composed of

solvents A(12.5 mMphosphate buffer, pH 6.4) and B

(acetonitrile in phosphate buffer, 50:50). The gradient con-

ditions were 0 to 15%Bfor 15 m mn 15 to45%B for 20 m i n ,

45 to 8 0 % G B for 1 min, and80%Bfor 2 min. Washing was

done with 100%Bfor 9 m i n . Equilibration was accomplished

with 100%Afor 8 m i n . The flow rate was 1.0 m I / m m n .Detection was at 245nmand 0.02 absorption units full scale

(AUFS). Peaks were integrated with model 2600 chromatog-

r a p h y s o f t w a r e ( P e r k i n - E l m e r N e l s o n S y s t e m s ) . S a m p l e s

were n o r m a l i z e d t o HPr ( i n t e r n a l s t a n d a r d ) a n d q u a n t i f i e d o n

t h e b a s i s o f p e a k area a n d r e t e n t i o n t i m e o f e x t e r n a l amino

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

o n t h e b a s i s o f l i p i d - p h o s p h a t e c o n t e n t . L i p i d s were e x -

t r a c t e d b y t h e method o f W h i t e e t a l . ( 2 4 ) , and p h o s p h o l i p i d -

p h o s p h a t e ( P L P ) was a n a l y z e d b y t h e method o f va n V e l d -

h o v e n a n d M a n n a e r t s ( 1 9 ) . Biomass i n b o t h s a n d s t o n e s w a si n t h e same o r d e r o f m a g n i t u d e : t h e c o u r t h o u s e s a m p l e

c o n t a i n e d 4 9 nmol o f PL P g - 1 ( 1 6 a ) ; t h e A n t a r c t i c s a m p l e

c o n t a i n e d 2 9 nmol o f PL P g - ' . T he A n t a r c t i c b i o m a s s v a l u e

was i n t h e range r e p o r t e d f o r s i m i l a r s a n d s t o n e s a m p l e s ( 2 0 ) .T h e r e i s no s i m p l e c o r r e l a t i o n between b i o m a s s b a s e d on

PL P a n d t h a t b a s e d o n c e l l n u m b e r s . White ( 2 3 ) h a s c a l c u -

l a t e d t h a t a p p r o x i m a t e l y 1 nmol o f p h o s p h a t e r e p r e s e n t s

a b o u t 1 0 8 b a c t e r i a t h e s i z e o f E s c h e r i c h i a c o l i . The c r y p t o e n -

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

l i c h e n s , a l g a e , a n d b a c t e r i a . T h u s , o u r PL P r e s u l t s c a n n o t b e

e x t r a p o l a t e d t o c e l l n u m b e r s .

A n a l y s i s o f t h e tw o s a n d s t o n e s a m p l e s showed c o n s i d e r -

a b l e d i f f e r e n c e s i n t h e t y p e s a n d q u a n t i t i e s o f SFAA ( F i g . 1 ) .T a b l e 1 s h o w s t h e c o n c e n t r a t i o n s o f i d e n t i f i e d a m i n o a c i d s

n o r m a l i z e d t o i n s i t u b i o m a s s ( n a n o m o l e s o f P L P ) . Thec o n c e n t r a t i o n s o f SFAA were 2 t o 4 6 t i m e s h i g h e r i n t h e

c o u r t h o u s e s a m p l e t h a n i n t h e A n t a r c t i c s a m p l e . A r g i n i n e ,

a l a n i n e , a n d g l y c i n e were t h e most a b u n d a n t amino a c i d s ( 4 8m o l % ) i n t h e c o ur t h o u s e s a mp l e , a n d a n u n i d e n t i f i e d amino

compound ( p e a k 7 ) was p r e s e n t i n l a r g e a m o u n t s . A s p a r t i c

a c i d , t h r e o n i n e , h i s t i d i n e , t y r o s i n e , v a l i n e , i s o l e u c i n e , l e u -

c i n e , a n d p h e n y l a l a n i n e were a b s e n t i n t h e A n t a r c t i c s a m p l e .

D i f f e r e n c e s b e t w e e n t h e A n t a r c t i c a n d t h e c o u r t h o u s e

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

t h e tw o e c o s y s t e m s . M i c r o o r g a n i s m s c o u l d u t i l i z e d i f f e r e n ta m i n o a c i d s t o d i f f e r e n t e x t e n t s ( 2 5 ) . F o r e x a m p l e , H a g s t r o me t a l . ( 8 ) h a v e d e mo n s t r at e d t h a t m a r i n e b a ct e r i a p r ef e re n -

t i a l l y u t i l i z e g l u t a m i n e a n d s e r i n e . A l s o , p r i m a r y p r o d u c e r s

c a n d i f f e r i n t h e i r a b i l i t y t o e x c r e t e ( 1 0 ) o r t a k e u p ( 2 2 ) a m i n o

a c i d s ; n o p h o t o t r o p h i c o r g a n i s m s were f o u n d i n t h e c o u r t -h o u s e s t o n e ( 1 6 a ) . F u r t h e r m o r e , a u t o l y s i s or e x o e n z y m a t i c

a c t i v i t i e s i n t h e c o m m u n i t i e s c o u l d d i f f e r ( 1 2 ) . S o r p t i o n

r e a c t i o n s i n t h e s t o n e a l s o p l a y a r o l e i n a m i n o a c i d a v a i l -a b i l i t y ( 2 1 ) , a n d i n m a r i n e s e d i m e n t s , t h e d i s s o l v e d f r e ea m i n o a c i d p o o l i s b a l a n c e d w i t h a n e x c h a n g e a b l e , a b s o r b e d

p o o l o f a m i n o a c i d s ( 2 ) .F r i e d m a n n e t a l . ( 5 ) h a v e c a l c u l a t e d t h a t a m axim um o f 2 9

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

A nt a r ct i c c ry p t o e n do l i t h i c e c o s y s t e m . T h i s l o w t o t a l a c t i v i t yc o u l d e x p l a i n t h e l o w a m i n o a c i d c o n c e n t r a t i o n i n A n t a r c t i c

s a m p l e s . I n v e s t i g a t i o n s o n o t h e r c o l o n i z e d A n t a r c t i c s a n d -

s t o n e s a m p l e s y i e l d e d s i m i l a r r e s u l t s ( 4 t o 1 1 n m o l o f

SFAA/nmol o f PLP [ d a t a n o t s h o w n ] ) . T h u s , t h e l o w SFAAc o n c e n t r a t i o n was n o t t h e r e s u l t o f a n a t y p i c a l s a m p l e . A l s o ,

i t i s u n l i k e l y t h a t t h e a m i n o a c i d s i n t h e A n t a r c t i c s a m p l ewere d e g r a d e d d u r i n g t h e l o n g s t o r a g e p e r i o d b e c a u s e t h e

PLP ( k n o w n t o d e g r a d e r a p i d l y [ 2 4 ] ) c o n c e n t r a t i o n i n t h e

T A B L E 1 . C o n c e n t r a t i o n s o f f r e e a m i n o a c i d s i n t w o c o l o n i z e d s a n d s t o n e s a m p l e s

n m o l / n m o l o f PL PS a m p l e

A s p G l u S e r G l y T h r A l a P r o H i s A r g T y r V a l Met I l e L e u P h e L y s

C o u r t h o u s e 0 . 2 0 . 8 1 . 4 4 . 6 1 . 4 5 . 7 2 . 8 1 . 8 6 . 5 2 . 0 1 . 3 1 . 8 0 . 6 1 . 7 1 . 0 0 . 9A n t a r c t i c 8 4 5 / 2 3 9 0 . 3 0 . 2 0 . 1 1 . 2 0 . 1 1 . 2 0 . 5 0 . 4

8 8 0 N O T E S



N O T E S 8 8 1

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

( 2 0 ) .W i t h t h e h e l p o f t h i s f a s t , s e n s i t i v e , a n d i n e x p e n s i v e

p r e c o l u m n PITC d e r i v a t i z a t i o n p r o c e d u r e , i t may b e p o s s i -

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

m i c r o o r g a n i s m s a c t i v e i n b i o l o g i c a l r o c k - w e a t h e r i n g p r o -

c e s s e s .

We t h a n k E . I . F r i e d m a n n ( F l o r i d a S t a t e U n i v e rs i t y , T al l a h as s e e)

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

E . I . F r i e d m a n n w a s s u p p o r t e d b y N a t i o n a l S c i e n c e F o u n d a t i o n

g r a n t DPP 8 3 - 1 4 1 8 0 . T h i s r es e ar ch w a s s u p p o r t e d b y a g r a n t t o P . H .

f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . R . J . P . w a s s u p p o r t e d

b y a H u m b o l d t f e l l o w s h i p a n d a g r a n t f r o m t h e S c h l e s w i g - H o l s t e i n

K u l t u s m i n i s t e r i u m .

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

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I n P r o c e e d i n g s o f t h e V I t h I n t e r n a t i o n a l C o n g r e s s on D e t e r i o -

r a t i o n a n d C o n s e r v a t i o n o f S t o ne . N i c h o l a s C o p e r n i c u s U n i v e r -

s i t y P r e s s , T o r u n , P o l a n d .

VOL. 5 7 , 1 9 9 1

analysis of free amino acids in microbially colonized sandstone by precolumn pitc

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