CAPÍTULO IV: MARCO PROPOSITIVO
4.4. RESULTADOS DE LA APLICACIÓN
4.4.1. Encuesta aplicada a los asociados
CARBON B Y
Th i s c h a p t e r i s c o n c e r n e d w i th e s t im a t i ng t h e amou n t o f b a c t e r i a l and m i c r o a l g a l c a r bon a s s i m i l a t ed b y Amp h i bol a
o n a d a i l y b a s i s . An e x pe r i m e n t a l m e a s u r e o f t h e a s s im i l a t i on o f b ac t e r i a l c a r b o n w i l l b e pr e s e n t ed , a n d t h i s f i gu r e w i l l b e u s e d t o c a l c u l a t e t h e d a i l y a s s im i l a t i o n o f b a c t e r i a l c a r b o n b y t h e two s t u d y po pu l a t i o n s o f Amph i bol a . Th e a s s im i l a t i o n o f m i c r o a l g a l c a r b o n wa s n o t m e a s u r ed , b u t i t w i l l b e e s t im a t ed a s t h i s i n fo rma t i o n i s r e q u i r e d i n C h a p t e r 6 . Th e r e su l t s pr e s e n t ed i n t h e pr e v i o u s c h a p te r s ugg e s t t h a t a s ig n i f i c a n t p r o po r t i o n o f t h e n e t pr od u c t i o n o f m i c r o a l g a l c a r b o n i s c o n s umed b y Amphi bo la.
5 . 1 . M E THODS A N D MATE H l ALS
1:) . 1 . ( i) A s s i m i l a t i o n t. f' t" 1 c.: i e n c y fo r lla c t e r i a l CcJ r b o n T e a s s im i l a t i o n o f b a c t e r i c1 l c a r bo n b y 1-\rnp rn b o l a w a s me a s u r e d u s i n g 1 4 C-labe ll ed b aci:. e r i a . 8 a c i:. e r i a we r e l a b e l l e o b y Avo n-He ai:.hc o te 1 C -i;luc osc a s c:Jo d <:: c.J t, o s u r r a c e s c d i 1n e n t w i l, l l '1 4 C-g l uco se . s e i i 1 1 t:; n 1., s l u r r y fro111 � .L X (. S e l ! I J (.. I i i, d i u t e d 1 : 1 w i L h fi l t, e r eu s e: cJ a\.,er ) . Ti � 111 i x t u r 1.. c, S :s 1 1 aK � 1 i n t h e d a r k fo r 4 h o u r s a t C ' a n d t 1 e n w a s h e u ci r l U c en tri f u�e d w i t h fi lt ci� eJ scc.: \.Jdt e r t. o r e1,1 o v e u n a s s 1 111 1 l a -c eu 1 4 c -g luco s e. c e n t r i f u i; i n g H a d i o a c t i v i l, y i n t r1 e s u µ e r n a t ,rn t. 1 o 1 1 o w 1 n g l, 1 1 e a c c o u n t e cJ f o r l c s s t, 11 u n 1 7o o
r
t I I e l, o t. a l i n t 1 1 e- 1 06 -
s e d imen t . I n o r d e r to p r e v e n t e x c e s s i v e l o s s o f ac t i v i t y t h r o u g h b ac t e r i al r e sp i r a t i o n , t h e s ed ime n t wa s s t o r ed o v e r n ight a t 5° C u n t i l the feed i ng e x per imen t s we r e c a r r i ed
o u t . F i f t e e n s n a il s , o f s im i l ar s i ze ( 1 8-20 mm s h e l l
l en g t h ) wh i c h had been s t a r v e d fo r 1 2 hour s , w e r e a l lowed t o f e e d f o r 20 m i n u t e s o n the sed iment c on t a i n i ng 1 4 c - l a b e l l e d b a c te r i a . The e n t i r e d i g e s t i v e t r a c t s o f t e n o f the s e s n a i l s wer e then d i s s e c t ed o u t a n d i nd iv i du a l l y d i g e st e d for 2 h o u r s at 60° C in 1 . 2 ml of a pe r c h l or i c a c i d -hyd r og e n
p yr o x i d e m i x t ur e ( Ma h i n and Lo fbe r g , 1 97 0 ) . F i f t e e n m l o f s c i n t i l l at io n cockt a i l ( 7 0% To l u en e , 30% Tr i t o n -X - l OU , � . O g/ 1 PPO ) was added to e a c h 1 . 2 ml o f d ig e s t a n d 1 4
c
a c t i v i tyc o u n ted i n a s c i n t i l l a t i o n cou n t e r . An e x t e r n a l s t a n a a r d
w a s u s ed fo r quench c o r r e c t i o n . Co un t i ng e f f i c i e n c y r anged from 6 4 - 9 5 % .
I t w a s assumed th a t the m e a n 1 4c - a c t i v i t y i n the � u t s o f t h e 1 0 d i s s e c t ed sn a i l s w a s d i rec t l y proµo r t i o t1 a l to l,11e
amoun t o f b a c t e r i a l c a r bo n i ng e s ted ( l(o fo ed , 1:J ( :, ) .
Bac t e r i cl l c a rbon e g e s t ed was e s L irn a t ed b y c o l l e c t i n g f a e c e s f r om t h e r em a i n i ng f i v e s n a i l s o v e r t h e sub seque n t l e' 110 ur per i o d a n d m e a su r i ng e g e s t ed 1 4
c .
faeces we r e co l l e c t c: ' ho u r 1 y a n d i nw1 e d i a t e 1 y p r e p c1 r e d f o r s c 1 n t i 1 1 a 1., i o n c o u 11 1., 1 n g b y t h e t e ch n i q u e d e sc r i b ed abov e . Loss o f ·1 4 C b y b a c t e r i a lr e s p i r a t i o n w a s checked b y 111on i t.o r i ng t h e r ad i o c.1 c t i v i t.y 0 1
t h e 1 a b e 1 1 e d s e d i ni e n t o v e r t h e 1 c'. -r1 p e r 1 o d t' o 1 1 o w 1 n g feed i n g . Bac t e r i a l c a r bo n a s s i m i l a t ed b y A1nph i bo l a W e.J S
c a l c u l ated a s t h d . f f e 1 e r e n c e e we e n b t th e m e a n 1 4
c ·
- 1 ng e s t e da n d t h e mean 1 4 C - e g e s t ed . The e f f i c i e n c y w i th wh i c h Amph i bo l a as s im i l ated b a ct e r i al 1 4c ( and pr e s umabl y a l l b a c te r i al c a r bon ) w a s c a l cu l a t ed a s :
1 4 c-Assimilated
1 4 C-Ing est ed X 1 0 0
5 . � . ( i i ) Da i l y Co n s umpt i o n o f Ba c t e r i al C a r b o n b y Amph i bo l a
T h e amoun t of b ac t e r i al c arbon c o n s umed d a i l y b y t h e t wo s t u d y po pu l at i o n s wa s e s t im a t ed by : a ) C a l c u l a t i ng t h e r a t e a t wh i c h Amph i b o l a i n g e s t ed b a c t e r i al ce l l s . b )
De t e r m i n i ng the a v e r ag e c a r bo n con t e n t of a s ed i m e n t b ac t er i a l ce l l . c ) U s i n g the f i gu r e s o b t a i n e d a bov e to c a l c u l at e the r a t e at wh i c h Amph i bo l a i ng e s t e d b a c t e r i a l c arbon , and then mu l t i pl i n g th i s f i 6 u r e by t h e me a s u r ed
{ { ' a s s im i l at ion e fvi c i e n c y .
5 . 1 . ( i i ) ( a ) I n g e s t i o n o f 8cJ c ·e, e r i al Ce l l s . 'fn e n o u r l y r a t e o f sed iment i ng e s t i o n by Arnµh i ll o l a popu l a t i o n s 1 n e a ch s t u d y a r e a was c a l c u l a ted b y mu l t l µl y i n � t n e r a t e o f i n g e s t i o n o f t r1 e cl v e r a g e s i z e s n a i l ( set: :::: . 1 ) b y L t l e s n c:1 1 1
d e n s i t y ( see A p p e nd i x 1 ) . · t1e a o i l y r a t e o f S\::O iwu1 t i n � e s t ion w a s then c · l c u l a t ed u s s u111 i n 6 a s i x i10 ur I'eeu i ng pe r i od ; see 2 . 2 . The r e su l t i ng f i gu r e w a s t t 1 e n rn u l t 1 µ l 1 eu b y s ed iment b a c t e r L.i l nu1n be r s . Th i s g cJ v e i:l d a i l y r o Le o r
- 1 0 8 -
ingestion of bacterial cel ls . Bacter i al cell numbers used
here were those. given as the control values
CAGES exper iments described in Chapter j ,
i n the 5. 1 . ( ii ) ( b ) Carbon Content of a Bacter i a l Cell.
Bacterial cel l volume was determined from Scanning Electron Microscope ( SEM ) observation of sediment bacter ia , and bacterial carbon content was calcul ated using a f i gure of
1 . 2 1 x 1 0-1 3 g C/um3 ( Watson et al . 1 '-;J'( '( ) . Bacte r i a
examined were from surface sediment col lected at the Avon-Heathcote Estuary study site . Sedirnenr, was f ixed in :> � glutaraldehyde i n cacody late buffer (pH 1 , b ) ana 1 wl al iquots of a 1 0 00 : 1 di lution were fi lte red onto U . t:'. urn Nuclepore fil ters. Samples were desalted whi l e sti l l in t11e fil ter apparatus by floodi ng successively for five minu tes with 2 ml each of 7 5 ) , 5 0 j and 2 � ) filtered seawa ter ana f i l tered disti l led water , and then dr awing the solut ions through by vacu um. The samples on the f il t e rs wer e 1.,t1en dehydr ated by washi ng in the same manner wi th a ser i e s of pref i l tered ethanol solutions of � � , l u � , cU � , j U ) , ? U � , 7 5 % , 85% , 9 0 % , 9 :J % and 1 U U '/4 etl1 anol . F i l 1:. e r s w e r e held overni ght in l U U ¼ ethanol befo r e c r i ticc.tl p o i n t d ry .L n g, w 1. cn
co
2 and coating with gol d. B l ank fil t e rs we r e also µrepdr eu to check fo r con tam ination of the w a s hing solu t ions .F i lter s were v i ewed with a Cdinb r i d i:;e Ste r eosc dn o u u S E M at a ma15ni f ication of :> U U U x and the u i 1ne11sions of' b act er i a 1 c e 1 1 s we r e tn ea s u r e d on the SI:: M sc r e en u s in is d
t r a n s p a r e n t r u l e . C e l l s wer e c l a s s i f i ed a s e i th e r rod s o r c o c c i , a n d me asur em e n t s we r e mad e o f the d i am e t e r o f c o c c i a n d t h e l enght a n d d i am e t e r of rods . Ce l l v o l umes w e r e c a l cu l a t e d a s sum i n g the c o c c i t o be s pher i c al a n d the r o d s t o b e c y l i nd r i c a l . Al l c e l l s i n each o b s e r v a t i o n a l f i e l d