Indirect ImpedimetryCO2 Measurement

7/28/2019 Indirect ImpedimetryCO2 Measurement

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Appl Microbio l Biotechnol (1994) 42:232-238 Springer-Verlag 1994

T . D 6 z e n c l o s M . A s c o n - C a b r e r a D . A s c o nJ . - M. L e b e a u l t - A . Pa u s sO p t im isa t ion o f the ind irec t im peda ncem et ry techn ique ;a h a n d y t e ch n i q u e f o r m ic r o b ia l g r o w t h m e a s u r e m e n t

Received: 1 December 1993/Received revision: 2 March 1994/Accepted: 11 March 1994

A b s t r a c t In t h e i n d ir e c t i m p e d a n c e m e t r y t e c h n i q u e ,t h e C O 2 p r o d u c e d d u r i n g b i o l o g i c a l a c t i v i t y r e a c t sw i t h p o t a s s i u m h y d r o x i d e ( K O H ) s o l u t io n , p r o v i d i n gn e g a t i v e c o n d u c t a n c e v a r i a t i o n . I n t h is w o r k , t h i s te c h -n i q u e w a s o p t i m i z e d , o n a r a p i d a u t o m a t e d b a c t e r i a li m p e d a n c e t e c h n i q u e ( R A B I T ) a p p a r a t u s , d e v e l o p e db y D o n W h i t l ey Sc . L t d ( U K ). T h e K O H c o n c e n t r a t io na n d v o l u m e , a s w e l l a s t h e t e m p e r a t u r e w e r e t e st e d, T h ed y n a m i c s o f C O 2 a b s o r p t i o n a n d t h e r a t i o b e t w e e n t h ec o n d u c t a n c e v a ri a t io n a n d t h e a m o u n t o f C O 2 p r o d u -c e d w e r e e x a m i n e d . A f t e r i n j e c t i o n o f CO 2 e i t h e r d i r -e c t ly i n t h e K O H s o l ut i on , o r a b o v e t h e K O H s o l ut i on ,t h e b e s t r e s u l t s w e r e o b t a i n e d w i t h a K O H v o l u m ec o r r e s p o n d i n g t o i m m e r s i o n o f t h e e l e c tr o d e s( 0 .7 - 1. 2 m l ), a n d w i t h K O H c o n c e n t r a t i o n s o f u p t o7 g / l , a l t h o u g h 5 - 6 g / 1 i s p r e f e r r e d . D e c r e a s e o f2 8 0 g S / g m o l C O 2 w a s o b t a i n e d a t 2 7 C fo r a K O Hc o n c e n t r a t i o n r a n g i n g f r o m 0 .5 t o 8 g /1 . A l l th e s e r e s u l t sw e r e s l i g h t l y a f f e c t e d b y t e mp e r a t u r e . H o w e v e r , i tw o u l d b e p r e f e r a b l e f o r t h e C O 2 p r o d u c e d t o b eb u b b l e d d i r e c tl y i n t o t h e K O H s o l ut i on , i n o r d e rt o d e c r e a s e t h e d y n a m i c r e s p o n s e o f th e s y s t e m(gaseous t rans fer ) .

I n t r o d u c t i o n

I m p e d a n c e m e t r y is n o w r e c o g n i z e d a s a p r a c ti c a l a n du s e fu l m e t h o d o f m e a s u r i n g m i c r o b i a l g r o w t h o r a c t iv -i ty . T h e f i r st i m p e d a n c e m i c r o b i o l o g y e x p e r i m e n t w a sa c h i e v e d i n 1 8 9 9 b y S t e w a r t . H e d e s c r i b e d t h e i mp e -d a n c e v a r i a t i o n s d u r i n g m i c r o b i a l m e t a b o l i s m o nb l o o d a n d s e r u m , a n d s u g g e s t e d t h a t b a c t e r i al g r o w t h sc o u l d b e m o n i t o r e d b y e l e c tr i c al m e a n s .

T. D6zenclos - M. Ascon-Cabrera - D. AsconJ.-M. Lebeault A. Pauss (~ )Division des Proc6d6s Biotechnologiques, Universit6 de Tech-nologie de Compi~gne, B.P. 649, F-60206 Compi~gne cedex, France.FAX: 33-44 20 48 13

T h e i m p e d a n c e o f a c o n d u c t i n g m a t e r i a ls s u c h a sb i o l o g i c a l me d i u m c a n b e s i mp l y d e f i n e d a s t h e r e s i s t -a n c e t o f l o w o f a n a l t e r n a t i n g c u r r e n t a s i t p a s s e st h r o u g h a c o n d u c t i n g m a t e r i a l. C o n s i d e r i n g t h e s y s t e ma s a s e ri e s c o m b i n a t i o n , t h e a p p l i c a t i o n o f a n a l t e r n a t -i n g s i n u s o i d a l p o t e n t i a l w i ll p r o d u c e a r e s u l t a n t c u r r e n tt h a t i s d e p e n d e n t o n t h e i m p e d a n c e ( Z ) o f t h e s y s t em ,w h i c h i n t u r n i s a f u n c t i o n o f it s c o n d u c t a n c e ( G ),c a p a c i t a n c e ( C ) a n d a p p l i ed f r e q u e n c y ( F ) as s h o w n i nF i g . 1 ( F i r s t e n b e r g - E d e n a n d E d e n ! 9 8 4) .I n b i o l o g y , m i c r o - o r g a n i s m s m a y p r o d u c e i o n i z e dme t a b o l i t e s d u r i n g g r o w t h ; t h e s e me t a b o l i t e s w i l lc h a n g e t h e m e d i u m ' s c o n d u c t i v it y . F ig u r e 2 s h o w s t h er e l a t i o n s h i p b e t w e e n t h e c o n d u c t a n c e v a r i a t i o n , t h ei o n i c c o n c e n t r a t i o n a n d t h e b a c t e r i a l c o n c e n t r a t i o n( F i r s t e n b e r g - E d e n a n d Z i n d u l is 1 9 8 4) . T h e d e t e c t i o nt i m e ( D T ) c o r r e s p o n d s t o t h e p o i n t w e r e t h e c o n d u c -t a n c e v a r i a t i o n r a t e i s b i g g e r t h a n a p r e d e t e r m i n e dv a l u e ( e .g . 60 g S / mi n ) . T h e D T o f t h e i n s t r u m e n t ( i .e .t i m e f o r le a s t v a r i a ti o n o f c o n d u c t a n c e a n d / o rc a p a c i t a n c e me d i a ) i s t h u s c o r r e l a t e d w i t h t h e i n i t i a lm i c r o b i a l c o n t e n t . T h e s m a l l e r t h e c o n t a m i n a t i o n , t h el o n g e r t h e d e t e c t i o n t i m e . T h i s p h e n o m e n o n c a n b eu s e d t o m e a s u r e g r o w t h o r b a c t e r i a l a c t iv i ty . I n p r a c -t i c e , m i c r o b i a l g r o w t h o r a c t i v i t y c a n b e f o l l o w e dt h r o u g h c o n d u c t a n c e , c a p a c i ta n c e , o r i m p e d a n c e v a r i -a t i o n s . T h e e x i s t i n g a p p a r a t u s f a v o u r s o n e o r o t h e r o ft h e s e p a r a m e t e r s . T h e a p p a r a t u s t h a t i s u s e d i n o u rl a b o r a t o r y is t h e r a p i d a u t o m a t e d b a c t e r i a l i m p e d a n c et e c h n i q u e ( R A B I T ) d e v e l o p e d b y D o n W h i t le y S c . L t d .( U K ) . I t p l o t s c o n d u c t a n c e a s a f u n c t i o n o f ti me ( S i ll e y1991b).T h e d i r e c t i m p e d a n c e m e t r y t e c h n i q u e is u s e d a sa t o o l f o r q u a l i t y c o n t r o l i n t h e f o o d ( E a s t e r a n dG i b s o n 1 98 9) a n d d a i r y i n d u s t r i e s ( F i r s t e n b e r g - E d e n1986) . I t was u t i l i zed , fo r ins t ance , fo r es t imat ing con-t a m i n a t i o n ( S i l v e rm a n a n d M u n o z 1 9 79 ; N o b l e e t al.1 99 1) , t o d e t e r m i n e t h e i n i ti a l c o n t a m i n a t i o n o f m i l k( G n a n a n d L u e d e c k e 1 9 8 2 ) , o r c o n t a m i n a t i o n a f t e rp a s t e u r i z a t i o n ( Bo s s u y t a n d W a e s 1 9 8 3 ) , f o r a r a p i d


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D T T i m eF i g . 2 R e l a t i o n s h i p b e t w e e n b a c t e r i a l g ro w t h , c o n c e n t r a t i o n o f i o n sa n d b a c t e r i a l c o n c e n t r a t i o n ( F i r s t e n b e r g a n d Z i n d u l i s 1 9 84 ) (LAGe n d o f la g p e r i o d , DT d e t e c t i o n t i m e )s c r e e n i n g o f m i l k m i c r o b i a l c o n t e n t ( C a d y e t al . 1 97 8),t o d e t e r m i n a t e t h e p o t e n t i a l s h e l f- li fe o f p a s t e u r i z e dw h o l e m i l k ( B i s h o p e t a l. 1 98 4) , f o r t h e d e t e c t i o n o fs o m e p a t h o g e n s , t h a t i s s o m e E n t e r o b a c t e r i a ( C o u s i n sa n d M a r l a t t 1 99 0) L a n c e f i e l d g r o u p D c o c c i i n s k im -m e d m i l k p o w d e r ( N e a v e s e t a l . 1 9 8 8 ) , S a l m o n e l l a( B l a c k b u r n 1 9 9 1 ; E a s t e r a n d G i b s o n 1 9 8 5 ) , Clos t r id iumb o t u l i n u m ( G i b s o n 1 9 8 7 ) , Lis t er ia s p p . ( P h i l l i p s a n dG r i f f it h s 1 9 89 ), a n d f o r t h e k i n e t ic s a n d i n t e r a c t i o n s o fl a c t i c a c i d b a c t e r i a ( A s c o n e t a l . 1 9 9 3 a ) .

2 3 3H o w e v e r , th e d i r e ct i m p e d a n c e m e t r y te c h n i q u e c a nn o t b e u s e d w i t h a l l m e d i a , a s t h e y n e e d t o b e o p t i m i z e dt o o b t a i n u s a b l e e l e c t r i c s i g n a l ( n e i t h e r t o o l o w o r t o oh i g h a s a l t c o n c e n t r a t i o n ) . M o r e o v e r , s o m e m i c r o -o r g a n i s m s c a n n o t b e d e t e c t e d w i t h t h i s t e c h n i q u e ,b e c a u s e t h e i r g r o w t h d o e s n o t i n d u c e a s u f f i c i e n t c o n -d u c t a n c e c h a n g e ( d u e , f o r i n s t a n c e , t o t h e f o r m a t i o n o f

u n i o n i z e d p r o d u c t s ) ( O w e n s a n d W a c h e r - V i v e r o s1 9 8 6 ) , o r b e c a u s e t h e y r e q u i r e s e l e c t i v e g r o w t h m e d i at h a t n o t f u l f i l t h e i o n i c r e q u i r e m e n t s .T h e p r o b l e m c o u l d b e o v e r c o m e b y u s i n g a n i n d i r e cti m p e d a n c e m e t r y t ec h n i q u e . T h i s t e c h n i q u e w a s f i rs td e s c r i b e d b y O w e n s e t al . (1 98 9) ; i t h a s b e e n d e v e l o p e df o r t h e R A B I T . I n t h i s t e c h n i q u e , t h e C O 2 - p r o d u c i n gm e d i u m is p l ac e d o n a s m a l l t u b e l o c a t e d a b o v e t h ee l e ct r o d e s a n d a K O H s o l u t i o n (F i g. 3 ). T h e a c i d - b a s er e a ct io n b e tw e e n C O 2 a n d K O H l ea d s t o a n i m p o r t a n tn e g a t i v e c o n d u c t a n c e c h a n g e . T h i s t e c h n i q u e c a n b eu s e d f o r a ll C O 2 - p r o d u c i n g m i c r o - o r g a n i s m s , w h a t e v e rt h e i r m e t a b o l i s m , a n d w h a t e v e r t h e c u l t u r e m e d i a( i o n i c c o n t e n t , v i s c o s i t y , s u b s t r a t e n a t u r e , e t c . ) .R a p i d d e t e c t i o n r at e s c a n b e a c h i e v e d f o r o r g a n i s m ss u c h a s Salmone l la , L i s t e r ia , y e a s t s a n d m o u l d s ( l e s st h a n 1 0 y e a s t s / g c a n b e d e t e c t e d w i t h i n 2 t o 3 d a y s ,w h i c h i s q u i c k e r t h a n c o n v e n t i o n a l t e c h n i q u e s ) ( S i l l e yf 9 9 1 a ) . T h e i n d i r e c t i m p e d a n c e m e t r y t e c h n i q u e h a sb e e n p e r f e c t ly a d a p t e d f o r t h e d e t e c t i o n o f S t a p h y l o c o c -c u s a u re u s, L . m o n o c y t o g e n e s , E n t e r o c o c c u s f a e c a li s ,Bac i l lus subt i l i s , Escher ich ia co l i , Pseudomonasaeruginosa , Aeromonas hydrophi la a n d Salmone l la s p p .( B o l t o n 1 99 0, 1 99 1). T h e d e t e c t i o n o f m a n y v e g e t a lp a t h o g e n s ( P . s yr ingae , X an thom ona s campes t r i s , e tc . ) i sp o s s ib l e w i th a s h o r t d e t e c t i o n t im e ( F r a n k e n a n d v a nd e r Z o u w e n 1 99 3) . T h i s t e c h n i q u e m a y b e a l s o u s e d fo rm e a s u r e m e n t s o f t h e C O 2 p r o d u c e d b y S t r e p t o c o c c u sthermophi lus i n p u r e a n d m i x e d c u l t u r e w i t h L a c -tobacillus ( A s c o n e t a l . u n p u b l i s h e d d a t a ) , o r f o r p o p u -l a t io n e s t i m a t i o n o f S. thermophi lus (A sco n e t a l. 1993b) .T h e d e g r a d a t i o n a c t i v it y o f a d h e r e d a n d s u s p e n d e dP s e u d o m o n a s c e l l s c u l t u r e d o n 2 , 4 , 6 - t r i c h l o r o p h e n o lw a s m e a s u r e d b y t h e i n d i r e c t i m p e d a n c e m e t r y t e c h -n i q u e ( A s c o n - C a b r e r a a n d L e b e a u l t 1 99 4).

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m e n t a l c o n d i t i o n s f o r C O 2 d e t e c t io n w i t h a R A B I Ts y s t e m . W e a l s o f oc u s o n t h e r e la t i o n s h i p b e t w e e n t h ea b s o r b e d C O 2 a n d t h e c o r r e s p o n d i n g c o n d u c t a n c ec h a n g e , e x p r e s s e d i n m i c r o s i e m e n s p e r ~ t m ol e o f C O 2 .

Mat er ia ls an d m et h o d s

ReactantsAll reactants (hydrochloric a cid, sodium carbon ate, K OH ), were ofanalytical grade (Prolabo, France). CO2 was of pure quality grade(Air Liquide, France). The K OH solutions were kept at 4C betweenthe experiments.

Impedance measurementsThe conductances were monitored using a RABIT developed byDo n W hitley Sc. Ltd. (UK). The apparatus can handle from 1 to 16modules containing 32 electrode tubes that can each be analysedsimultaneously. This is a completely multiworker system. The tem-perature range was up to 4C above ambient temperature up to55 + 0.005C. The R AB IT system may b e used in the classical directimpedancemetry mode or the indirect impedancemetry mode.

Experimental proceduresIndirect impedancemetry was calibrated and optimized through themon itoring of conductance change of the K O H solution with CO2absorption into this solution. Conductance changes were recordedduring 24 h 6-rain intervals. In practice, a K O H solution was placedin the electrode tubes. These tubes were tightly closed with rubberbungs, and placed in the RA BIT incu bato r block. After stabilisation(1 h), a needle was pricked thro ugh the rubber bungs. Tw o com para-tive procedures we re then followed:1. Exce ss CO2 was bubbled into the K O H solution, and thenconverted into carbonate; the final quantity of COz absor bed wasdetermined by the initial quantity of KOH (1 mol CO2 reacts with2 m o l O H - ) ,2. Known CO2 volumes were injected through the rubber bungsdirectly into the KOH solution.

The K OH concentration (1 to 10 g/l), the K O H volume (0.3 to5 ml), the test temp eratu re (27-57C) and the co rresponding dy-namics w ere optimized. The initial conductance value (BS), themagnitude change (BS), and the relationship between the latter a ndthe absorbed CO2 (gS/gmol), were measured and calculated.

Microbial growthG row th o f E. coli was performed under sterile conditions, in parallelin the impedancem etry tubes (3 ml as final volume) and in no n-stirred erlenmeyer flasks (100 ml final volume) a t 3 0C. The culturemedium contained 10 g tryptone, 5 g mea t extract, and 5 g sodiumchloride per litre (Nutrient Broth, Biokar, France). The inoculumconcentration was 4.105 colony-forming units (cfu)/ml, as deter-mined b y serial dilution on petri dishes filled with the sam e growthmedium added w ith 1.5% agar.

Resul ts

O p t i m a l K O H c o n c e n tr a t io nF i g u r e 4 s h o w s a t y p i c a l c u r v e o f t h e K O H c o n d u c -t a n c e w h e n e x c es s C O 2 w a s b u b b l e d . T h e c o n c e n t r a -t i o n w a s i n c r e a s e d f r o m 1 g / l to 1 0 g /1 w i t h 1 m l K O Hs o l u t io n . T h e s e f ir s t e x p e r i m e n t a t i o n s w e r e c a r r i e d o u ta t t h e l o w e r s t a b l e t e m p e r a t u r e , i . e . 2 7 C . T h e i n i t i a lc o n d u c t a n c e a n d t h e m a g n i t u d e c h a n g e a f te r C O 2 a d -d i t i o n i n c r e a s e d l i n e a r l y u p t o 8 g /1 ( F ig . 5 a) . T h e yv a r i e d f r o m 4 , 2 0 0 p S t o 3 2 ,7 0 0 p S , a n d f r o m 2 , 5 0 0 p S t o2 0 , 5 0 0 g S , r e s p e c t i v e l y .

A b o v e 8 g/1 t h e i n it i al c o n d u c t a n c e o f K O H s o l u t i o nw a s c o n s t a n t ( 3 2 ,7 0 0 B S) . T h i s v a l u e r e p r e s e n t e d t h em a x i m u m r e a d ab l e i m p e d a n c e b y th e R A B I T . T o e s-t a b l i s h th e r e l a t i o n s h i p b e t w e e n t h e c o n d u c t a n c e v a r i -a t i o n a n d t h e a b s o r b e d C O 2 , i t w a s a s s u m e d t h a t a l lK O H m o l e c u l es h a d r e a c te d w i th t h e e x ce s s C O > T h ea b s o r b e d C O 2 w a s th u s c a l c u la t e d v ia t h e a m o u n t o fi n t r o d u c e d K O H a n d t h e s t o i c h i o m e t r i c co e f fi c ie n t(1 m o l C O 2 r e a c ts w i t h 2 m o l K O H ) . F i g u r e 5 b s h o w e dt h a t t h is c o e f f ic i en t w a s c o n s t a n t f o r a c o n c e n t r a t i o nr a n g i n g b e t w e e n 1 a n d 8 g /1 . I t w a s e q u a l t o2 8 0 + 7 p S / g m o l C O 2 a t 2 7 C . A b o v e 8 g /1 t h i s c o e ff i -c i e n t w a s d e c r e a s e d .

T h e v e r y g o o d r e p r o d u c i b i l i t y o f t h is t e c h n i q u e m u s tb e e m p h a s i z e d : a ll t h r e e p a r a m e t e r s o b s e r g e d p r e s e n t e dv e r y s h a r p s t a n d a r d d e v i a t io n s .

O p t i m a l K O H v o lu m eT h e v o l u m e o f a 6 g K O H / 1 s o l u t i o n w a s i n c r e a s e df r o m 0 .3 u p t o 5 m l a t 2 7 C . T h e i n i ti a l c o n d u c t a n c ea n d t h e m a g n i t u d e o f c h a n g e i n c r e a s e d l i n e a r l y w i t hv o l u m e a u g m e n t a t i o n u p t o 1 .2 m l . F r o m 1.5 m l t h o s ev a l u e s w e r e c o n s t a n t a t 3 1 , 5 0 0 g S a n d 2 0 , 0 0 0 g S( F i g . 6 a ) r e s p e c t i v e l y . F u r t h e r m o r e , t h e c o e f f i c i e n t b e -t w e e n t h e c o n d u c t a n c e c h a n g e a n d t h e a b s o r b e d C O 2w a s m a x i m a l f o r a v o l u m e r a n g e o f 0 . 7 - 1 . 2 m l (F ig . 6 b) .

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1 5 0 , j , L ,0 2 4 6 8 I 0 1 2

b K O H c o n c e n t r a t i o n ( g/ I)Fig. 5 a Changes in the initial conductance (B) and in conductancechange (A) as a function of the K OH concentration, w hen excessCO2 was bubbled into K OH (27C, 1 ml KO H). b Variation in theconductance chan ge to absorbed CO 2 ratio as a function of theKO H concentrat ion, when excess COz was bubbled into K OH(27C, 1 ml K OH )

T h i s c o e f fi c i en t w a s C o n f i r m e d b y i n j e c t i o n o f v a r i -a b le C O 2 v o l u m e f o r 1 .1 m l o f 6 g K O H / 1 a t 3 7 C( F ig . 7 ). T h e c o n d u c t a n c e c h a n g e l i n e a r l y i n c r e a s e dw i t h t h e C O 2 v o l u m e , p r o v i d i n g a c o ef f i ci e n t o f2 5 7 p S / p m o l . T h e d i f f e r en c e b e t w e e n t h e p r e v i o u s c o e f -f i c i e n t ( 2 8 0 g S / p m o l ) a n d t h e l a t t e r i s d u e t o t h e d i f f e r -e n t t e m p e r a t u r e a n d K O H v o l u m e us ed .

T e m p e r a t u r e e f f e c tI n 1 m l o f a 6 g K O H / 1 s o l u t i o n , o u r t h r e e p a r a m e t e r sw e r e m o n i t o r e d w i t h b u b b l i n g e x ce ss C O 2 i n t h e r a n g e2 7 - 4 7 C . F i g u r e 8 a s h o w s t h a t t h e i n i ti a l c o n d u c t a n c ew a s s e n s i t i v e t o t e m p e r a t u r e v a r i a t i o n ( a b o u t2 5 0 ~ t S / C ) , a s w e l l a s t h e c o n d u c t a n c e c h a n g e , a n d t h ec o n d u c t a n c e t o C O z c o e f f i c i e n t ( F i g . 8 b ). A l l t h r e e i n -c r e a s e d b y a b o u t 1 . 5 % p e r C .

D y n a m i c r e s p o n s e o f t h e c o n d u c t a n c e v a r i a t i o n sA s i n d i c a t e d b y F i g. 3, t h e C O g - p r o d u c i n g s o l u t i o n i su s u a l l y l o c a t e d i n a s m a l l t u b e p l a c e d a b o v e t h e e l e c -t r o d e s . T h e C O g p r o d u c e d a p p e a r s i n t h e g a s p h a s e ,w h i c h m u s t d i s p l a ce t h e i n i t ia l g a s p h a s e b e f o r e r e a c -

4 0 0 0 0

235

30000 ,

20000 ,

10000,

a 0 ,

K =

i "

e

i i i i 1

30 0 -250 -200 -15 0 -

0i i i i i

1 2 3 4 5K O H v o l u m e ( m l )

Fig. 6 a Changes in the initial conductance (, , ) and in conductancechange (a) as a function of the K O H volume, when excess COz wasbubbled into K OH (27C, 6 g KO H/1). b Variation in the conduc-tance change to absorbed CO2 ratio as a function of the KOHvolume, when excessCO2 was bubbled into K OH (27C, 6 g KOH /1)

12000 , ~ z , , ,100008000

=~ 60004000

L ) 2 0 0 0

i I , I , I , I i I , I ,

5 10 15 20 25 30 35 40C O 2 ( g m o l )

F i g . 7 C o n d u c t a n c e v a r i a t i o n o f 1 .1 m l o f a 6 g K O H / 1 s o l u t i o n f o rincreasing amount of gaseous CO2 volu met i n g w i t h th e K O H s o l u t i o n . T o m e a s u r e t h i s g a s t r a n s -f e r, d i f f er e n t e x p e r i m e n t a l m e t h o d s w e r e p u r s u e d . T h e yw e r e c o m p a r e d t o t h e u s u a l b u b b l i n g o f e x ce s s C O 2i n t o K O H s o l u t i o n ( 6 g /l ). F i r s t ly 1 m l o f 0. 26 8 m m o lc a r b o n a t e / 1 w a s s e t i n t h e i n n e r t u b e . E x c e s s h y d r o c h l o -r i c a c i d w a s a d d e d o n c e , t o c r e a t e s u d d e n C O 2 p r o d u c -t i o n . S e c o n d l y , ex c e s s g a s e o u s C O 2 w a s i n j e c t e dt h r o u g h t h e n e e d le a b o v e t h e K O H s o lu t io n T h i rd l y ,k n o w n C O 2 v o l u m e s w e r e i n je c t ed t h r o u g h t h e r u b b e rb u n g s d i r e c tl y i n t o th e K O H s o l ut i on .


5/7

3500030000-250o0-=:1.20000-

"~ 15000-10000-5000 -

a . 0

38 0 i i

. ~ 3 6 0~ 340

32 0~ N 300

28 026 0

2 3 6

~ , . - - . . . . . . . . . . . . . . . ~ . . . . . . .

. . . . . t ~ . . . . . . . ' fi ' . . . . . . . . . . . . . . . . . " . . . . . .

100-~ " 8 0 -' ~ 6 0 -

4 0 -2 0 -

0 - ~ i i i i

0 50 100 150 200T i m e ( m i n )

25 0

Fig. 9 Reaction achievement between KOH and COz as a functionof time, for direct injection of CO2 into KOH solution (,), COzabove the KOH solution (c~), and for CO2 production in the centraltube when HC1 is injected in to NazCO3 solution (n)

40

I , I 3025 35 45

T e m p e r a t u r e ( C ) ~ 20~ 7F i g . 8 a C h a n g e s i n t h e i n i t i al c o n d u c t a n c e ( m ) a n d i n c o n d u c t a n c e ~ 10

c h a n g e (z x) a s a f u n c t i o n o f t e m p e r a t u r e , w h e n e x c e ss C O 2 w a sb u b b l e d i n t o K O H (1 m l o f 6 g K O H / 1 ) . b V a r i a t i o n i n th e c o n d u c -t a n c e c h a n g e t o a b s o r b e d C O 2 r a t i o a s a f u n c t i o n o f t e m p e r a t u r e ,w h e n e x c e s s C O 2 w a s b u b b l e d i n t o K O H (1 m l o f 6 g K O H / 1 )

T h e r e s u l t s o f F i g. 9 d e m o n s t r a t e t h a t t h e C O 2 m a s st r a n s f e r w a s n o t i n s t a n t a n e o u s . A b o u t 2 5 _+ 3 a n d2 4 5 _ + 1 6 m i n w e re n e e d e d t o o b t a i n , r e s p e c t i v e l y , 5 0 %a n d 9 5 % o f t h e to t a l c o n d u c t a n c e c h a n g e w h e n t h ep r o d u c e d C O 2 d o e s n o t di r ec t ly b u b b l e in t o t h e K O Hs o l u t i o n ( a t 2 7 C w i t h 6 g K O H / 1 ) , H o w e v e r , w h e na r e l a ti v e l y l a r g e fl u x o f C O 2 a r r i v e d a b o v e t h e K O Hs o l u t i o n , t h e s e d e l a y s w e re l e ss i m p o r t a n t , i .e . 1 2 + 4a n d 2 4 m i n fo r , r e s p e c ti v e ly , 5 0 % a n d 9 5 % . W h e n C O 2a r r i v e d d i r e c tl y i n t o t h e K O H s o l u t io n , t h e re a c t i o nw a s i m m e d i a t e , t h a t i s l e s s t h a n t h e l e a s t i n t e rv a l o f' ti m e r e c o rd i n g .

G r o w t h m e a s u r e m e n tA s a n e x a m p l e , E . c o l i g r o w t h w a s p e r f o r m e d a t 3 0 C i np a r a l l e l i n t h e i m p e d a n c e m e t r y t u b e s a n d i n n o n - s t i r -r e d e r l e n m e y e r f l a s k s . G r o w t h w a s f o l l o w e d b y o p t i c a ld e n s i t y a t 5 4 0 n m a n d b y c o n d u c t a n c e c h a n g e . A ne x c e l l e n t c o r r e l a t i o n w a s fo u n d , a s i l l u s t r a t e d i nF i g . 10 . T h e t o t a l c o n d u c t a n c e c h a n g e c o r r e s p o n d s t oa C O 2 p r o d u c t i o n o f 33 .3 g m o l , u s i n g 3 05 S / m o l a scoeff ic ien t .I t i s e m p h a s i z e d t h a t t h e u s u a l o p t i c a l d e n s i t ym e a s u r e m e n t s w e r e m o r e t i m e - c on s u m i n g , b u t a r e a l som o r e s u b j e c t to e x p e r i m e n t a l e r r o r s, a s s h o w n b y t h e

12000,1 0 0 0 0

~ 8 0 0 06000

.~ 40002 0 0 0

0 1

i i i i

'' v - - - ' - ~

1.00,8 :0 .60.4 .~0.2 ",~00 20 40 60 80

T i m e ( h )Fig. 10 Escherichia co li growth curve mea sured with the indirecti m p e d a n c e m e t r y t e c h n i q ue (continuous l ine) , a n d b y o p t i c a l d e n s i t ya t 5 4 0 n m (circles a n d d o t t e d l i n e )d i s p e r s i o n o f t h e e x p e r i m e n t a l v a l u e s a l o n g t h e g r o w t hc u r v e , i n c o n t r a s t t o t h e c o n d u c t a n c e v a l u e s , w h i c hw e r e a u t o m a t i c a l l y r e c o r d e d .

D i s c u s s i o n

T h e i n d i r e c t i m p e d a n c e m e t r y t e c h n i q u e c a n b e u s e d f o ra n y C O 2 - p r o d u c i n g m e d i u m . W i t h t h e R A B I T s y s t e m ,t h e o p t i m a l c o n d i t i o n s o f t h is t e c h n i q u e w e r e d e t e r -m i n e d a s f o ll o w s : a K O H c o n c e n t r a t i o n o f 6 - 7 g/1 a n da K O H v o l u m e o f 0 . 7 - 1. 2 m l. T h e K O H c o n c e n t r a t io ni s l i m i t e d b y t h e e l e c t r ic d a t a o f t h e R A B IT . A 3 2,76 8 p Sc o r r e s p o n d s t o a r e s i s t a n c e o f 30 .5 f~, t h e m a x i m a lr e a d a b l e v a lu e b y t h e R A B I T . T h e K O H v o l u m e c o r-r e s p o n d s t o t h e v o l u m e d e f i n e d b y t h e e l e c t r o d e h e ig h t .F o r K O H v o l u m e s g r e a t e r t h a n 1 .2 m l , t h e " e x c e d e n t "K O H i s n o t " 's ee n " b y t h e e l e c tr o d e . A n e f fe c ti v e m a x i -m a l v o l u m e i s t h u s d e f i n e d b y t h e i n n e r t u b e d i a m e t e ra n d t h e e l e c t r o d e h e i g h t . T h e o p t i m a l o p e r a t i n g c o n d i -t i o n s u s i n g R A B I T a r e s u m m a r i z e d i n T a b l e 1 .A s s h o w n i n t h e p r e v i o u s f i g u re s , t h e s e v a l u e s a r eh i g h l y i : e p ro d u c i b l e . O n t h e o t h e r h a n d , w i t h i n t h e


6/7

T a b l e 1 O p e r a t i n g c o n d i t i o n sfo r ind i rec t impedancem e a s u r e m e n t s w i t h t h e r a p i da u t o m a t e d b a c t e r i a l i m p e d a n c etechn ique (RABIT) underop t im al cond i t ions (1 ml o fg K O H / 1 )

P a r a m e t e r sIn i t i a l conduc tan ce (mS)C o n d u c t a n c e c h a n g e ( m S)C o n d u c t a n c e c h a n g e t o

absorb ed CO2 (S /mol )

T e m p e r a t u r e ( C )27 30 37 40 47

24.50 25.25 26.05 27.05 31.2015.00 16.35 17.15 17.15 " 19.60

280 305 320 320 367

23 7

T a b l e 2 C a c u l a t e d r e a d a b l e CO 2 p r o d u c t i o n b y t h e R A B I T a n des t imated CO2 prod uc t ion by d i f fe ren t b io log ica l r eac t ions . Thees t ima t ions were base d on the fo l lowing hypo thes i s : ( i) 3 ml reac t ionvo!ume; ( i i) s to ich iome t r ic r eac t ions and comp le te t r ans fo rmat ionsof ~he subs t ra tes in to CO2, the o the r p roduc t s and m ic ro -organ ism s(gt 'owth yield of , respetively, 0 .5, 0 .1, and 0.1 for th e a erob ic cultureand the e tha no l ic and m ethanogen ic fe rmenta tions ) , w i th in the usua l

temp era tu re range o f these th ree fe rmenta t ions ; ( ii i) fo r the ae rob icfe rmenta t ion , the CO2 i s exc lusive ly de r ived f rom the d i s so lved O2 ,i.e. 8.25, 8.08, 7.81, 7.00, mg/1, respec tively , at 25, 27, 30, 37C ; (iv) inthe e than o l ic f e rmenta t ion , 10 g /1 o f g lucose a re t r ans fo rmed in toe thano l , CO2 and yeas t s ; (v ) 3 g /1 o f ace ta te a re t r ans fo rme d in tom e t h a n e a n d C O 2 d u r i n g t h e m e t h a n o g e n i c fe r m e n t a t i o n

T e m p e r a t u r e ( C )P a r a m e t e r s 25 27 30 37 47M i n i m u m r e a d a b l e C O 2 p r o d u c t i o nM a x i m u m r e a d a b l e G O 2 p r o d u c t i o nM a g n i t u d e o r d e r o f CO 2 p r o d u c t i o n o f :ae rob ic cu l tu reehano l ic f e rmenta t ionmethanogen ic fe rmenta t ion

(gm ol) 0:036 0.036 0.033 0.031 0.028(gl) 0.89 0.89 0.82 0.79 0.74(gmol) 53.8 53.6 53.6 53.6 53.4(gl) 1315 1320 1330 1365 1400(I.tmol)(btmol)(gmol )

0.39 0.38 0.37 0.33300 300 300135 135

2 7 - 4 7 C r a n g e , t h e y a r e s l i g h t ly a f fe c t e d b y t e m p e r -a t u r e , a b o u t a 1 . 5 % v a r i a t i o n f o r a I C i n c r e a s e . T h i si n d ic a t e s t h a t C O 2 p r o d u c t i o n c a n b e d i r e c t ly q u a n t i-f ie d b y t h e c o n d u c t a n c e c h a n g e o f t h e C O 2 a b s o r b e n ts0 lu t ion .A s s u m i n g , f i rs t, th a t t h e c o n d u c t a n c e p a r a m e t e r s a r et h e v a l u e s r e p o r t e d i n T a b l e 1 , a n d s e c o n d l y t h a t t h em i n i m u m r e a d a bl e c o n d u c t a n c e ch a n g e b y th e R A B I Ti s a b o u t 1 0 b tS , t h e n t h e m a x i m u m a n d l e a s t a b s o r b a b l eC O 2 c a n b e c a l c u l a t e d f o r d i f f e r e n t t e m p e r a t u r e s( T a b l e 2 ). I n T a b l e 2 , th e s e r e s u l t s a r e c o m p a r e d t ot h r e e t h e o r e t i c a l e s t i ma t e d CO g p r o d u c t i o n s o f d i ff e r -e n t c a s e - s t u d y b i o l o g i c a l me d i a . T h e d e t a i l s a r e g i v e n int h e t a b l e l e g e n d . ~A c c o r d in g to T a b l e 2 , a n d w i t h t h ep r e s e n t c a p a c i t i e s , t h e R A B I T c a n b e s u c c e s s fu l l y u s e dt o me a s u r e d a e r o b i c g r o w t h o r a c t i v it y . Co n v e r s e l y , t h ep o t e n t i a l C O 2 p r o d u c t i o n o f a n a e r o b i c f e r m e n t a t i o n si s t o o g r e a t f o r i t s c a p a c i t y . H o w e v e r , e ' e n : i n t h o s ef e r m e n t a t i o n s t h e R A B I T c a n b e u s e d to h a n d l e t h ed y n a m i c s o f m e t h a n o g e n i c f e r m e n t a t i o n . F o r i n s t a n c e,i t c a n a c c u r a t e l y fo l l o w CO 2 p r o d u c t i o n u n t i l t h e s a t u -r a t i o n o f K O H ( i. e. 4 0 % o f t h e t o t a l p o t e n ti a l p r o d u c -t ion).T h e C O 2 d y n a m i c t r a n s fe r i n t h e tu b e w a s s h o w nv e r y i mp o r t a n t i n t h i s s t u d y . T h e c l a ss i c a l d e s i g n o f t h ei n d i r e c t i m p e d a n c e m e t r y t e c h n i q u e ( as i n d i c a t e d i nF i g . 2 ) c a n i n d u c e a p e r v e r s e e f fe c t. T h e r e s u l ts s h o w ni n : F ig . 9 d e m o n s t r a t e t h a t t h e C O 2 m a s s t r a n sf e r:W o uld n o t b e i n s t a n t a n e o u s , a n d t h a t t h e d y n a m i c s o f

fas t bac te r i a l C O 2 p r o d u c t i o n c a n b e m a s k e d b y t h i sma s s t r a n s f e r .T h e E. co l i g r o w t h c u r v e s h o w s t h a t t hi s m e t h o d c a nb e a c t u a l l y u s e d f o r g r o w t h m e a s u r e m e n t . O t h e rg r o w t h e x a m p l e s m e a s u r e d w i t h t h is t e c h n i q u e C a n b ef o u n d i n A s c o n - C a b r e r a a n d L e b e a u l t ( 1 9 9 4 ) , A s c o ne t al . ( 19 93 b) , F r a n k e n a n d v a n d e r Z o u w e n (1 99 3) a n dO w en s et , al . (1989).Acknowledgement The au th ors g rea t ly acknow ledge M r J . P . Duva l( from AES com pany , F ren ch dea le r o f the R ABIT) fo r h is p rec ioushe lp and adv ice .

ReferencesA s c o n - C a b r e r a M , L e b e a u l t J M ( 1 9 9 4 ) D e g r a d a t i o n a c t i v it y o fa d h e r e d a n d s u s p e n d e d Pseudomonas cells cul tured on 2,4,6-t r i ch lo ropheno l , measured by ind i rec t impedancemen(y . ApplE n v i r o n M i c r o b i o l i n p r e s sAscon D, Coche t N , Lebeau l t JM (1993a) Kine t ic s and in te rac t ionsof l ac t i c ac id bac te r ia measured by impedancemet ry . In : ( eds )VI th E urope an C ongress on Bio techn01ogy, F lo rence , I t a ly ,13-17 June 1993. 1 , MO 223A s c o n D , C o c h e t N , L e b e a u l t J M ( 19 9 3b ) P o p u l a t i o n e s t i m a t i o n o fStreptococcus thermophilus by ind i rec t im pedancem et ry . In : ( eds )Fo rum or Appl ied Bio techno logy , Gen t (Be lgium), 30 Sep tem-ber -01 .Oc tober 1993 (in p ress )B i s h o p J R , .W h i t e C H , F i r s t e n b e r g - E d e n R ( 1 98 4 ) R a p i d i m p e d i -met r ic m eth od fo r de te rm in ing the po ten t ia l she l f- li fe o f pas -t e u r i z e d w h o l e m i l k . J F o o d P r o t 4 7 : 4 7 1 - 4 7 5


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238Blackburn C (1991) Detection of S a l m o n e l l a in foods using impe-dance. Eu Food Drink Rev Winter: 35-40Bolton FJ (1990) An investigation of indirect conductimetry fordetection of some food-borne bacteria. J Appl Bacteriol69:655-661.Bolton FJ (1991) Conductance and impedance methods for detect-ing pathogens. In: Vaheri A, Tilton RC, Balows A (eds) Rapidmethods and automation in microbiology and immunology.

Springer-Verlag, Berlin, pp 176-181Bossuyt RG, Waes GM (1983) Impedance measurements to detectpost-pasteuriza tion cont amina tion of pasteurized milk. J Food Prot 46:622-624Cady P, Hardy D, Martins S, Dufour SW, Kraeger SJ (1978) Auto-. mated impedance measurements for rapid screening of milkmicrobial content. J Food Prot 4l:277-283Cousins DL, Marlatt F (1990) An evaluation of a conductanacemethod for the enumeration of Enterobacteriaceae in milk.J Food Prot 53:568-570Easter MC, Gi bson DM (1985) Rapid and automated detection ofS a l m o n e l l a by electrical measurements. J Hyg 94:245-262Easter MC, Gibson DM (1989) Detection of microorganisms byelectrical measurements. Rapid methods in food microbiology.Prog Ind Microbiol 26:57-100Firstenberg-Eden R, Eden G (1984) Impedance microbiology. Wiley,New YorkFirstenberg-EdenR (1986) Food-borne microorganisms and theirtoxins. In: Pierson MD, Stern NJ (eds) Developing methodology.Dekker, New York, pp 129-144Firstenberg-Eden R, Zindulis J (1984) Electrochemical changes inmedia due to microbial growth. J Microbiol Methods 2:103-115Franken AAJM, Zouwen PS van der (1993) Direct and indirectconductimetry for identification and detection of pl ant patho-~ enic bacteria. J Appl Bacteriol 74:234-242

Gibson AM (1987) Use of conductance measurements to detectgrowth of Cl o s t r i d i u m b o t u l i n u m in a selective medium. Lett ApplMicrobiol 5:19-21Gnan S, Luedecke LO (1982) Impedance measurement in rawmilk as an alternative to the standard plate count. J Food Prot45:4-7Neaves P, Waddell M J, Prentice GA (1988) A medium for thedetection of Lancefield Group D cocci in skimmed milk powderby measurement of conductivity changes. J Appl Bacteriol65:437-448Noble PA(Ashton E, Davidson CA, Albritton WL (1991) Hetero-trophic'plate counts of surface water samples by using impedancemethods. Appl Environ Mierobiol 57:3287"3291Owens JD; Wacher-Viveros MC (1986) Selection of pH buffers foruse in conductimetric microbiological assays. J Appl Baeteriol60:395-400Owens JD, Thomas DS, Thompson PS, Timmerman JW (1989)Indirect conductimetry: a novel approach to the conductimet ricenumeration of microbial populations. Lett Appl Microbiol9:245-249Phillips JD, Griftiths MW (1989) An electrical method for detectingL i s t e r i a spp. Lett Appl Microbiol 9:129-132Silley P (1991a) Rapid automated bacterial impedance technique(RABIT). Soc Gen Microbiol Quarter 18:48-52

Silley P (1991b) "RABIT" in the food industry. Nutr FoodSci 131:2-3Silverman MP, Munoz EF (1979) Automated electrical impedancetechnique for rapid enumeration of fecal coliforms in effluentsfrom sewag e treatment plants. Appl Environ Microbiol37:521-526Stewart G N (1899) The changes produced by the growth of bactriain the molecular concentration and electrical conductivity ofculture media. J Exp Med 4:235-243

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