Fundamentals and Applications of Chemical Sensors - American

20 Microbial Sensors for Process and Environmental Control

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 18, 2016 | http://pubs.acs.org Publication Date: May 29, 1986 | doi: 10.1021/bk-1986-0309.ch020

I. Karube Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 227, Japan

Many organic and inorganic compounds are present in fermentation process and waste waters. Rapid and sensitive on-line monitoring of these compounds is required. Microbial sensors composed of immobilized microorganisms and an electrochemical device are suitable for the determination of these compounds. A microbial sensor for glucose consisted of immobilized whole cells which utilize mainly glucose and an oxygen electrode. The steady state current obtained depended on the concentration of glucose. The microbial sensor could be used for the determination of glucose in fermentation media. Furthermore, alcohols, acetic acid, ammonia, and methane were determined by microbial sensors based on the same principle. On the other hand, potentiometric microbial sensors have been developed for fermentation process. A microbial sensor for glutamic acid consisted of immobilized Escherichia coli having glutamate decarboxylase activity and a carbon dioxide gas-sensing electrode. The concentration of glutamic acid in some fermentation broths were determined by the sensor. Cephalosporines were also measured by a microbial sensor system. Furthermore, microbial sensors were applied to the determination of organic pollution. A microbial sensor for BOD consisted of Trichosporon cutanium and an oxygen electrode. The sensor could be used for a long time for the estimation of BOD. Nitrite and mutagens were also determined by microbial sensor systems. Many u s e f u l compounds such as amino a c i d s , n u c l e i c a c i d s , a l c o h o l s , v i t a m i n s , a n t i b i o t i c s , f o o d s , e t c . a r e produced i n ferment a t i o n i n d u s t r i e s . Furthermore, many o r g a n i c and i n o r g a n i c compounds are p r e s e n t i n waste waters. The d e t e r m i n a t i o n o f these compounds i s r e q u i r e d f o r c o n t r o l o f f e r m e n t a t i o n and environment. A n a l y s i s of these compounds can be done by s p e c t r o p h o t o m e t r i c methods. Howe v e r , c o m p l i c a t e d procedures and l o n g r e a c t i o n times a r e r e q u i r e d , 0097-6156/ 86/ 0309-0330S06.00/ 0 © 1986 American Chemical Society

In Fundamentals and Applications of Chemical Sensors; Schuetzle, Dennis, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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Furthermore, samples are not usually o p t i c a l l y c l e a r . O n the other hand, e l e c t r o c h e m i c a l m o n i t o r i n g o f these compounds can be done by b i o s e n s o r s . Many r e p o r t s on enzyme s e n s o r s have been p u b l i s h e d f o r c l i n i c a l and food a n a l y s e s ( 1 , 2 ) . The enzyme sensors c o n s i s t e d o f the i m m o b i l i z e d enzyme and an e l e c t r o c h e m i c a l d e v i c e . However, enzymes are u n s t a b l e and e x p e n s i v e . T h e r e f o r e , the enzyme sensors are not s u i t a b l e f o r i n d u s t r i a l p r o c e s s and e n v i r o n m e n t a l c o n t r o l . On the o t h e r hand, m i c r o b i a l s e n s o r s composed of i m m o b i l i z e d l i v i n g microorganisms and an e l e c t r o c h e m i c a l d e v i c e have been developed f o r p r o c e s s and e n v i r o n m e n t a l c o n t r o l ( 3 - 5 ) . L i v i n g microorganisms are used as a m o l e c u l a r r e c o g n i t i o n element i n t h i s sensor. A s s i m i l a t i o n of o r g a n i c compounds by microorganisms i s monitored by the r e s p i r a t i o n a c t i v i t y change or the amount of m e t a b o l i t e s , produced, which can be measured d i r e c t l y w i t h v a r i o u s e l e c t r o d e s . The m i c r o b i a l s e n s o r s a r e v e r y s t a b l e and can be used f o r a l o n g t i m e . M i c r o b i a l s e n s o r s f o r f e r m e n t a t i o n and e n v i r o n m e n t a l c o n t r o l are d e s c r i b e d i n t h i s chapter. M i c r o b i a l Sensors

f o r Fermentation Process

Glucose s e n s o r . The d e t e r m i n a t i o n o f g l u c o s e i s i m p o r t a n t f o r p r o c e s s c o n t r o l . A s s i m i l a t i o n o f g l u c o s e by microorganisms can be determined from the r e s p i r a t i o n a c t i v i t y by u s i n g an oxygen e l e c t rode. T h e r e f o r e , a m i c r o b i a l sensor f o r g l u c o s e c o n s i s t e d o f immobil i z e d whole c e l l s which u t i l i z e m a i n l y g l u c o s e and an oxygen e l e c t rode(6). I m m o b i l i z e d whole c e l l s of Pseudomonas f l u o r e s c e n s were used f o r the g l u c o s e s e n s o r . The m i c r o b i a l sensor has been a p p l i e d to the c o n t i n u o u s d e t e r m i n a t i o n of g l u c o s e i n molasses. A schematic diagram o f the m i c r o b i a l sensor i s i l l u s t r a t e d i n F i g u r e 1. The sensor c o n s i s t e d o f double membranes of which one l a y e r was the b a c t e r i a - c o l l a g e n membrane ( t h i c k n e s s 40yum) , the o t h e r an oxygen permeable T e f l o n membrane ( t h i c k n e s s 27jam), an a l k a l i n e e l e c t r o l y t e , a p l a t i n u m cathode, and a l e a d anode. The double membrane i s i n d i r e c t c o n t r a c t w i t h the p l a t i n u m cathode and i s t i g h t l y secured w i t h rubber r i n g s . The m i c r o b i a l sensor was i n s e r t e d i n t o a sample s o l u t i o n o f g l u c o s e i n water and the sample s o l u t i o n was s a t u r a t e d w i t h d i s s o l v e d oxygen a t i t s p a r t i a l p r e s s u r e i n a i r and s t i r r e d m a g n e t i c a l l y w h i l e measurements were t a k e n . The temperature o f the sample s o l u t i o n was m a i n t a i n e d a t 30+0.1C. The c u r r e n t was measured by a m i l l i a m m e t e r . The c u r r e n t a t time z e r o was t h a t o b t a i n e d i n a sample s o l u t i o n s a t u r a t e d w i t h d i s s o l v e d oxygen. The b a c t e r i a began t o u t i l i z e g l u c o s e i n a sample s o l u t i o n when the sensor was p l a c e d i n i t . Then, consumption of oxygen by the b a c t e r i a i n the c o l l a g e n membranen began. Consumption o f oxygen by the bacteria caused a decrease i n d i s s o l v e d oxygen around the membrane. As a r e s u l t , the c u r r e n t of the s e n s o r markedly decreased w i t h time u n t i l a steady s t a t e was reached. The steady s t a t e i n d i c a t e d t h a t the consumption of oxygen by the b a c t e r i a and the d i f f u s i o n of oxygen from the s o l u t i o n t o the membrane were i n e q u i l i b r i u m . The s t e a d y s t a t e c u r r e n t was a t t a i n e d w i t h i n 10 min a t 3Cfc. The steady s t a t e c u r r e n t depended on the c o n c e n t r a t i o n of g l u c o s e . 'Current' means the steady s t a t e current hereafter. A l i n e a r r e l a t i o n s h i p was observed between the c u r r e n t and


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6

QQ

O

-Pi

Ο ι

=o

CH 0H + NAD 3

+

+ H 0 2

The methane c o n c e n t r a t i o n was determined w i t h a m i c r o b i a l sensor c o n s i s t i n g of i m m o b i l i z e d Mj_ f l a g e l l a t a and an oxygen e l e c t rod e( 15 L6) . Mj_ f l a g e l l a t a were i m m o b i l i z e d i n a c e t y l c e l l u l o s e f i l t e r s w i t h agar. The m i c r o b i a l sensor system i s s c h e m a t i c a l l y i l l u s t r a t e d i n F i g u r e 7. Methane containing gas introduced into both r e a c t o r s by a pump a t a c o n t r o l l e d f l o w r a t e . The p a r t i a l p r e s s u r e of oxygen i n each l i n e was monitored w i t h an oxygen e l e c t r o d e . The d i f f e r e n c e between t h e o u t p u t c u r r e n t s o f t h e two e l e c t r o d e s i s r e l a t e d t o t h e amount o f methane i n t h e f l o w l i n e s . The response time r e q u i r e d f o r t h e d e t e r m i n a t i o n o f methane gas was 1 min. A l i n e a r r e l a t i o n s h i p was observed between t h e c u r r e n t d i f f e r e n c e of t h e e l e c t r o d e s and t h e c o n c e n t r a t i o n o f methane (below 6.6 ir\mol']/ The minimum c o n c e n t r a t i o n f o r t h e d e t e r m i n a t i o n was 13.1μπιο1·1 The c u r r e n t decrease was r e p r o d u c i b l e w i t h i n ±5 % i n 25 experiments w i t h sample gas c o n t a i n i n g 0.66 mmol»l methane The c u r r e n t output o f t h e sensor system was almost c o n s t a n t f o r more than 20 days and 500 a s s a y s . The m i c r o b i a l sensor c a n , t h e r e f o r e , be used t o assay methane over a l o n g p e r i o d o f time. In t h e same experiment t h e c o n c e n t r a t i o n o f methane was determined by both t h e e l e c t r o c h e m i c a l sensor and t h e c o n v e n t i o n a l method (gas chromatography). A good c o r r e l a t i o n was o b t a i n e d between t h e methane c o n c e n t r a t i o n s determined by t h e two methods ( c o r r e l a t i o n c o e f f i c i e n t 0.97). JJ

4

M i c r o b i a l Sensors f o r Environmental

Control

BOD Sensor. The b i o c h e m i c a l oxygen demand (BOD) t e s t i s one o f the most w i d e l y used and important t e s t s i n t h e measurement o f orga n i c p o l l u t i o n . S i n c e t h e BOD t e s t measures biodegradable o r g a n i c compounds i n waste waters, i t r e q u i r e s a l o n g i n c u b a t i o n p e r i o d (5 days a t 20C). Therefore a simple and r e p r o d u c i b l e method f o r e s t i m a t i o n o f 5-day BOD i s r e q u i r e d f o r p o l l u t i o n c o n t r o l ( 1 7 ) . S i n c e b a c t e r i a a r e known t o u t i l i z e o r g a n i c compounds such as c a r b o h y d r a t e s and p r o t e i n s , t h e b i o f u e l c e l l system u s i n g immobi l i z e d C^ b u t y r i c u m c o u l d be a p p l i e d t o t h e e s t i m a t i o n o f t h e BOD of waste w a t e r s ( 1 8 ) . C. b u t y r i c u m was i m m o b i l i z e d i n p o l y a c r y l a m i d e g e l membrane and t h e i m m o b i l i z e d whole c e l l s were f i x e d on t h e anode. A l i n e a r r e l a t i o n s h i p was o b t a i n e d between t h e s t e a d y - s t a t e c u r r e n t and t h e BOD from 0 t o 250 ppm. The s t e a d y - s t a t e c u r r e n t was r e p r o d u c i b l e w i t h i n ±7 % o f r e l a t i v e e r r o r , when t h e standard s o l u t i o n (50 mg 1*1 g l u c o s e , 50 mg 1"! glutamate) was measured r e p e a t e d l y . The s t a n d a r d d e v i a t i o n was 2 ppm.



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Figure 6. The microbial sensor system for ammonia. 1. Electrolyte (NaOH); 2. Cathode (Pt); δ. Immobilized cells; 4. Magnetic stirrer; 5. Gas permeable Teflon membrane; 6. Teflon membrane; 7. Anode (Pb)

Figure 7. Schematic diagram of the methane gas sensor. 1. Pump; 2. Gas sampler; 3. Sample gas; 4. Cotton filter; 5. Reference reactor; 6. Methane oxidizing bacterial reactor; 7. Oxygen electrode; 8. Amplifier; 9. Recorder


342



The m i c r o b i a l sensor was a p p l i e d t o the e s t i m a t i o n o f the BOD of waste w a t e r s . Three k i n d s o f i n d u s t r i a l u n t r e a t e d waste water — s l a u g h t e r - h o u s e , food f a c t o r y , and a l c o h o l f a c t o r y — were employed i n the e x p e r i m e n t s . R e l a t i v e e r r o r o f BOD e s t i m a t i o n o f i n d u s t r i a l wastewaters was w i t h i n 10% campared t o 5-day BOD t e s t . Assay c o u l d be done w i t h i n 20 min. No d e c r e a s e i n c u r r e n t output was observed over a 30-day p e r i o d . A second s e n s o r employed T r i c h o s p o r o n cutaneum, w h i c h was u t i l i z e d f o r wastewater t r e a t m e n t ( 1 9 ) . The s t e a d y - s t a t e c u r r e n t depended on the BOD o f t h e sample s o l u t i o n . A l i n e a r r e l a t i o n s h i p was observed between the c u r r e n t d i f f e r e n c e (between i n i t i a l and s t e a d y s t a t e c u r r e n t ) and the 5-day BOD o f the s t a n d a r d s o l u t i o n below 60 mg 1~1 . The minimum measureable BOD was 3 mg l" . The c u r r e n t was r e p r o d u c i b l e w i t h i n ±6 % o f r e l a t i v e e r r o r when BOD o f 40 m g - l " ^ "the s t a n d a r d s o l u t i o n was emp l o y e d . The s t a n d a r d d e v i a t i o n was 1.2 mg-1" f BOD experiments. The c u r r e n t means the c u r r e n t d i f f e r e n c e s h e r e i n a f t e r . The m i c r o b i a l sensor was a p p l i e d t o e s t i m a t i o n o f 5-day BOD f o r u n t r e a t e d waste waters from a f e r m e n t a t i o n f a c t o r y . The 5-day BOD o f the waste waters was determined by the J I S method (Japanese I n d u s t r i a l S t a n d a r d Committee, 1974). Good comparative r e s u l t s were o b t a i n e d between the BOD e s t i m a t i o n by the m i c r o b i a l s e n s o r and those determined by the J I S method. The r e g r e s s i o n c o e f f i c i e n t was 1.2 i n 17 experiments and the r a t i o s (BOD e s t i m a t e d by the m i c r o b i a l sensor/5-day BOD determined by J I S method) were i n the range from 0.85 t o 1.36. T h i s v a r i a t i o n might have been caused by changes i n c o m p o s i t i o n o f o r g a n i c waste water compounds. S t a b l e responses to the s t a n d a r d s o l u t i o n (20 mg-1' BOD) were observed f o r more than 17 days (400 t e s t s ) . F l u c t u a t i o n s o f the c u r r e n t and the base l i n e (endogeneous l e v e l ) were w i t h i n ±20 % and 15 % r e s p e c t i v e l y f o r 17 days. The m i c r o b i a l s e n s o r c o u l d be used f o r a l o n g time f o r the e s t i m a t i o n o f BOD. A c o n t i n u o u s BOD e s t i m a t i o n system i s now c o m m e r c i a l i z e d i n Japan ( F i g u r e 8 ) . 1

1

1

i

n

1

0

Q

1

N i t r i t e Sensor. The p r i n c i p a l gaseous o x i d e s o f n i t r o g e n o f i n t e r e s t i n a i r p o l l u t i o n sampling and a n a l y s i s a r e n i t r i c o x i d e (NO), and n i t r o g e n d i o x i d e ( N O 2 ) . N i t r o g e n d i o x i d e i s t h e most r e a c t i v e o f the gaseous o x i d e s o f n i t r o g e n and i s a p r i m a r y a b s o r b e r o f s u n l i g h t i n p h o t o c h e m i c a l atmospheric r e a c t i o n s t h a t produce photochemical smog. T h e r e f o r e , the d e t e r m i n a t i o n o f n i t r o g e n d i o x i d e i s i m p o r t a n t i n e n v i r o n m e n t a l and i n d u s t r i a l p r o c e s s a n a l y s e s . N i t r o b a c t e r s p . u t i l i z e n i t r i t e as the s o l e source o f energy and oxygen i s consumed by t h e r e s p i r a t i o n as f o l l o w s : _ 2N0~ + 0

Nitrobacter sp. 2

> 2N0~

T h e r e f o r e , NO2 generated i n the b u f f e r (pH 2.0) can be determined by the m i c r o b i a l sensor u s i n g i m m o b i l i z e d N i t r o b a c t e r sp. and an oxygen e l e c t r o d e ( 2 0 , 2 1 ) . The scheme o f the m i c r o b i a l sensor system i s i l l u s t r a t e d i n F i g u r e 9. When the sample s o l u t i o n c o n t a i n i n g sodium n i t r i t e was i n j e c t e d i n t o the system , n i t r o g e n d i o x i d e was produced i n the f l o w c e l l



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14 Figure 8. Schematic diagram of Continual Measuring System for BOD. 1. Sensor unit; 2. Recorder unit; 3. Data processing unit; 4. Flow line selector unit; 5. Sampling unit; 6. Amplifier; 7. Microbial sensor; 8. Pump; 9. Incubator; 10. Flow meter; 11. Air pump; 12. Buffer tank; 13. Selector contrôle; 14. Standard solution; 15. Filter; 16. Waste

Figure 9. Schematic diagram of the nitrite sensor system. l.Air(280mh/ min); 2. Pump; 3. Buffer (pH 2.0); 4. Sample solutioni 5. Peristalt%c pump; 6. Pump; 7. Valve; 8. Incubator (30 C); 9. Microbial electrode; 10. Waste; 11. Amplifier; 12. Recorder; 13. Electrolyte (30% sodium hydroxide); 14. Teflon membrane; 15. Buffer (pH 2.0) containing NO2 gas; 16. Immobilized whole cells; 17. Gas permeable membrane; 18. cell space (1.0 ml); 19. Teflon cup; 20. Waste; 21. Rubber rings; 22. Platinum cathode; 23. Lead anode; 24. Insulator




344

and permeated through t h e gas permeable membrane. N i t r i t e was form ed i n t h e b a c t e r i a l l a y e r and a s s i m i l a t e d by t h e i m m o b i l i z e d b a c t e ria. The s t e a d y - s t a t e c u r r e n t i s o b t a i n e d w i t h i n 10 min. The d i f f e rences between t h e i n i t i a l and s t e a d y - s t a t e c u r r e n t s were d i r e c t l y p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f sodium n i t r i t e . A linear rela t i o n s h i p was observed between t h e c u r r e n t decrease and the sodium n i t r i t e c o n c e n t r a t i o n below 0.59 mM ( c u r r e n t decrease 0.63^uA). At g r e a t e r than 0.65 mM sodium n i t r i t e , a l i n e a r r e l a t i o n s h i p was not observed between t h e c u r r e n t and c o n c e n t r a t i o n s . The minimum c o n c e n t r a t i o n f o r t h e d e t e r m i n a t i o n o f sodium n i t r i t e was 0.01 mM ( s i g n a l t o n o i s e , 20; r e p r o d u c i b i l i t y , ±5 %). The c u r r e n t d e c r e a s e was r e p r o d u c i b l e w i t h i n ±4 % o f r e l a t i v e e r r o r and the standard d e v i a t i o n was 0.01 mM i n 25 experiments when a sample s o l u t i o n con t a i n i n g 0.25 mM o f sodium n i t r i t e was employed. The sensor d i d n o t respond t o v o l a t i l e compounds such as a c e t i c a c i d , e t h y l a l c o h o l , and amines ( d i e t h y l a m i n e , propylamine, and b u t y l a m i n e ) o r t o n o n v o l a t i l e n u t r i e n t s such as g l u c o s e , amino a c i d s , and metal i o n s (potassium and sodium i o n s ) . T h e r e f o r e , the s e l e c t i v i t y o f t h i s m i c r o b i a l sensor was s a t i s f a c t o r y i n t h e presence o f these d i f f e r e n t substances. The c u r r e n t o u t p u t o f the sensor was almost c o n s t a n t f o r more than 21 days and 400 a s s a y s . The m i c r o b i a l sensor can be used t o assay sodium n i t r i t e f o r a l o n g p e r i o d . I n the same experiments t h e c o n c e n t r a t i o n o f sodium n i t r i t e was d e t e r mined by both t h e sensor proposed and t h e c o n v e n t i o n a l method (dime thyl-α -naphty lamine method). A good c o r r e l a t i o n was o b t a i n e d between the sodium n i t r i t e c o n c e n t r a t i o n s determined by t h e two methods ( c o r r e l a t i o n c o e f f i c i e n t 0.99). Mutagen Sensor. Long-term c a r c i n o g e n i c i t y t e s t s w i t h l a b o r a t o r y mammals a r e n o t o n l y time-consuming b u t a l s o demanding o f r e s o u r c e s . The mutagenic a c t i v i t y o f c a r c i n o g e n s has r e c e n t l y been confirmed i n a g r e a t number o f cases. The e x i s t e n c e o f a h i g h c o r r e l a t i o n between t h e m u t a g e n i c i t y and c a r c i n o g e n i c i t y o f c h e m i c a l s i s now e v i d e n t . The use o f m i c r o b i a l systems i s i m p o r t a n t f o r a survey of mutagenic c h e m i c a l s . R e c e n t l y , a number o f m i c r o b i a l methods f o r d e t e c t i n g t h e v a r i o u s types o f mutagens have been developed. A method named " r e c - a s s a y " u t i l i z i n g B a c i l l u s s u b t i l i s has a l s o been proposed f o r s c r e e n i n g chemical mutagens and c a r c i n o g e n s (22), However, t h e " r e c - a s s a y " s t i l l r e q u i r e a l e n g t h y i n c u b a t i o n o f b a c t e r i a and c o m p l i c a t e d procedures. An e l e c t r o d e c o n s i s t i n g o f t h e a e r o b i c r e c o m b i n a t i o n - d e f i c i e n t b a c t e r i a and t h e oxygen e l e c t r o d e was a p p l i e d t o t h e p r e l i m i n a r y s c r e e n i n g o f c h e m i c a l mutagens and carcinogens(23-25). The m i c r o b i a l sensor system i s shown i n F i g u r e 10. The e l e c t rode system c o n s i s t e d o f two m i c r o b i a l e l e c t r o d e s : t h e e l e c t r o d e of B. s u b t i l i s Rec" (Rec~ e l e c t r o d e ) and t h e e l e c t r o d e o f B^_ s u b t i l i s R e c * ~ ( R e c e l e c t r o d e ) . Each e l e c t r o d e was composed o f i m m o b i l i z e d b a c t e r i a and an oxygen e l e c t r o d e . I f s u f f i c i e n t n u t r i e n t s (e.g., 0.3 g l ' g l u c o s e ) a r e p r e s e n t i n a sample s o l u t i o n , a c o n s t a n t c u r r e n t i s o b t a i n e d from t h e e l e c t r o d e . The c u r r e n t depends on t h e t o t a l r e s p i r a t i o n a c t i v i t y of i m m o b i l i z e d c e l l s . T h e r e f o r e , t h e t o t a l r e s p i r a t i o n a c t i v i t y of b a c t e r i a , t h e c u r r e n t , depends on t h e number o f v i a b l e c e l l s i m m o b i l i z e d onto t h e a c e t y l c e l l u l o s e membrane. The r e l a t i o n s h i p between t h e c u r r e n t and t h e c e l l numbers on t h e a c e t y l c e l l u l o s e f

1



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Figure 10. Schematic diagram of the electrode system for rapid de tection of chemical mutagen. (I) Rec electrode; (II) Rec~ electrode; 1. Bacillus subtilis Rec"; 2. Bacillus subtilis Rec+; δ. Membrane filter; 4. Teflon membrane; 5. Ft cathode; 6. Pb anode; 7. Recorder +


346

FUNDAMENTALS AND APPLICATIONS OF CHEMICAL SENSORS 8

membrane was l i n e a r i n the range over 0.1-3.0 χ 1 0 c e l l s . Conse q u e n t l y , 2.7 χ 1 0 c e l l s o f s u b t i l i s Rec" and R e c were immobi l i z e d , t h e r e a f t e r , on the membrane o f the e l e c t r o d e . When the Rec" and R e c e l e c t r o d e s were i n s e r t e d i n t o the g l u c o s e - b u f f e r s o l u t i o n (0.3 g»l~ glucose), steady-state currents were o b t a i n e d . Then, AF-2, well known mutagen, was added t o the s o l u t i o n . A f t e r 24-40 min, the c u r r e n t o f the Rec" e l e c t r o d e began t o i n c r e a s e g i v i n g a s i g m o i d a l c u r v e . On the o t h e r hand, the c u r r e n t of the Rec* e l e c t r o d e d i d not i n c r e a s e . The r a t e o f c u r r e n t i n c r e a s e i s a measure o f the mutagen c o n c e n t r a t i o n and i s most e a s i l y measured as the l i n e a r s l o p e a t the midpoint o f the s i g m o i d a l curve. When c h e m i c a l mutagens such as AF-2, mitomycin, Captan, 4NQ0, N-methyl-N'-nitro-N-nitrosoguanidine, and a f l a t o x i n B i were added to the g l u c o s e - b u f f e r s o l u t i o n , the r a t e s o f the c u r r e n t i n c r e a s e of the Rec" and R e c e l e c t r o d e s were measured. The c u r r e n t o f the Rec* e l e c t r o d e markedly i n c r e a s e d when these reagents were added t o the system. T h e r e f o r e , the m u t a g e n i c i t y o f c h e m i c a l s can be e s t i m a t e d w i t h the sensor system. L i n e a r r e l a t i o n s h i p s were o b t a i n e d i n the range over 1.6-2.8 g ml" f o r AF-2 and 2.4-7.3 g ml" f o r mitomycin. T h i s m i c r o b i a l sensor system i s based on the i n h i b i t o r y a c t i o n of the mutagens on the r e s p i r a t i o n o f EL s u b t i l i s Rec~. Bj_ s u b t i l i s M45 (Rec~) i s g e n e t i c a l l y d e f i c i e n t i n the DNA r e c o m b i n a t i o n enzyme system, whereas B^ s u b t i l i s H17 (Rec ") i s a w i l d s t r a i n which has the a b i l i t y t o r e p a i r damaged DNA. The subsequent death o f Rec b a c t e r i a i s preceded by the decrease o f r e s p i r a t i o n . As a r e s u l t , the number o f Rec" c e l l s on the s u r f a c e o f the oxygen e l e c t r o d e decreased and the c u r r e n t o f the Rec" e l e c t r o d e i n c r e a s e d . On the o t h e r hand, the damaged DNA o f R e c b a c t e r i a i s r e p a i r e d w i t h the r e c o m b i n a t i o n system. T h e r e f o r e , the number of Rec* c e l l s d i d not change and the c u r r e n t o f the R e c e l e c t r o d e d i d not i n c r e a s e . S i n c e the r e s p i r a t i o n o f b a c t e r i a l c e l l s i s d i r e c t l y and immediately c o n v e r t e d t o an e l e c t r i c s i g n a l , the p r e l i m i n a r y s c r e e n i n g o f mu tagens i s p o s s i b l e w i t h i n 1 h. Moreover, the m i c r o b i a l sensor system employs a homogeneous s u s p e n s i o n . Consequently, the s e n s i t i v i t y of the m i c r o b i a l sensor i s h i g h e r than the " r e c - a s s a y " . The minimum measurable mutagen c o n c e n t r a t i o n i s 1.6 jug ml" by the m i c r o b i a l sensor and 5.0 / i g ml" by the " r e c - a s s a y " and 10 jdg ml" by the Ames t e s t (26) f o r AF 2. On the o t h e r hand, S a l m o n e l l a typhimurium TA 100 r e q u i r e s h i s t i dine f o r growth. When the membrane f i l t e r - e l e c t r o d e c o n t a i n n i n g v a r i o u s amounts o f Sj_ typhimurium r e v e r t a n t was i n s e r t e d i n t o the g l u c o s e - b u f f e r s o l u t i o n s a t u r a t e d w i t h oxygen, g l u c o s e was a s s i m i l a t e d by the b a c t e r i a . However, the r e v e r t a n t o f t h i s s t r a i n can grow on the h i s t i d i n e - f r e e medium. The e l e c t r o d e response d e pends on the numbers.of v i a b l e b a c t e r i a r e t a i n e d on the membrane filter. Then, v a r i o u s amounts o f AF-2 were added t o the medium. The c u r r e n t was measured a t 2-h i n t e r v a l s . A f t e r 8 h o f i n c u b a t i o n , the c u r r e n t decreased w i t h i n c r e a s i n g i n c u b a t i o n time because t h e r e v e r t a n t s grew above the minimum d e t e c t a b l e numbers o f c e l l s by the e l e c t r o d e . A 10-h i n c u b a t i o n gave the g r e a t e s t s e n s i t i v i t y . On the o t h e r hand, t h e r e was no decrease i n c u r r e n t from the medium i n the absence o f AF-2. The c u r r e n t decrease became l a r g e r w i t h i n c r e a s i n g AF-2 c o n c e n t r a t i o n . The minimum measurable c o n c e n t r a t i o n of AF-2 was 0.001 jig ml" . The c u r r e n t decrease was r e p r o d u c i b l e 8

+

+


1

+

1

1

1

4

+

1

1

1

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within 5 % of relative error when a sample solution containing 0.006 /ig ml"1 of AF-2 was employed. When Sj_ typhimurium was incubated with chemical mutagens such as N-methyl-N1-nitro-N-nitrosoguanidine, nitrofurazone, methyl methanesulfonate, and ethyl methanesulfonate, for 10 h the current decrease of the electrode was measured. The response of the electrode increased with increasing concentration of chemical mutagens. Therefore, the mutagenicity of chemicals can be estimated with the microbial electrode. In this study, a homogeneous bacterial suspension was employed and mutagens were added to the homogeneous suspension. The complete medium containing amino acids, vitamins, and mineral salts was used for experiments. Furthermore, employment of the electrode system makes possible a large injection of bacterial suspension. As a result, the time required for mutagen test was shortened to 10 h. The time is longer than that (1 h) of the micribial electrode system described above. The sensitivity of the microbial electrode system was higher than the convetional Ames test. The minimum measurable mutagen concentration was 0.001 /ig ml"1 by the microbial electrode, and 10jdg ml"1 by the Ames test for AF-2. Acknowledgment The author thank Professor Shuichi Suzuki, The Saitama Institute of Technology, for his encourage during experiments. Literature Cited 1.

Guilbault, G. G. "Hand-book of Enzymic Analysis"; Dekker: New York, 1976. 2. Karube, I.; Suzuki, S. "Ion-Selective Electrode Reviews"; Pergamon Press: Oxford, 1984; Vol. 6, p. 15. 3. Karube, I.; Suzuki, S. "Annual Reports on Fermentation Processes"; Academic Press: New York, 1983; p. 203. 4. Karube, I. "Biotechnology & Genetic Engineering Reviews"; Intercept Ltd.: Newcastle upon Tyne, 1983; Vol. 2, p. 313. 5. Karube, I. "Biotechnology Handbook"; Butterworth/Ann Arbor Sci. Pub. Co.: New Jersey, 1985; p. 134. 6. Karube, I.; Mitsuda, S.; Suzuki, S., Europ.J. Appl. Microbiol. Biotechnol. 1979. 7, 343. 7. Hikuma, M.; Kubo, T.; Yasuda, T.; Karube, I.; Suzuki, S., Biotechnol. Bioeng. 1979. 21, 1845. 8. Hikuma, M.; Kubo, T.; Ysauda, T.; Karube, I.; Suzuki, S., Anal. Chim. Acta 1979. 109,33. 9. Matsunaga, T.; Karube, I.; Suzuki, S., Europ. J. Appl. Microbiol. Biotechnol. 1980. 10, 235. 10. Hikuma, M.; Obana, H.; Yasuda, T.; Karube, I.; Suzuki S., Anal. Chim. Acta 1980. 116, 61. 11. Hikuma, M.; Yasuda, T.; Karube, I.; Suzuki, S., Ann. New York Acad. Sci. 1981. 369, 307. 12. Karube, I.; Okada, T.; Suzuki, S., Anal. Chem. 1981. 53, 1852. 13. Okada, T.; Karube, I.; Suzuki, S., Anal. Chim. Acta 1982. 135, 159. 14. Hikuma, M.; Kubo, T.; Yasuda, T.; Karube, I.; Suzuki, S., Anal. Chem. 1980. 52, 1020. American Chemical Society Library 1155 15th St., N.W. In Fundamentals and Applications of Chemical Sensors; Schuetzle, Dennis, et al.; Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

348 15. 16. 17. 18. 19.


20. 21. 22. 23. 24. 25. 26.


Okada, T.; Karube, I.; Suzuki, S., Europ. J. Appl. Microbiol. Biotechnol. 1981. 12, 102. Karube, I.; Okada, T.; Suzuki, S., Anal. Chim. Acta 1982. 135, 61. Karube, I.; Matsunaga, T.; Mitsuda, S.; Suzuki, S., Biotechnol. Bioeng. 1977. 19, 1535. Karube, I.; Matsunaga, T.; Suzuki, S., J. Solid phase Biochem. 1977. 2, 97. Hikuma, M.; Suzuki, H.; Yasuda, T.; Karube, I.; Suzuki, S., Europ. J. Appl. Microbiol. Biotechnol. 1979. 8, 289. Karube, I.; Okada, T.; Suzuki, S., Europ. J. Appl. Microbiol. Biotechnol. 1982. 15, 127. Okada, T.; Karube, I.; Suzuki, S., Biotechnol. Bioeng. 1983. Kada, T.; Tutikawa, K.; Sadaie, Y., Mutat. Res. 1972. 16, 165. Karube, I.; Matsunaga, T.; Nakahara, T.; Suzuki, S., Anal. Chem. 1981. 53, 1024. Karube, I.; Nakahara, T.; Matsunaga, T.; Suzuki, S., Anal. Chem. 1982. 54, 1725. Karube, I., Trends in Analytical Chemistry 1984. 3, 40. Ames, B.N.; Lee, F.D.; Durston, W.E., Proc. Natl. Acad. Sci. USA 1972. 70, 782.

RECEIVED February 6, 1986


Fundamentals and Applications of Chemical Sensors - American

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