ELSEVIER ADVANCED TECHNOLOGy

Biosensors & Bioelectronics Vol. 11. No. 12, pp. 1215-1219, 1996 © 1996 Elsevier Science Limited

Printed in Great Britain. All fights reserved 0956-5663/96/$15.00

Detection of chlorinated and brominated hydrocarbons by an ion

sensitive whole cell biosensor

J. Peter, W. Hutter, W. St611nberger & W. Hampel

Institute for Biochemical Technology and Microbiology, University of Technology, Vienna, Getreidemarkt 9/172, 1060 Vienna, Austria

Tel: [43] (1) 588014706 Fax: [43] (1) 5874835

(Received 4 December 1995; accepted 16 April 1996)

Abstract: A microbial biosensor was formed by applying immobilized cells of Rhodococcus sp. DSM 6344 to the surface of ion selective potentiometric electrodes. The bacterium contains the enzyme alkyl-halidohydrolase (EC 3.8.1.1), which transforms appropriate halogenated hydrocarbons into the corre- sponding alcohols and halogen ions, the latter being detectable by ion sensitive electrodes. Several matrices for immobilization (alginate, agarose, carrageenan, polyacrylamide, etc.) were tested, the most effective being the direct formation of a catalytic layer on the electrode surface by alginate gels.

Influence of the specific activity of the catalytic layer and the effect of temperature and pH on sensor performance were tested. Reproducible results were obtained within a time period of 5 min. Calibration with 1-chlorobutane and ethylenebromide showed a non-linear dependence and a good sensitivity, e.g. 0.22 and 0-04 mg/1, respectively. The relative standard deviation was determined as 7.8% by carrying out five consecutive experiments. The sensor can be stored at 277 K in dry form for 1 week and is easily rehydrated in calcium nitrate solutions. © 1996 Elsevier Science Limited

Keywords: Rhodococcus sp., whole cell biosensor, halogenated hydrocarbons

INTRODUCTION

Biosensors incorporate biological sensing elements such as enzymes, antibodies, receptors or higher integrated systems (e.g. organelles, tis- sue slices, microbial cells) which are coupled to a suitable transducer element (e.g. Clark oxygen electrode, carbon dioxide electrode, optrodes). In this way a biochemically generated signal will be converted into an electric signal that can be pro- cessed and quantified by a measuring device.

In most cases enzymes are used in immobilized form as biological sensing elements. Nevertheless,

they often become inactivated during the measur- ing procedure, an effect that is caused by inhibi- tory or destabilizing substances in the sample. The use of whole cells as biocatalysts has several advantages as compared to isolated enzymes, the most important being increased stability and pro- tection from interfering substances (Brooks, 1994). Consequently, microbial biosensors are preferred for measurements in contaminated samples.

Traditional biological assays have a long response time, which can be reduced considerably by the introduction of biosensors. The application

1215

J. Peter et al. Biosensors & Bioelectronics

of biosensors for the specific estimation of halo- genated organic compounds is described by sev- eral authors. Rawson et al. (1987) used the elec- tron transfer chain of the photosynthetic microorganism Synechococcus sp. to detect chlor- toluron. A biosensor using Escherichia coli cells as the biological component was introduced for the detection of pentachlorophenol by its effect on respiration (Gaisford et al., 1991).

A bioassay for the determination of halogen- ated hydrocarbons was described by Hutter et al. (1995). It is based on the liberation of halogen ions by the action of the enzyme alkyl-halidohyd- rolase (EC 3.8.1.1) present in cells of Rhodoc- occus sp. DSM 6344 and on an ion selective potentiometric electrode as transducer element. This paper describes the construction of a com- pact biosensor by applying immobilized cells of the bacterium close to the sensitive membrane of the potentiometric electrode and the characteristic features of such a device.

EXPERIMENTAL

Microorganism and cultivation

Rhodococcus sp. DSM 6344, which was isolated by Leisinger (1983) was grown aerobically in a closed 50 1 PE bottle at 303 K containing a nutri- ent medium (51) of composition: 5.37g/1 Na2HPO4-12H20; 1.36 g/1 KH2PO4; 0.5 g/l (NH4)2504; 0-2 g/l MgSO4-7H20; 1 mg/l biotin; 20 mg/1 thiamine.HC1; 5 ml trace element solution (Wolin et al., 1963); 0.1% of 1-chlorobutane was added as sole carbon source. After 30 h growth, the cells were collected by centrifugation (9000 g; 25 min; 277 K).

Determination of cellular activity

Bacterial cells in free (750 mg wet weight) or immobilized (2-00 g wet weight) form were sus- pended in 20 ml phosphate buffer (50 mM; pH 8-5) containing 10 mM 1-chlorobutane as a sub- strate. The stirred mixture was incubated at 303 K for 60 min. After removing the cells by centrifu- gation (9000g; 25 rain; 277 K) Ag0)chromate (0-03 g) was added and the content of chloride ions was determined in the supernatant according to Issacs (1922).

1216

Immobilization

Different techniques of cell immobilization were applied for the preparation of bioactive layers.

Direct deposition of Rhodococcus cells on a membrane was used in a first approach. A total amount of 0-2 g wet cells was loaded on a cellu- lose acetate filter membrane (12mm diameter; 0.2/~m pore size; Type 13400; Sartorius AG, Grttingen, Germany). The filter was attached to the electrode with the cell loaded surface exposed to the sensing layer of the electrode and covered by a cupropham membrane which was fixed by a rubber O-ring.

Further catalytic layers were prepared by entrapping bacterial cells in different matrices. A total amount of 2 g wet cells was suspended in 4g of 1% agarose, 2% carrageenan and 15% polyacrylamide (0.8% N,N-methylenebisacryla- mide, 0-5% N,N,N' ,N'- te tramethylenethylene- amide); gel discs were formed with a thickness of 0.5 mm and a diameter of 10 mm. For better mechanical stability the discs were placed on a metal sieve plate (Type 3206004; Ingold AG, Urdorf, Germany). The disc/metal sieve plate was kept in place by a fine nylon mesh and fixed with a rubber O-ring.

In another attempt the catalytic layer was for- med directly on the sensing membrane of the ion selective electrode. A total of 2 g wet cells was suspended in 4g of a 1-5% aqueous sodium alginate solution and a cell alginate layer was formed by dipping the working electrode into the solution. The layer was stabilized by bathing it in a stirred 2% calcium nitrate solution for 1 h.

Electrodes

Chloride and bromide electrodes with ion selec- tive AgX-/Ag2S membranes in combination with a Ag/AgC1 reference electrode were used as described by Hutter et al. (1995).

Measuring procedure

The assays on the biosensor's response character- istics and for performing calibrations were done in the following way: 0-15 ml of 5 M calcium nitrate solution was added to 20 ml of water and the electrodes immersed in the liquid. After the potential of the electrode attained a constant value the substrate was added. The decrease in potential was registered and used for the calculation of

Biosensors & Bioelectronics

results. All experiments 293 K.

were carried out at

RESULTS AND DISCUSSION

Fresh cells of Rhodococcus harvested from the cultivation broth by centrifugation showed a specific activity of 82 nkat/g with 1-chlorobutane as a substrate on basis of bacterial dry weight.

Catalytic active layers were formed by entrap- ment of bacterial cells in agarose, carrageenan, polyacrylamide or alginate. For estimating the efficiency in immobilization, the specific activity of small discs (10mm diameter, 0.5mm thickness), which can be applied directly to the ion selective electrode, was determined. The best result (1.38 nkat/g layer) was achieved with bac- terial cells entrapped in an alginate layer (equal to 100%). Only a slightly reduced activity was found with cells immobilized in agarose (97%) and carrageenan (95%), whereas for immobiliz- ation with acrylamide a clear reduction was observed (74%). This may be caused by the toxicity of the matrix monomers to the bacterial cells during polymerization.

These discs as well as bacterial cells deposited on a filter membrane were applied directly to the sensing part of an ion selective electrode, thus forming a compact biosensor for halogenated hydrocarbons. Moreover, a rather thin catalytic layer was formed by dipping the sensing part of the potentiometric electrode into the alginate solution containing bacterial cells, which was sub- sequently stabilized in a calcium nitrate solution. In several experiments the response of these kinds of biosensors was assayed by the addition of 1- chlorobutane (final concentration, 0-16mM) to ionically stabilized water samples. Figure 1 shows typical response curves from these experiments. The quickest response was achieved with the electrode, where the catalytic layer was formed directly on the sensing membrane. All other sen- sors showed only a reduced response; the change in electromotoric force within a period of 5 min after substrate addition was determined for aga- rose, polyacrylamide and carrageenan layers with 36, 23 and 19% of that of the thin alginate layer. The biosensor, where the bacterial cells were deposited on a filter membrane, showed the slow- est response (7%). According to these results further experiments were performed only with the alginate biosensor.

Detection of hydrocarbons by a whole cell biosensor

260,

256.

~ 254.

~ ' 252

m 2~ 248

246

244 0

I I I I I

2 4 6 8 10 Time [rain]

Fig. 1. Response characteristics of the biosensor. (~(l~) Alginate; (11) agarose; (&) polyacrylamide; (X) carra- geenan. 1-Chlorobutane (16 raM) was added at 2 min.

See text for preparation of the membrane.

The influence of the specific activity of the catalytic layer on the response characteristics was tested in further experiments (Fig. 2). There is almost no improvement in the response, if the specific activity of the catalytic layer is increased, but there is a drastic reduction in response if the specific activity is reduced below 0.5 nkat/g.

Calibrations were established for 1-chlorobut- ane and a chloride ions sensitive electrode, as well as for ethylenebromide and a bromide ions sensitive sensor. It was done by plotting the decrease of the electrode potential within a time period of 5 rain against the concentration of the halogenated organic hydrocarbon in the sample. Figure 3 shows the results from these exper- iments. Non-linear calibration graphs were obtained due to the non-linearity of the chloride and bromide electrodes in the range close to the detection limit. The detection limits for ethyl-

100 T ~ e - - - - - - - - •

80

~ ..~, 60

~-'4o

o I I

0 1 2 3 Speci f ic act iv i ty [nkat/g]

Fig. 2. Influence of specific activity of the catalytic layer on the response of the alginate biosensor. The specific activity of the catalytic layer was varied by

varying its bacterial cell density.

1217

J. Peter et al. Biosensors & Bioelectronics

20 18

~'16 E 14 ~12

-~-8 w. 6 S nl 4

2 t o O,Ol

100 T = =

/ t

20 ]- z O l I I

2 4 6 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I pH

0,1 1 10 100 Halogenated Hydrocarbon [mg/I] Fig. 5. Effect of pH on biosensor response.

Fig. 3. Calibration graph for (&) ethylenebromide and ( ~ ) 1-chlorobutane.

enebromide and 1-chlorobutane were 0.04 and 0.22 mg/1, respectively.

C h a r a c t e r i s t i c s o f t h e b i o s e n s o r

I 10

was no significant loss of specific activity in the tested layers.

CONCLUSION

The reproducibility of the alginate bionsensor was tested in five consecutive experiments measuring a sample solution containing 0.07 mM 1-chloro- butane. The relative standard deviation was deter- mined as 7.8%.

The effect of temperature and pH in the sample solution was investigated in several experiments. The influence of temperature was assayed between 293 and 318 K, showing an almost con- stant decrease of the change in electromotive force within a time period of 5 min with increas- ing temperature (Fig. 4). Variations in the pH had no effect in the range pH 5-9, but affected the sensor's response below pH 5 (Fig. 5).

The biosensor with a catalytic alginate layer can be stored under dry conditions for 1 week at 277 K. The rehydration was done by incubation at 293 K for 120 min in a solution containing 2% calcium nitrate. Applying this procedure, there

100 -

~. 8 0 -

~ ,~d, 60 -

P'2'40.

S 2 0 - o z 0 I I I I I I

290 295 300 305 310 315 320 T e m p e r a t u r e [K]

Fig. 4. Effect of temperature on biosensor response.

The detection of halogenated hydrocarbons (Keith & Telliard, 1979) can be done by the developed whole cell biosensor in a sensitive and very inexpensive way. Cells of Rhodococcus sp. DSM 6344 forming an alkyl-halidohydrolase (EC 3.8.1.1), can easily be immobilized as a very active catalytic layer on the surface of a trans- ducer electrode by entrapment in calcium alginate. The intracellular hydrolytic dehalogenase of the organism enables the dehalogenation of several 1-halo-n-alkanes, t~-to-dihaloalkanes and of some halogenated aromatic compounds. Ion selective potentiometric electrodes for both chloride and bromide ions are applicable as a specific trans- ducer element, thus forming a very compact biosensor for halogenated hydrocarbons in water samples.

The advantage of the biosensor incorporating Rhodococcus sp. DSM 6344 cells as compared to the microbial bioassay developed by Hutter et al. (1995) is the simpler handling and the long storage period without significant loss of enzyme activity. Disadvantageous are the enlarged detec- tion limits and a preincubation period of 30 min before the electrode potential attains stability.

ACKNOWLEDGEMENT

The authors are obliged to the Jubil~iumsfonds der Oesterreichischen Nationalbank (Project No. 5654) for financial support.

1218

Biosensors & Bioelectronics Detection of hydrocarbons by a whole cell biosensor

REFERENCES

Brooks, C.E.A. (1994). Whole cell biosensors for monitoring toxic water pollutants. SGM Quarterly, 21, 100-102.

Gaisford, W. C., Richardson, N. J., Haggett, B. G. D. & Rawson, D.M. (1991). Microbial biosensors for environmental monitoring. Biochem. Soc. Trans., 19, 15-18.

Hutter, W., Peter J., Swoboda, H., Hampel, W., Rosen- berg, E., KrOner, D. & Kellner, R. (1995). Devel- opment of a microbial bioassay for chlorinated and brominated hydrocarbons. Anal. Chim. Acta, 306, 237-241.

Isaacs, M.L. (1922). A colorimetric determination of blood chlorids. J. Biol. Chem., 53, 17.

Keith, L.H. & Telliard, W.A. (1979). Priority pol- lutants: I - -A perspective view. Environ. Sci. Tech- nol., 13, 416--423.

Leisinger, T. (1983). Microorganisms and xenobiotic compounds. Experientia, 39, 1183-1191.

Rawson, D. M., Willmer, A. J. & Cardois, M. F. (1987). The development of whole cell biosensors for on- line screening of herbicide pollution of surface waters. Toxic. Assess., 2, 325-340.

Wolin, E.A., Wolin, M.J. & Wolfe, R.S. (1963). Formation of methane by bacterial extracts. J. Biol. Chem., 238, 2882-2886.

1219