BillTECHNOLOGY TECHNIQUES Volume 10 No.3 (March 1996) pp.183-188 Received 25th January.

Detection of halogenated hydrocarbons by a microbial sensorsystem using a stop-flow-technique

J. Peter, W. Hutter, W. Stellnberger, W. Hampel

Institute of Biochemical Technology and Microbiology, University of Technology

Getreidemarkt 9/l 72, 1060 Vienna, Austria

ABSTRACT To improve the applicability and efficiency of the microbial assay developed by W. Hutter, J. Peter, H. Swoboda, W. Hampel, E. Rosenberg, D. K&ner, and R. Kellner (1995) a stop-flow-technique was developed for the determination of halogenated hydrocarbons in water samples. Cells of Rhodocuccus sp. DSM 6344 were imniobilized in alginate beads and placed in a stirred flow-through reactor. The time of incubation, the bacterial cell density and the amount of alginate beads in the reactor on the response of the system as determined by the drop in EMF was investigated. Optimal conditions were achieved with 2 g beads containing a bacterial cell concentration of 0,l g cells wet wt/g matrix and an incubation time of 20 min. Calibrations with chlorinated and brominated substrates like ethyl bromide, 1 ,Zdl&romopropane, isobutyl bromide, 1 -chlorobutane and 1,5- dichloropentane showed a non-linear dependence at low substrate concentrations. The detection limits for ethyl bromide and l-chlorobutane were estimated as 0.02 mgA and 0.45 mg/l, respectively; the relative standard deviation was below 10 %. The great advantage of the stop-flow-technique compared to discontinuous measurements can be seen in a simplified handling and an increase of sample capacity.

INTRODUCTION

Halogenated hydrocarbons in water may be specifically determined by a

biochemical assay with the enzyme halido-hydrolase [E.C. 3.8.1.11. The reaction

products of hydrolytic dehalogenation are the corresponding alcohol and the halide

(Scholz et al., 1988) according to the equation:

R-X + Hz0 + R-OH + H’ + X- The halogen ions are easily quantified by an ion selective electrode. Hutter at al.

(1995) described a discontinuous assay with immobilized cells of Rhodococcus sp. DSM 6344 which showed excellent specifity but had some time-consuming

disadvantages due to the complicated procedure of washing the immobilizate after each measurement.

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An improvement in this respect can be achieved by the application of continuous

techniques. The commonly used Flow Injection Analysis (FIA) is considered to be

inadequate, as the enzymatic reaction needs a long incubation period for the

release of measurable amounts of halogen ions.

So the stop-flow-technique was used as an alternative. It allows to manipulate the

incubation time resulting in different concentration profiles, which makes this

method very flexible (Krug et al., 1986). A summary of further characteristics can

be found elsewhere (Ruzicka and Hansen, 1978). Moreover, this technique is

frequently performed in combination with spectrometric methods (Willis et al.,

1970; Beckwith and Crouch, 1972).

This paper describes the application of a stop-flow-technique for the estimation of

halogenated hydrocarbons in water samples in an assay with immobilized cells and

ion selective electrodes.

MATERIALS AND METHODS

Microorganism and cultivation The bacterium Rhodococcus sp. DSM 6344, which was isolated by Leisinger (1983), was cultivated aerobically in media (5 I) as described elsewhere (Hutter et al., 1995). After a cultivation period of 30 h the cells were collected by centrifugation (9000 x g; 25 min; 4 “C).

Determination of dehalogenase activity The activity of the immobilized cells was assayed by the incubation of 2 g immobilizate in 20 ml phosphate buffer (50 mM; pH 8.5) containing 40 mM l- chlorobutane at 30 “C for a period of 60 min. After removing the immobilizate by centrifugation (9000 x g; 25 min; 4 “C) Ag(l)chromate (0.03 g) was added to 2 ml solution. The chloride content was determined in the supernatant according to lsaacs (1922). The activity of cells was assayed in the same way using 0.075 g cells (wet weight) and 10 mM substrate in 2 ml buffer solution.

Immobilization Bacterial cells (2 g; wet weight) were suspended in 20 g of an aqueous sodium- alginate solution (1.5 %). Uniform beads were prepared in a way as described by Hutter et al. (1995).

Electrodes For the detection of released halogen ions in the solution ion-selective electrodes (SC 9417; Orion Res. Inc., Boston, USA; SBR 9435; Seibold Kappl AG, Vienna, Austria) were used in combination with a Ag/AgCI reference electrode (CSZ 246749; Seibold Kappl AG, Vienna, Austria), which were interfaced with a pH- lonometer (G 154; Seibold Kappl AG, Vienna, Austria).

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Measuring procedure The configuration of the analytical system is presented 7

in Figure 1. A volume of 1.5 ml of 5 M calcium nitrate 5

solution was added to 200 ml * of water sample. The stirred solution was delivered with constant flow (15 ml per min) to the analytical system by a 1 peristaltic pump (EYELA Micro tube pump MP-3, Tokyo, Rikakikai Co.). The

8

I stirred reaCtOr (5) contained F lgure 1: Arrangement of the biological essay (1, sample solution;

in a total volume of 20 ml the 2. peristaltic pump; 3,4. threeway valve; 5, reactor (volume = 20 ml);

Rhodococcus immobilizate (2 6, magnetic stirrer; 7, working electrode; 8, reference electrode)

g) and the sample solution, which were incubated for 20 minutes. During the incubation a blank stream passed the electrodes and provided the baseline. By switching the two three-way valves (3,4) simultaneously into the opposite position, the incubated sample was passed to the sensing electrodes (7) for the detection of released halogen ions. The signal resulting from the decrease in the electrode potential in reference to the baseline was used for the calculation of results.

RESULTS

Cells of Rhodococcus were cultivated in media containing 1-chlorobutane as the

sole carbon source. The specific activity of the cells collected by centrifugation was

82 nkat/g dry biomass. The cells were entrapped in alginate beads, the specific

activity of the immobilizate being 1.27 nkat/g alginate beads. The beads (2 g) were

placed in a thermostated reactor, which was connected by two three-way-valves to

a continuous sample stream. After filling the reactor with the sample, the position of

the two valves was changed simultaneously and the sample was incubated for 20

min. In the meantime, the remaining sample was passed continuously and directly

to the ion selective electrodes, thus providing the baseline signal for the

measurement. After applying a new sample to the delivery system, the valves were

put into the original position, which resulted in a flow of the old reacted sample to

the ion selective electrode and a replacement of the old sample by the new one in

the reactor. The decrease in the electromotoric force (EMF) of the electrode

system was monitored and the lowest value taken for the calculation of results.

Figure 2 shows a typical response for a sample solution containing 0.07 mg/l ethyl bromide.

In several experiments calibrations were performed with water samples containing

different chlorinated and brominated hydrocarbons, e.g. ethyl bromide, 1,2-

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dibromopropane, isobutyl bromide, I-chlorobutane and 1 ,Sdichloropentane. The

results are shown in Figure 3.

228

226

5 224

= 222

3 220

g 218

216

214

212

4J’ / - ,I: , ,,syff/&5yg ,,(,,

0

0,001 0,Ol O,l 1 10

Halogenated Hydrocarbon [ mg/l]

Figure 2: Response of the branide sensitive indicator Figure 3: Calibration graph for isobutyi bromide (+), electrode in a sample solution containing 0.07 mg!l 1.2-dibromopropane (k). ethyl bromide (A),

ethyl bromide using 20 min incubation time I-chlorobutane (0) and 1 ,S-dichloro(Jentane (a)

The graphs were prepared by plotting the maximum decrease in EMF against the

concentration of the halogenated hydrocarbon in the sample. Non-linear graphs

‘were obtained due to the non-linearity of the chloride and bromide electrodes in the

concentration range close to the detection limit. According to the 3 (T criterion, the

detection limit for ethyl bromide and I-chlorobutane was calculated with 0.02 mg/l

and 0.45 mgll, respectively. The reproducibility of the microbial assay was tested in

six consecutive experiments measuring a

sample solution containing 1.5 mg/l l- 45 -

chlorobutane; from the results of these

experiments the relative standard

deviation of 5.3 % was calculated.

In additional experiments the effect of

variations in the length of incubation of

the sample with the immobilizate, the cell

density in the immobilizate and the total

amount of immobilizate in the reactor

0,001 0,Ol O,l 1

I ,2-Dibromopropane [mgA]

were investigated. The incubation period Figure 4: Effect of the incubation tima on the respcnse of the microbial assay (+ 5 min,

for the sample with the immobilized cells n i0 min, A 40 min)

in the reactor was studied between 5 to 40 min. Calibration graphs obtained with

1,2-dibromopropane as a substrate are given in Fig. 4 for incubations of 5, 20 and

40 min, respectively. There is no linear correlation of the incubation time to the

response of the potentiometric electrode as determined by the maximum change in

EMF. It is caused by the fact that the amount of halogen ions liberated by the immobilized cells is governed by enzyme kinetics.

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At low cell densities in the immobilizate, an increase in the number of active cells

also increased the response of the measuring system. At higher concentrations the

increase was limited by diffusion barriers to the encapsulated cells, which resulted

in an almost constant response as shown in Fig. 5; the effect was studied with a

sample solution containing 0.9 g/l I-chlorobutane and an incubation time of 5 min.

Increasing the amount of immobilizate in the reactor leads to an increased

response of the measuring system. There is no linear increase of the measuring

signal in correlation to the total amount of immobilizate as kinetic principles are

ruling the enzymatic conversion of the halogenated substrate. Experiments

performed on this matter were done with 0.9 g/l I-chlorobutane and an incubation

time of 5 min with immobilizate variations in the range of 0.05 to 0.25 g/ml; the

results are given in Fig. 6.

0,05 0,1 0,15

Cells wet weight/Alginate [g/g]

160

140

120

100

80

60

40

20

0

0

I

0,05 0,I 0,15 0,2 0,25

Immobilisate [g/ml]

Figure 5: Effect of the bacterial cell density on the response of the microbial assay using 5 min incubation

Figure 6: Effect of the immobiliie amount on the current

time and 0.9 g/l I-chlorobutane as substrat respcfise of the microbial assay

For measuring samples of environmental origin, it is necessary to remove chloride

ions from the sample in order to achieve optimal sensitivity. This can be easily

done by passing the sample through a column containing an ion exchanger. In

addition, this procedure will stabilize the pH of the sample at pH 7. Other interfering

ions like bromide, iodide, sulfide, ammonium and cyanide can be removed

(Frenzel, 1989) by using the CISA solution recommended by Orion (Orion Res.

Inc., Boston, USA, Cat. No. 941709).

DISCUSSION

The bacterium Rhodococcus sp. DSM 6844 is known as an effective biodegrading

microorganism for several chlorinated and brominated hydrocarbons (Scholz et al.,

1988) of interest in environmental pollution. Encapsulation of bacterial cells in

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polymer beads enables the determination of halogenated hydrocarbons in

aqueous solutions by ion selective potentiometric electrodes. in a microbial

bioassay as described by Hutter et al. (1995). The permanent addition and retrieval

of immobilized cells to and from the sample solutions forms a severe handicap for

the described bioassay. In order to improve its applicability, a stop-flow-technique

was developed and tested for its efficiency. Compared to the discontinuous

method, the stop-flow-technique showed increased detection limits but was easier

to handle, which increased the sample frequency. As an almost complete

conversion of the halogenated substrate would need a long incubation period, a

kinetic procedure was used. Consequently, there were effects of incubation time as

well as of the specific activity and the total amount of the immobilizate on the

reaction rate. According to the fact that nonlinear correlations were observed, the

method needs recalibration if one of these parameters is to be changed.

ACKNOWLEDGMENTS

The authors are obliged to the “Jubillumsfonds der Oesterreichischen

Nationafbank” for financial support (Project Nr. 5654).

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