Acta Biotechnol. 17 (1997) 2, 123-130 Akademie VerIag

Characteristics of a Microbial Assay for the Detection of Halogenated Hydrocarbons Using Cells of an Actinomycete-like Organism as a Biological Component

PETER, J.. BUCHINGER, W., KARNER, F., HAMPEL, w

Technische Universitiit Wien Institut fiir Biochemische Technologie und Mikrobiologie Getreidemarkt 911722 1060 Wien, Austria

Summary

Cells of an Actinomycete-like bacterium, strain GJ70, with the ability to degrade several haloalkanes were used as a biological component in a discontinuous microbial bioassay for the detection of 1,3-dichloropropene and 1.2-dibromoethane in wakr. The ceils were entrapped in different matrices such as calcium alginate, carrageenan, chitosan. polyacrylamide-hydrazide and chitosan-carboxy- methyl cellulose; the specific dehalogenating activity of the immobilized cells was highest with the two last matrices. By the addition of small beads of immobilized cells to a stirred sample solution and by the use of an ion selective electrode (ISE) for the quantification of enzymatically released halogen ions, the concentration of halogenated hydrocarbons could be estimated by determining the change of electrode potential within a period of 5 min. The detection limits for 1,3-dichloropropene and 1,Zdibromoethane were below 100 pg/I and 25 pgA, respectively; the relative standard deviation was c 10%. In addition, several chlorinated and brominated hydrocarbons were converted by the bacterial cells at a reduced rate e.g. 1,2-dibromopropane, 1-bromoethane, 1,5-dichloropentane, etc. Moreover, temperatures of between 20 and 40 "C did not affect the enzymatic activity of the cells, and a pH of between at 5 and 9 had little influence. Several organic substances and non-metabolizable compounds did not affect the conversion, whereas some heavy metal ions acted as inhibitors.

Introduction

When using enzymes as catalytic components in an analytical assay for the specific determination of toxic compounds in environmental analysis, a lot of inherent difficul- ties emerge. Most of the enzymes, specifically converting these substances, are in general not available commercially. Consequently, a lot of labour and money must be invested to get appropriate starting material and to isolate and purify the enzyme needed. Moreover, most of the enzymes are inactivated during the analytical proce- dure, an effect which is caused by inhibiting and destabilizing substances of the sample. Some of these drawbacks can be avoided by the use of whole cells instead of purified enzymes [l]. In this respect, microbial cells have the great advantage of being

Acta Biotechnol. 17 (1997) 2 124

simply and easily available and of being propagated effectively by standard proce- dures, as specifically required. In addition, microbial cells show various levels of sensitivity and selectivity for the converted substrate and exhibit better resistance towards inhibiting and inactivating compounds. Various halogenated hydrocarbons are converted by the enzyme halidohydrolase [E.C. 3.8.1.1.1 which liberate halide ions. By applying a specific procedure for the detection of these ions, a bioassay for halogenated hydrocarbons can be formed. An estimation of the level of these substances in water can be easily made by using a combination of immobilized microbial cells and an ion selective electrode as a sensor, as described in [2]. The type of microbial cells greatly influences the selectivity of the bacterial assay. Cells of Rhodococcus sp. preferably convert long chain 1 -halo-n-alka- nes and a,o-dihaloalkanes [3]; but most of these halogenated hydrocarbons are rarely found as pollutants in the environment. A potent dehalogenating bacterium was isolated from a pilot-scale activated sludge unit by JANSSEN et al. [4]. The organism, strain GJ 70, was first identified as an Acinetobucfer species and was later reclassified as a gram-positive Actinomycete-like organism [5] , exhibiting a very broad spectrum for the dehalogenation of halogenated hydrocarbons. The enzyme was isolated and purified and showed high activity with several environmentally relevant halogenated hydrocarbons e.g. 1,2-dibrornoethane, 1 ,2-dibromopropane, 1 -bromoethane, 3-chloro- propene, 1,3-dichloropropene, etc. [5] For testing the applicability of strain GJ 70 cells in a discontinuous microbial bioassay and for the detection of environmentally relevant halogenated hydrocarbons in water, several experiments were performed in order to get detailed information on the charac- teristics of such a bioassay. The results of these investigations are reported in this article.

Materials and Methods

Microorganism and Cultivation

The Actinomycete-like organism, strain GJ 70. was cultivated aerobically in a sealed polyethylene bottle (volume 50 1) at 30 OC using minimal salt medium. The medium (5 1) contained per litre: 5.2 g Na2HPO4 x 3 H2O; 1.4 g KH2Po4; 0.5 g (NH4)2S04; 0.2 g MgS04 x 7 H20; 0.2 g yeast extract and 5 ml salt solution [6]. I-Chlorobutane (0.146) was added as the sole carbon source. The cells were harvested by centrifugation (9.000 X g; 10 min; 4 "C) comprising of 18-2096 dry biomass.

Determination of the Dehalogenating Activity of Cells

The activity of the immobilizate was assayed by the incubation (30 "C; 60 min) of 2 g immobilizate (wet weight) in 20 ml phosphate buffer (10 mM; pH 8.5) containing 50 mM I-chlorobutane as a sub- strate. After removing the immobilizate by centrifugation (4,000 x g; 10 min; 4 "C). silver@-chro- mate (0.03 g) was added to 2 ml of solution. The chloride content was determined as described by ISAACS [7]. One unit is defined as the activity that catalyzes the formation of 1 m o l e halidelmin under the conditions used. The activity of resting cells was assayed in the same way using 15 mg cells (dry weight) and 10 mM substrate in 2 ml phosphate buffer (10 mM, pH 8.5).

P E E R , J. er af., Microbial Assay for the Detection of Halogenated Hydrocarbons 125

Immobilization

Cells were immobilized by entrapment in a gel matrix. Several standard matrix substances for the immobilization of microorganisms were tested. Bacterial cells (0.5 g wet weight/g matrix) were mixed with different matrix substances such as calcium alginate (1.8%). carrageenan (1.5%). chitosan (2%), polyacrylamide-hydrazide (5%) and chitosan-carboxymethyl cellulose (3%). The preparation of the matrices was carried out as described in other literature: calcium alginate [8]. carrageenan [91, chitosan I lo], polyacrylamide-hydrazide I 111, chitosan-carboxymethyl cellulose [ 121. Uniform beads were prepared by a procedure described in [3].

Microbial Bioassay on Organohalides

The contents of organohalides of aqueous solutions were measured by a procedure described in more detail in [3]: 0.3 ml of 5M N a N q solution was added to 20 ml of the sample. The chloride or bromide selective electrode (Model SCL 9417 or SBR 9435; SEIBOLD AG, Vienna, Austria) in combination with an Ag/AgCI reference electrode were immersed in the solution. The electrodes were interfaced with the pH ionometer G 154 (SEIBOLD AG). The potential of the electrode was allowed to stabilize for approximately two minutes, and 2 g of celI immobilizate was added to start the conversion. When monitoring the formation of halogen ions, 5 min proved to be a sufficiently long period to obtain a significant decrease in electrode potential. All experiments were carried out at 25 "C.

Results and Discussion

To mediate the dehalogenation of chlorinated and brominated hydrocarbons in water samples, an active cellular biocatalyst was used. The specific activity of the cells was 5.4 U/g dry biomass using I-chlorobutane as a substrate. This cellular activity is com- parable to the data published by JANSSEN et af. [4]. In the first step of developing the microbial assay, several natural and synthetic polymers such as calcium alginate, carrageenan, chitosan, polyacrylamide-hydraide (PAAH) and chitosan-carboxymethyl cellulose were tested for the encapsulation of the dehalogenating cells. High specific activity of the immobilizate was achieved by using PAAH as a matrix substance, resulting in a specific activity of 61 mU/g immobilizate using I-chlorobutane as a substrate. With a substantially increased amount of dehalogenating cells of Rhodococcus sp. and with calcium alginate as a matrix, H ~ R et al. [2] achieved a specific activity of 114 mU/g immobilizate performing the assay with 1-chlorobutane as a substrate. Only slightly reduced activities of the immobilizate were observed using other matrix substances (substrate: 1 -chlorobutane, 50 mM). Clear differences were found by comparing the mass recovery of the immobilizate (Tab. 1). PAAH showed both a good recovery of matrix substances and of biomass. In contrast, immobilization of cells with calcium alginate, carrageenan or chitosan resulted in a considerable loss of materials, which was caused by the formation of a large amount of fines during preparation. Using chitosan-carboxymethyl cellulose as a matrix, there was a total yield of immobi- lizate of 107%, which was caused by swelling of the beads and osmotic effects during the immobilization procedure; this effect has already been described by YOSHIOKA et al. 1121. In addition, the mean diameter of the beads increased during the swelling process and attained almost 1.7 mm after 72 h. Investigations into long-term stability were per-

126 Acta Biotechnol. 17 (199'7) 2

formed by storing the beads at 4 "C for 12 weeks in a solution of 2% sodium phosphate, pH 7.0 (chitosan, PAAH, chitosan-carboxymethyl cellulose) or ammonium sulphate, pH 5.4 (carrageenan) or calcium nitrate, pH 5.2 (calcium alginate). There was a similar decrease of about 10% in the dehdogenating activity of all immobilizates.

Tab. 1. Immobilization of strain GJ 70 cells in different matrices

Matrix Mean bead Recovery of Recovery of diameter immobilizate dehalogenating

activity [mml 181 [%I

Calcium alginate 1.1 59 64 Carrageenan 1.2 57 60 chitosan 0.9 24 24 PAAH 1.1 86 95

Chitosan~arboxy- 1.3 methyl cellulose

107 100

Immobilized cells in PAAH were used as components of a bioassay with chloride or bromide selective potentiometric electrodes as sensors, as already described above. A comparable technique was used by H U - I T E R ~ ~ al. [2] with immobilized cells of Rhodococcus sp. in alginate beads. Aqueous solutions containing 1.2-dibromoethane or 1,3-dichloropropene were used for calibration. The results are given in Fig. 1 ~ showing non-linear graphs. According to the 3 a criterion, detection limits of 100 pg /1 and 25 pg / 1 were estimated for 1,3-dichloropropene and 1,2-dibromomethane, respec- tively. The reproducibility of the microbial assay was tested in five consecutive experi- ments measuring a sample solution containing 3 mgA 1,3-dichloropropene. The relative standard deviation was calculated as 5.9%.

30 T

0 0.0 1 0.1 1 10

Substrate [mgA J

Fig. 1. Calibration graphs for the bioassay Immobilized cells of strain GJ 70 (0.1 g/ml) were incuhated at 25 "C with aqueous solutions containing 1,2-dibromoethane (+) or 13-dichloropropene (B) in known amounts; liberated halide ions were detected by the ion selective potentiometric elec- trodes.

PETER, J. er of.. Microbial Assay for the Detection of Halogenated Hydrocarbons 127

Tab. 2. Relative degradation of halogenated hydrocarbons by immobilized cells of suain GJ 70 ~~~

Substrate Relative degradation rate [%I

1.3-Dichloropropene 100

1.5-Dichloropentane 63 1 -Chlorobutane 45

I-Chlorohexane 36

1.2-Dichloroethane 9 Trans- 1 ,2-dichloroeth ylene 2

Dichlorornethane 0

Trichloroeth ylene 0

1.2-Dibromoethane 100

1 .ZDibromopropane 85 Ethyl bromide 83

Isobutyl bromide 60

Measurements were carried out with PAAH-immobilized cells (0.1 g/ml) with a sub- strate concentration of 10 mM. The activities are expressed as percentages of the rate found with 1.3-dichloropropene (electrode SCL 9417) or 1,Zdibromoethane (electrode SBR 9435).

The initial rates for the conversion of several halogenated hydrocarbons by immobi- lized cells were determined using 10 mM substrate. The results are summarized in Tab. 2 in relation to the conversion rate of 1,3-dichloropropene for chlorinated hydro- carbons and to 1,Z-dibromoethane for brominated hydrocarbons; the data are means of duplicate assays showing a standard deviation of 3-596. 1-Haloalkanes (l-chloro- butane, I -chlorohexane) and a,o-dihaloalkanes (1.2-dibromopropane, 1,5-dichloro- pentane) were converted at a reduced rate, whereas a very slow rate for the liberation of halogen ions was found for 1,Zdichloroethane. Considering a standard deviation of 3-5% for the assay, the substrate trans-l,2-dichloroethylene was clearly not trans- formed by the immobilized cells. Dichloromethane and trichloroethylene showed no detectable conversion. The results agree with data on substrate specificity obtained with the purified halidohydrolase from GJ 70 cells, as reported by JANSSEN et al. [ 5 ] . There was obviously no shift in the substrate specificity of the dehalogenating activity, when immobilized cells were used instead of the pure enzyme.

Physical and Chemical Parameters

The effect of several physical and chemical parameters on the dehalogenating activity of strain GJ 70 cells was investigated using 10 mM 1-chlorobutane as a substrate and using an incubation period of 60 min. Results given are means of duplicate determi- nations showing a standard deviation in the range of 3-5%. The influence of

128 Acta Biotechnol. 17 (1997) 2

temperature was assayed between 20 and 70 "C at pH 8.5 (Fig. 2). There was almost no effect on cellular dehalogenating activity at temperatures below 40 "C. Variations in the pH assayed at 30 "C did not considerably affect the dehalogenating cellular activity in the range of pH 5-9 (Fig. 3).

20 30 40 50 60 70 Tempemtuz ["C]

Fig. 2. Effect of temperature on dehalogenating cellular activity Cells of strain GJ 70 (7.5 mglml) were incubated for 60 min at pH 8.5. The activities are expressed as percentages of the rate found with 10 m M l-chloro- butane at 30 "C.

0 2 4 6 a 10 12

PH

Fig. 3. Effect of the pH on dehalogenating cellular activity Cells of strain GJ 70 (7.5 mgfml) were incubated at 30 OC for 60 min. The activities are expressed as percentages of the rate found with 10 mM I-chloro- butane at pH 8.5.

The inhibition of the dehalogenating cellular activity by several heavy metal ions, organic substances and non-metabolizable compounds was studied at 30 "C and pH 8.5 (Tab. 3). Considering the reproducibility of the assay (3-5%), there is almost no effect of organic substances and non-metabolizable compounds on the dehalogenating activ- ity of the cells, whereas several heavy metal ions act only as inhibitors at unusually high concentrations.

PETER, J. et QL, Microbial Assay for the Detection of Halogenated Hydrocarbons 129

Tab. 3. Inhibition of the dehalogenating activity of strain GJ 70 cells

Concentration of potential inhibitors [mmole] Inhibition

Glucose Urea Sodium citrate Sodium acetate CU"

Feu Zn" Pb" Cd" Ba" Dichloromethane Trans-l,2-dichlorethylene Pb" Cd" Ba"

1 .o 1.0 1 .o 1 .o 1 .o 1.0 1 .o 1.0 1.0 1.0 1 .o 1.0 0.1 0.1

0.1

0 2

0 5

11 8

13 72 58 49 0 0

9 7 2

Cells (7.5 mg/ml) were incubated at 30 "C and pH 8.5 for 60 rnin. Inhibition is given as percentages of the cite found with 10 rnM I-chlorobutane.

Conclusion

Cells of an Actinomycete-Eke bacterium, strain GJ 70, entrapped in polyacrylamide- hydrazide gels allow, in a discontinuous assay, the detection of some chlorinated and brominated aliphatic hydrocarbons. These are included in the toxic pollutant list pub- lished by the American Toxic Pollutant Agency [13]. In contrast to the microbial bioassay developed by HU~TER et al. [Z] with cells of Rhodococcus sp., the microor- ganism used in this study allows the detection of environmentally important compounds such as 1,2-dibromoethane, 1,3-dichIoropropene and ethyl bromide. Nevertheless, several important halogenated hydrocarbons, which are frequently found as environmental pollutants, are not converted by strain GJ 70 cells i.e. trichloro- ethylene, dichloromethane, trans- 1,2-dichloroethylene, etc. Compared to the stop-flow- technique [14], the discontinuous method showed better detection ranges and a shorter incubation period, but needed more active handling. Nevertheless, the established bioassay wiI1 reduce the cost of determination of halogenated environmental pollutants. It will also be resistant to the effects of inhibition of high heavy metal ion concentrations and variations in pH and temperature.

Acknowledgement

The authors would like to express their gratitude to the "JubilSurnsfonds der Oesterreichischen Nationalbank" for supporting this project (No. 5654).

130 Acta Biotechnol. 17 (1997) 2

Received 13 February 1997 Received in revised form 28 April 1997 Accepted 29 April 1997

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