A novel microbial sensor for the determination of cyanide

AluLYTIcA CHIMICA

ELSEVIER

ACTA Analytica Chimica Acta 313 (1995) 69-74

A novel microbial sensor for the determination of cyanide Jeong Im Lee, Isao Karube

*

Research Center for Advanced Science and Technology, Uniuersity of Tokyo, 4-6-l Kornaba, Meguro-ku, Tokyo 153, Japan Received 21 February 1995; revised 5 April 1995; accepted 27 April 1995

Abstract A novel cyanide biosensor was developed using cyanide-degrading microorganisms immobilized in membrane and an oxygen electrode. Immobilized Pseudomonas fruorescens NCIMB 11764 specifically oxidized cyanide thereby consuming oxygen without reacting with other toxic materials (like Cr3+, Cd’+, Pb 2+, LAS). A change in oxygen concentration was detected using a Clark oxygen electrode. Cyanide could thus be determined at concentrations between 0.1 and 1 ppm in 50 mM phosphate buffer (pH 8). The optimum working conditions of the sensor are pH 8 at 3O”C, resulting in a response time of less than 2 min. This sensor was unaffected by chloride and heavy-metal ions. The relative standard deviation was estimated to be about 8%. With its specificity, rapid response time and ease of operation, this cyanide sensor has a potential commercial application for the monitoring and sensing of cyanide in industrial wastes. Keywords: Biosensors; Oxygen consumption; Environmental analysis; Cyanide degrading bacteria; P. fluorescens NCIMB11764

1. Introduction In recent years, protection of the global environment gets much attention, and has become recognized world-wide as a problem. Monitoring of environmental pollution is therefore one of the most important controlling options. One of these pollutants, cyanide, is extremely toxic to living systems by inactivating respiration, due to its strong binding to cytochrome c oxidase and to a lesser extent to other metalloproteins [l]. Cyanide waste is generated by industries involved in metal plating, coke production, plastics manufacture, and ore leaching. The determination of cyanide concentration, therefore, is

* Corresponding author.

of fundamental importance in environmental monitoring, controlling and in industrial waste analysis. Conventional methods for the determination of cyanide are a calorimetric method and the use of a cyanide electrode. The widely used calorimetric method involves toxic reagents and is subjected to many interfering factors [2]. In addition, the response time is long and the method is complicated. The commercially available cyanide electrode is vulnerable to sulfide and iodide interferences [3,4]. Recently, many biosensors have been developed for environmental monitoring, such as those for the determination of ammonia [5,6], phosphate [7] and biological oxygen demand (BOD) [8,9]. Microbial biosensors are very suitable for environmental monitoring since they are relatively inexpensive and simple to operate. A number of reports on the microbial degradation

0003-2670/95/%09.50 0 1995 Elsevier Science B.V. AII rights reserved SSDI 0003-2670(95)00232-4

70

J.I. Lee, I. Karube/Analytica cyanide

oxidase

CN’ + 02 fjNADH,

OCN’ + Hz0 NAD+

OCN’ + H20 ‘=NHs+ Fig. 1. The overall stoichiometry cyanide.

CO2

of the oxidative

degradation

of

Chimica Acta 313 (1995) 69-74

phate buffer (pH 7.0), and subsequently resuspended in the same buffer. A 0.3 ml portion of the suspension (40 mg) was added dropwise onto a cellulose nitrate membrane (2 mm diam., 0.45 pm pore size, Advantec) upon slight suction. The membrane was washed with the 50 mM phosphate buffer (pH 7.0) and stored at 4°C. 2.3. Construction

of cyanide wastes have appeared. Harris and Knowles [lO,ll], for example, described the isolation of a number of cyanide utilizing Pseudomonas from soil, of which P. fluorescens NCIMB 11764 aerobically biodegrades cyanide as a sole nitrogen source. The overall stoichiometry of oxidative cyanide degradation by P. fluorescens NCIMB 11764 is shown in Fig. 1. Utilization of an oxygenase produces cyanate as an intermediate which would then be hydrolyzed by cyanase to carbon dioxide and ammonia. Oxygen uptake by bacteria can directly be determined by an oxygen electrode onto which the bacteria are immobilized. This is the first microbial sensor developed for the measurement of cyanide. In this paper, determination of cyanide with a microbial sensor using whole cells of P. ffuorescens NCIMB11764 and an oxygen electrode is described.

2. Experimental 2.1. Microorganism

and culture

P. fluorescens NCIMB11764 was cultured in a liquid medium on a reciprocal shaker at 30°C for 24 h. The medium consisted of 10 mM glucose in M9 minimum salt medium (per liter; 6 g Na,HPO,; 3 g KH,PO,; 5 g NaCl; 1 mM NI-I,Cl) [12] containing 1 ml of trace metals solution per liter (10.75 g MgO; 2.0 g CaCO,; 4.5 g FeSO,; 1.44 g ZnSO, .7H,O; 1.12 g MnSO,. 4H,O; 0.25 g CuSO, .5H,O; 0.28 g CoSO, * 7H,O; 0.06 g H,BO,; 51.3 ml cont. HCl) [13]. P. fluorescens NCIMB 11764 was preserved on nutrient agar plates at 4°C. 2.2. Cell immobilization The stationary phase growing cells were harvested at 3000 g, washed twice with 50 mM sodium phos-

of the cyanide biosensor

The experimental set-up of the cyanide biosensor is shown in Fig. 2. A Clark-type oxygen electrode (Model BO-Ul, Able, Japan) consisted of a PTFE membrane, a platinum cathode, an aluminum anode and a saturated potassium chloride electrolyte solution. The microorganism immobilized membrane is placed on the membrane and fixed in place using 200 mesh nylon and an O-ring. The cells were thus entrapped between two membranes. 2.4. Measurement procedures The microbial electrode was inserted into the detection chamber containing 30 ml of oxygensaturated 50 mM phosphate buffer (pH 8.0) or Watarase river water (Japan, August, 1994), while continuously stirring with a magnetic bar. The temperature of the detection chamber was maintained at 30°C. The current output of the oxygen electrode was measured using a digital multimeter (Keithley, 175) and with an electronic poly-recorder (TOA Electronics, Model EPR-151 A). After equilibrium was established, 30 ~1 of KCN solution was injected

III 0a

4

Fig. 2. Construction of the cyanide sensor. (1) Oxygen electrode, (2) resistor, (3) recorder, (4) digital multimeter, (5) thermostat, (6) magnetic stirrer, (a) F’TFE membrane, (b) microorganism immobilized membrane, (c) nylon mesh.

J.I. Lee, I. Karube/Analytica Chimica Acta 313 (1995) 69-74 Current

3. Results and discussion

* WI

71

,o -

3.1. Response and calibration sor

O.lppm

of the cyanide biosen-

/

01

washing

f’ Ti

-

0.3 w

-

0.5 ppm

+--

0.7 ppm

L

-

/

1

d

Fig. 3. Typical

response

l.Oppm

i

ix =t

curves for various

KCN concentrations.

into the detection chamber and the decrease in voltage was recorded on the chart recorder (Fig. 2). Selectivity of the microbial sensor to various toxic substances such as Pb2+, Cd2+ and Cr3+, a non-linear alkyl benzene sulphonate (LAS) as an anionic surfactant and simazine as a pesticide, were studied at pH 8.0 and 30°C. 2.5. Effect of heavy-metal sponse

ions on the sensor

Typical response curves of the cyanide sensor to different concentrations of KCN (0.1-l pg ml-‘) are shown in Fig. 3. A cyanide solution was injected when the current became steady (baseline current, C,). Consequently, the difference between the response current (C) and C, was determined as the sensor response (AC), reaching a maximum after 2 min and returning to the baseline within 10 min. The sensor response showed a linear relationship between current decrease and cyanide concentration for 0.1 to 1 pg ml-’ (Fig. 4). Above 1.5 pg ml-‘, the response did not increase (data not shown); it might be that formation of ammonia inhibited the cyanide degrading reaction [14]. Although the application concentration range is small the sensor response might be further increased by improvement of sensor construction and different immobilization techniques for the microorganism. The maximum permitted concentration of cyanide in waste water is defined as 1 pg ml-’ by the Water Pollution Control Law in Japan. Therefore, this sensor is practically useful for monitoring cyanide in rivers. For a 1 pg ml-’ cyanide solution, a reproducible response could be obtained within f8% relative error of the mean

-r-----l

re-

The microbial electrode was inserted into 50 mM phosphate buffer (pH 8.0 at 30°C) containing 1 pg ml-’ heavy metal ion (like Cu2+, Zn2+, Fe3+ or Mn2+). After equilibrium was established, 30 ~1 of KCN solution was injected and the current decrease measured.

KCN concentration

(ppm)

Fig. 4. Correlation between KCN concentration crease (50 mM phosphate buffer, pH 8.0, 30°C).

and current

de-

72

J.I. Lee, I. Karube /Analytica

o

0’

0

o

10

a

20

Temperature

3.2. Optimization conditions

deviation

40

(“C)

Fig. 5. Effect of temperature on the current phosphate buffer, pH 8.0, 1 pg ml-’ KCN).

value; the standard (n = 10).

’

30

decrease

(50 mM

was 0.07 pg

Chimica Acta 313 (1995) 69-74

resulting in a maximum current decrease at 30°C (Fig. 51, which was the optimum temperature for the growth of P. fluorescens NCINB 11764 [lo]. The optimum pH was investigated between pH 5 and 9. The response increased rapidly with pH to give a maximum at pH 8. Above pH 8 the response rapidly declined (Fig. 6). Such behavior is characteristic of all Pseudomonas species which grow well in a neutral or slightly alkaline environment [15]. The effect of the amount of immobilized microorganism on the sensor response was also examined. In Fig. 7, 40 mg (wet weight) of immobilized microorganism gives the maximum current decrease. Above 40 mg, the response declined, which may be related to the thickness of the immobilized membrane, resulting in a diffusion limitation of 0, and substrate (CN-).

ml-’ 3.3. Selectivity

of the cyanide biosensor operating

Optimum conditions of the sensor were determined. The effect of temperature and pH on the current decrease was investigated in 50 mM phosphate buffer, for 1 pg ml-’ KCN. The temperature was varied between 5 and 35°C

of the cyanide biosensor

The sensor response to other toxic substances was also examined between 0.1 and 1 pg ml-‘. The biosensor did not respond to LAS, Cd’+, Cr3+ and Pb2+ (Fig. 8), but gave a slight response to simazine, opposite in sign (data not shown). The negligible response to these toxic materials is important in that the present biosensor is specific and sensitive to cyanide.

l

2304

0’ 4

n

5

c

* 6

7

I 8

.

m

9

’

20

I 30

Cell

decrease

I 50

I 60

70

10

PH

Fig. 6. Effect of pH on the current KCN).

I 40

(3O”C, 1 pg ml-’

wet

weight

(mg)

Fig. 7. Influence of the amount of immobilized microorganism on the current decrease (50 mM phosphate buffer, pH 8.0, 3O”C, 1 pg ml-’ KCN).

J.I. Lee, I. Karube/Analytica

73

Chimica Acta 313 (1995) 69-74

400’

Table 1 Effect of heavy metal ions on the current decrease ml-l) Heavy metal ions (1 pg ml-‘)

Current decrease

aoo-

Control Cr3+ Fe3+ Mn*+ Cl?+ Zn*+

100 100 100 98 97 93

zoo100’

(KCN: 1 pg

(%I

0-

_,W’

.

0.0

I

,

,

,

.

.

0.2

0.4

0.6

0.8

1.0

1.2

the determination of cyanide in river water, which normally has a lower NaCl concentration.

Toxic matarid concentration (pg ml “)

Fig. 8. Correlation ~lr+~bpos~ ,

between toxic materials and current decrease buffer, pH 8, 3O’C). 0 = KCN, 0 = Cr3+, .

3.4. Effect of NaCl biosensor response

concentration

on the cyanide

River water contains materials that could effect the sensor response. One of these, chloride, is present in river waters. The effect of Cl- (NaCl) on the sensor response was investigated in 50 mM phosphate buffer (pH 81, for 1 pug ml-’ KCN. Fig. 9 shows that the sensor was not affected by NaCl up to 30 ,ug ml-‘. Therefore, the sensor can be applied for

_

4oor---l

0

10

NaCl

20

30

concentration

3.5. Effect of heavy biosensor response

50

60

(ppm)

Fig. 9. Effect of NaCl concentration on the current decrease mM phosphate buffer, pH 8.0, T = 3072, 1 pg ml-’ KCN).

(50

ions on the cyanide

Most river waters contain various heavy metal ions, like Cu2+, Zn2+, Fe3+ and Mn2’. Since the presence of these heavy metal ions in river water might interfere with the microorganisms activity, their effect at 1 pg ml-’ on the sensor response was investigated at 1 pg ml-’ CN-. The results in Table 1 show that Fe3+, Zn*+, Mnzf, Cr3+ and Cu*+ (1 ppm) have virtually no effect on the response of the cyanide sensor. In this study, 50 mM sodium phosphate buffer (pH 8, 30°C) was used as a control. Our results agree with results reported by Trevors et al. [16], that many bacteria, including several Pseudomonas species, are resistant to metal ions. 3.6. Application water analysis

40

metal

of the cyanide

biosensor

to river

An investigation of the effects of other substances present in a sample is extremely difficult because of variations between different rivers. The cyanide sensor was used for the determination of cyanide in river water. The sample was taken from the Watarase river (Japan) in August 1994 to which KCN was added. It was anticipated that many substances would be present in the Watarase river water because it is polluted with many heavy metal ions [17]. Fig. 10 shows that the response of the cyanide biosensor was satisfactory for Watarase river water containing 0.1 - 1 pg ml-’ cyanide. The sensor response for

74

J.I. Lee, I. Karube/Analytica

Chimica Acta 313 (1995) 69-74

The system was applicable to measuring cyanide concentrations in river water. The response time of the sensor was 2 min.

Acknowledgements The authors thank Dr. A.V. Elgersma ing the manuscript.

for correct-

References 0.0

0.2

0.4

0.6

KCN concentration

0.6

1.0

1.2

111L.P. Solomonson,

(ppm)

Fig. 10. The correlation between KCN concentration and current decrease in river water. (0) Watarase river water, (0) 50 mM sodium phosphate buffer (pH 7.01.

Watarase river water shows response in phosphate buffer.

no difference

for the

3.7, Stability of the cyanide biosensor The stability of the sensor was examined at 4°C over 30 days in 50 mM phosphate buffer (pH 8.0). The actual sensor response was measured at 30°C. The sensor did respond beyond 30 days, but the sensor response had declined to half of the initial response within 2 weeks. Since the stability of the sensor is short, further study regarding the stability of the sensor is required before practical environmental monitoring can be achieved.

4. Conclusions Since conventional methods for the detection of cyanide are subjected to many interferences and require long and complicated procedures, a fast responding and stable sensor is needed for cyanide monitoring. In this study, a cyanide sensor is described using whole cells of P. fi7uorescens NCIMB 11764, sensitive for cyanide with almost no interference. The oxygen uptake was linearly related to the cyanide concentration in the range 0.1-l pg ml- ‘.

in B. Vennesland et al. (Ed%), Cyanide in Biology, Academic press, New York, 1981, pp. 11-28. i21 M.A. Franson (Ed.), Standard Methods for the Examination of Water and Wastewater, 15th edn., Vol. 312, American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, DC, 1980, pp. 312-332. [31 MS. Frant, Plating, 58 (1971) 686. 141 MS. Frant, J.W. Ross and J.H. Riseman, Anal. Chem., 44 (1972) 2227. [51 M. Hikuma, T. Kubo, T. Yasuda, I. Karube and S. Suzuki, Anal. Chem., 52 (1980) 1020. 1611. Karube, T. Okada and S. Suzuki, Anal. Chem., 53 (1981) 1852. 171 T. Matunaga, S. Suzuki and R. Tomoda, Enzyme Microb. Technol., 6 (1984) 355. 181 K. Riedel, R. Renneberg, M. Kuhn and F. Scheller, Appl. Microb. Biotechnol., 28 (1988) 316. [91 S.E. Strand and D.A. Carlson, J. Water Pollut. Control Fed., 56 (1984) 464. [lOI R.E. Harris and C.J. Knowles, J. Gen. Microbial., 129 (1983) 1005. 1111 R.W. Harris and C.J. Knowles, J. FEMS Microbial Lett., 20 (19831 337. in Molecular Genetics, Cold 1121 J.H. Miller, in Experiments Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, p. 431. t131 T. Bauchop and S.R. Elsden, J. Gen. Microbial., 23 (1960) 457. 1141 D.A. Kunz, 0. Nagappan, J. Siva-Avalos and G.T. Delong, Appl. Environ. Microbial., 58 (1992) 2022. [I51 G. Phillips (Ed.), Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, DC, 1981, pp. 85-98. 1161J.T. Trevors, K.M. Oddie and B.H. Belliveau, FEMS Microbiol. Rev., 32 (1985) 39. [I71 River Bureau, Ministry of Construction, Japan, Annual Report on River Water Quality in Japan (Suishhitu Nenkanl, 1992.