- Email: [email protected]
Rapid physiological characterization of microorganisms by biosensor technique
Microbiol. Res. (1997) 152,233-237
Microbiological Research 1) Gustav Fischer Verlag
Rapid physiological characterization of microorganisms by biosensor technique Klaus RiedeF, Gotthard Kunze! I 2
Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany Dr. Bruno Lange GmbH Berlin, Willstatter Str. 11, D-40549 DUsseldorf II, Germany
Accepted: April 6, 1997
Abstract Eleven microorganisms, Arxula adeninivorans LS3, Candida boidinii DSM 70034, Candida lactis-condensi DSM 70635, Pichia jadinii DSM 2361, Pichia minuta DSM 7018, Kluyveromyces lactis DSM 4394, Pseudomonas putida DSM 50026, Alcaligenes sp. DSM 30002, Arthrobacter nicotianae DSM 20123 as well as Issatchenkia orientalis DSM 70077 and Rhodococcus erythropolis DSM 311 were characterized by the sensor technique by injection of 30 different substrates and substrate mixtures. Th,e obtained data wich are based on the determination of respiratory rate of microorganisms are similar to physiological characteristics obtained with conventional methods. In comparison to these conventional methods the sensor technique works much more rapid and permits quantification of the data. Therefore, the described technique provides an alternative method for the characterization of microorganisms. Key words: biosensor
Introduction Detailed physiological characterization is one of the important prerequisites for the genetical and molecular biological handling of a microorganism. The actual characterization technique of microorganisms is very time consuming work which needs precise standard conditions. In recent years many microbial sensors were developed (Karube 1989, Riedel et al. 1989c; Karube and Suzuki 1990; Racek 1995; Wittmann et al. 1997). In these sensors microorganisms are in direct contact Corresponding author: G. Kunze
with a transducer, which converts the biochemical signal into a quantifiable electrical response signal. This principle can be used for sensitive detemlination of a large spectrum of substances to monitor pollution. Each microorganism is able to recognize a specific set of substances which is exploited for the determination of complex variables, such as the sum of biodegradable compounds and mutagenic compounds in waste water (Neujahr 1984; Karube and Endo 1991; Riedel et al. 1989c, 1992; Riedel 1994a, b). Wild types as well as genetically engineered organisms can be used as microorganisms for such sensors (Riedel et al. 1989b). The biosensor technique, which uses in most cases a combination of microorganisms and an amperometric oxygen electrode, offers an elegant possibility for the characterization of microorganisms (Riedel et al. 1985, 1988 a, 1989 a; Riedel 1996, 1997 a; Karube and Suzuki 1990; Byfield and Abuknesha 1994; Wood and Gruber 1996). The signal reflecting the changes of the respiratory rate must arise without limitation by substrate diffusion. A very low "microbe loading" of the biosensor is a prerequisite for such a kinetically controlled respiration electrode (Riedel et al. 1988b; Riedel 1991). In the present paper we describe the production and use of 11 biosensors containing various microorganisms. The data obtained characterize these 11 microorgamsms.
Materials and methods Microorganisms strains and culture conditions. Issatchenkia orientalis DSM 70077, Candida boidinii DSM 70034, C. lactis-condensi DSM 70635, Pichia jadinii DSM 2361, P. minuta DSM 7018, Kluyveromyces lacMicrobiol. Res. 152 (1997) 3
233
tis DSM 4394, Rhodococcus erythropolis DSM 311, Pseudomonas putida DSM 50026, Alcaligenes sp. DSM 30002 and Arthrobacter nicotianae DSM 20 123 were obtained from the "German Collection of Microorganisms and Cell Cultures" Braunschweig, Gennany. The strain Arxula adeninivorans LS3 (Basionym: Trichosporon adeninovorans) isolated from wood hydrolysates in Siberia (Russia) was taken from the yeast collection of the "Institute of Plant and Crop Plant Research" Gatersleben, Germany (Gienow et al. 1990). The yeast strains were grown under nonselective and aerobic conditions using yeast-extract-peptone-medium (YEPD) with 1% glucose as carbon source at 30 DC for 24 h. All bacterial strains were cultured in PM medium (0.5% peptone, 0.3% meat extract) or a especial Arthrobacter medium (1 % casein peptone - tryptic digest, 0.5% yeast extract, 1.5% NaCl, 0.5% glucose) under aerobic conditions at 24 DC for 24 h. Immobilization. Cells were harvested from 50 ml culture suspension by centrifugation and the sediment was resuspended with 0.1 M phosphate buffer (pH 6.8) to get a biomass concentration of 0.2-1.0 g wet weight! m1 (corresponding to approximately 4 times the dry weight: 0.05 - 0.25 mg dry weight!ml). An aliquot of a 5% polyvinylalcohol (PVA) solution was added to the microbial suspension and mixed thoroughly. This suspension was dropped onto a capillary membrane Rotroc (pore size 0.6llm; Oxyphen GmbH Dresden, Germany) in a volume of 20 fll to cover an area of about 6 mm diameter. These microbial membranes could be kept at 4 DC and were used between one and two weeks. The data of measurements varied between individual membranes by ±5%. Microbial sensor. The membran with immobilized microorganisms is placed on the teflon membrane of an oxygen electrode (Au cathode with a diameter of 5 mm; Dr. Bruno Lange GmbH Berlin, Germany) and fixed in place using either a bored cap or "O"-ling. Subsequently, the sensor was placed in a measuring cell containing 2.5 ml 100 mM phosphate buffer (pH 6.8, 37 DC). The change in CUlTent was followed at - 600 mV against Ag/AgCI (Riedel et al. 1985, Riedel 1994a, b, 1997a). The measurement of the biosensor response to an added substrate followed with endpoint determination as well as with kinetic measurements (the first derivative of the cUlTent/time curve) by a Sensor BOD-system ARAS (Dr. Lange GmbH Berlin, Germany) (Riedel 1993). The latter method was used for the determinations reported below. It allowed response times of 1 min. A recovery time of approximately 5 min was required between measurements. 234
Microbiol. Res. 152 (1997) 3
Results and discussion A. adeninivorans LS3, C. boidinii DSM 70034, C. lactis-condensi DSM 70635, P. jadinii DSM 2361, P. minuta DSM 7018, Kl. lactis DSM 4394, Ps. putida DSM 50026, Alc. sp. DSM 30002, Arth. nicotianae DSM 20123, I. orientalis DSM 70077 and Rho. erythropolis DSM 311 were characterized by the sensor technique. A cell mass of 6.8 mg dry weight per cm2 was necessary for immobilization on the capillary membrane to achieve a basis level of CUlTent (3 flA). In contrast to A. adeninivorans, 1. orientalis and Rho. el},thropoiis the immobilization was difficult for the other 8 microorganisms tested. Cultures of C. lactis-condensi, C. boidinii and Ps. putida achieved only low cell densities in the stationary growth phase and had to be highly concentrated for the immobilization. The concentration of Art. nicotianae, P. jadinii, P. minuta, Kf. lactis and Alc. sp. cells by centrifugation was also difficult. In most cases the required cell concentration could not be achieved. Therefore, these sensors were incubated with culture medium for some hours. Under these conditions the cells fixed on the membrane of the sensor divide. This procedure was calTied out until a basis CUlTent level (3 flA) was achieved similar to those of A. adeninivorans, I. orientalis and Rho. erythropolis. The sensors with various microorganisms were studied by injection of 30 different substrates and mixtures. Each microorganism showed differential sensitivities to the tested substrates. With glucose as standard substrate highest signals were measured with P. jadinii, C. lactiscondensi and Kl. lactis sensors whereas C. boidinii, P. minuta, Alc. sp., Art nicotianae, Ps. putida and I. Olientalis sensors gave only weak signals (Tab. 1). These results showed that the oxygen consumption varied among the microorganisms. Differences could be detected between the used substrates. The sensor containing Art. nicotianae reacted only with 10 of the 30 substrates and mixtures. In contrast, the sensor containing P. minuta recorded signals with all 30 substrates. However, these signals were relatively low between 1 and 84 nAimin. The best results were obtained with the A. adeninivorans, C. lactis-condensi, P. jadinii and Kl. lactis sensors. These reacted with most substrates and the signals were relatively high. Furthermore, the spectrum of substrate signals registered by the microbial sensors was compared with physiological data from the seven yeast species obtained with conventional techniques (Barnett et af. 1990, Riedel et af. 1997b). Many results were similar, but some results diverged (Tab. 2). For example in contrast to our technique the conventional technique revealed no usage of sucrose and maltose by C. boidinii (Barnett et af. 1990). The reasons for these differences are the metho-
a::(S.
V1
W
N
w
~
-.)
'D 'D
N
-
.... Ul
:n
(D
:;>.:I
?:
cr"
.... 0
3 32 0 37 I
10 7 32 4 12 5
21 28 18 11 23 7 4
citric acid acetic acid methyl alcohol ethyl alcohol glycerol
alanine glycine glutamic acid lysine methionine tryptophan
butyric acid capron acid Na capryl acid Na caproic acid Na lauryl acid Na propionic acid oil
1
0 3
61 291 581 458 88 40 3
96 102 19 87 81 27
5 180 0 160 7
134 64 114 2 48 0 57 20 55 24
LS3
Substrate concentrations were 1 mM.
5 5
43 8 3 0 4 0 17 0 15 7
glucose fructose 'galactose ribose xylose sorbitol sucrose lactose maltose glucosamin
phenol benzoate
70077
vorans
I. orientalis A. adenini-
DSM-Nr
microorganism
2 3
10 11 23 17 57 8 7
13 45 133 42 13 1 1 1 7
10 4 6 8 6 2
0 11 3 6 19
41 22 5 1 7 0 43 4 51 2
70034
C. boidinii
78 41 29 16 18 25
0 97 0 93 10
195 59 4 1 5 3 231 7 34 0
70635
C. laetiseondensi
2 1
0 0
0 0 1 1 1 0 2
143 60 185 175 228 49 3
26 12 3
11
0 0 0 0 0 0
35 10
20 5 9 0 0 0
4 6 2 2 1 0 21 17 31 22 20 28
23 34 21 69 8 7
4 13
34 125 232 256 83 128 9
3 12
28 76 84 57 43 38 2
4 5
15 31
72 85 134 34 52 4 7
0 281 0 19 89
0 35 0 1 184 1 63 0 121 2
3 78 28 39 8
0 250 0 152 160
0 0 0 7 30
0 2 0 28 6
5
11
19 78 35 45 15 35 2
98 204 0 0 2 0 9 0 2 I
55 7 12 4 0 9 0 37 0 12
189 49 187 3 3 0 168 299 25 0
53 7 3 1 7 6 6 6
195 105 0 0 4 0 313 57 66 10
61 0 0 0 0 0 2 2 4 0
72 26 17 3 10 0 52 7 20 10
7018
5 5
346 446 551 495 316 216 11
24 9 51 2 13 0
311
30002
4394
K. lactis
R. erythropolis
P. minuta
Ale. sp.
P. jadinii
2361
Art. nieotianae 20123
50026
Ps. putida
Table 1. Selectivities of sensors containing I. orientalis, A. adeninivorans, C. lactis-eondensi, C. boidinii, Ps. putida, Arth. nieotianae, P. jadinii, P. min uta, Kl. laetis, Ale. sp. and R. erythropolis to various substrates and substrate mixtures (nA/min)l.
Table 2. Comparison between the sensor data and the results obtained by the conventional technique and used for the taxonomic classification of A. adeninivorans, C. lactis-condensi, C. bo idin ii, P jadinii, P minuta, Kl. lac tis and I. orientalis. A. adeninivorans
C. lactiscondensi
C. boidinii
P jadinii
P minuta
K. lactis
I. orientalis
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
+ + + + + D + +
+ + +,+ + + + +
+
+
+
+
+
+ +
+ +
+,+ + + +,-
citric acid acetic acid methyl alcohol ethyl alcohol
D
+,+
+
+
+
Abbreviations:
conventional data (conv.)
microorganism
glucos~
galactose ribose xylose sucrose lactose maltose glucosarnin
+
+ +,D
W
+
+ +,+,+,+ +,+
+,-
+ +,+,+,+ +,+ +,-
+ +
+,+,+
+ +
+
positive response negative response variable response delayed positive response weak positive response
Microbiol. Res. 152 (1997) 3
+ +
+
+
+ +
+,+ + +
-D + +,+
+ + +,+,+ +.
+ -D
+,+
+ -D
+,-
+
+ +,-
+ +,-
+,+
-W
+,+
+
+
+
+,-
sensor data (sens.) + +,-
dical principles. Whereas the conventional technique measured utilisation of substrates using standard conditions, the biosensor technique registered changes in the oxygen consumption during a very short time. Effects like adaptation of cells to the substrate induction are not taken into consideration by the biosensor technique. The advantage of our technique is the sensitivity and the quantitative registration of the signals which enables recording of very low signals. Based on these results the sensor technique is a suitable method for the rapid characterization of microorganisms and allows gather information about the substrate spectrum. The quantitative data enables comparisons of different strains, mutants of transformants of one and the same species or investigations of transport effects as well as the activation of cell metabolism by the substrate. The comparison of obtained data among the microorganisms showed that especially the A. adeninivorans sensor is relatively simple to prepare. It reacted with most of the tested substrates. Additionally this yeast is a thermoresistant and osmotolerant yeast. It can be cultured up to 48°C in media containing up to 20% NaCI (Wartmann et ai. 1995; Kunze and Kunze 1996). All these properties favour this yeast for the microbial sensor technique for the determination of complex parameters (biochemical oxygen demand [BOD], mutagenicity and toxicity - Riedel 1996). Investigations in this field are in progress. 236
+
+D +D
+ +,+,+,+,+,+ +,-
>10 nAirnin 1-10 nA/min o nAirnin
Acknowledgements We are grateful to Dr. 1. Philipps, Prof. F. Schauer and Prof. K. MUntz for helpful discussions and critical reading of the manuscript. We also thank R. Franz and C. Riedel for excellent technical assistance.
References Barnett, 1. A., Payne, R. W. , Yarrow, D. (1990): Yeasts characteristics and identification. Cambridge Univ. Press, Cambridge, New York, Porst Chester, Melbourne, Sydney. Byfield, M. P., Abuknesha, R. A. (1994): Biochemical aspects of biosensors. Biosensors Bioelectron. 9, 373-400. Gienow, U., Kunze, G., Schauer, F., Bode, R., Hofemeister, 1. (1990): The yeast genus Trichosporon sp. LS3; molecular characterization of genomic complexity. Zentralbl. MikrobioI. 145,3-12. Karube, I. (1989): Microorganisms based sensors. In: Biosensors: Fundamentals and application. (eds. Turner, A. P. F., Karube, I., Wilson, G. S.) Oxford University Press, Oxford, pp.13-29. Karube, 1., Suzuki, M. (1990): Microbial sensors. In: Biosensors: A practical approach. (ed. Cass, A. E. G.) Oxford University Press, New York, pp. 155 -170. Karube, I., Endo, H. (1991): Biosensors in biodegradation of wastes. In: Biological degradation of wastes. (ed. Martin, A. M.) Elsevier, London, pp. 103-131. Kunze, G., Kunze, 1. (1996): Arxula adeninivorans. In: Nonconventional yeasts. (ed. Wolf, K.) Springer Verlag, Berlin, Heidelberg pp. 389-409.
Neujahr, H. Y. (1984): Biosensors for environmental control. Biotechnol. Genetic Engineer. Rev. 1, 167-186. Racek, J. (1995): Cell-based biosensors. Technomic Publ. Co Inc., Lancester, Basel. Riedel, K., Renneberg, R., Liebs, P. (1985): An electrochemical method for detennination of cell respiration. J. Basic Microbiol. Bioenerg. 25,51-56. Riedel, K., Renneberg, R., Liebs, P. (1988 a): Biochemical basis of kinetically controlled microbial sensor. Bioelectrochern. Bioenerg. 19, 137-144. Riedel, K., Liebs, P., Renneberg, R., Scheller, E (1988b): Characterization of the physiological state of microorganisms using the respiration electrode. Anal. Lett. 21, 1305-1322. Riedel, K., Renneberg, R., Scheller, E (1989 a): Studies in peptide utilization by microorganisms using biosensor techniques. Bioelectrochem. Bioenergetics 22,113-125. Riedel, K., Naumov, A. v., Grishenkov, V. G., Boronin, A. M., Stein, H. J., Scheller, E, Mueller, H. G. (l989b): Plasmidcontaining microbial sensor for E-caprolactam. Appl. Microbiol. Biotechnol. 31, 502-504. Riedel, K., Renneberg, R., Wollenberger, U., Kaiser, G., Scheller, E (1989 c): Microbial sensors: Fundamentals and application for process control. J. Chern. Biotechnol. 44, 85-106. Riedel, K. (1991): Biochemical fundamentals and improvement of selectivity of microbial sensors - a minireview. Bioelectrochem. Bioenerg. 25, 19-30. Riedel, K., Neumann, B., Scheller, E (1992): Mikrobielle Sensoren auf der Basis von Respirationsmessungen. Chern. Ing. Techn. 64,518-528. Riedel, K. (1993): Schnellbestimmung des biochemischen
Sauerstoffbedarfs mit mikrobiologischen Sensoren - Ein Uberblick. Vom Wasser 81, 243- 256. Riedel, K. (l994 a) : Microbial sensors and their applications in environment. Exp. Techn. Physics 40, 63-76. Riedel, K. (1994 b): Whole-cell and tissue sensors. In: Food biosensor analysis. (eds. Wagner, G., Guilbault, G. G.) Marcel Dekker, Inc, New York, Basel, Hong Kong, pp.I23-150. Riedel, K. (1996) : Microbial BOD sensors: Problems of practical use and comparison of sensor BOD and BODs. In: Frontiers in biosensorics II. (eds. Scheller, E W., Schubert, E, Fedrowitz, J.) Birkhauser Verlag, Basel, Switzerland, pp.99-108. Riedel, K. (1997 a): Microbial biosensors based on oxygen electrodes. In: Protocolls in biosensors research. (eds. Mulchandanil, A., Rogers, K.) Humana Press, USA (in press). Riedel, K., Lehmann, M., Adler, K., Kunze, G. (l997b): Application of a microbial sensor in the physiological characterization of the yeast Arxula adeninivorans LS3. Antonie van Leeuwenhoek 71, 345-35l. Wartmann, T., KrUger, A., Adler, K., Bui, M. D., Kunze, I., Kunze, G. (1995): Temperature dependent dimorphism of the yeast Arxula adeninivorans LS3. Antonie van Leeuwenhoek 68, 215-223. Wittmann, C, Riedel, K., Schmid, R. D. (1997): Microbial and enzyme sensors for environmental monitoring. In: Handbook of biosensors and electronic noses. (ed. Kress-Rogers, E.) CRC Press, New York, pp. 299-332. Wood, K. v., Gruber, M. G. (1996): Transduction in microbial biosensors using multiplexed bioluminescence. Biosensors Bioelectron. 11,207-214.
Microbial. Res. 152 (1997) 3
237
Microbiological Research 1) Gustav Fischer Verlag
Rapid physiological characterization of microorganisms by biosensor technique Klaus RiedeF, Gotthard Kunze! I 2
Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany Dr. Bruno Lange GmbH Berlin, Willstatter Str. 11, D-40549 DUsseldorf II, Germany
Accepted: April 6, 1997
Abstract Eleven microorganisms, Arxula adeninivorans LS3, Candida boidinii DSM 70034, Candida lactis-condensi DSM 70635, Pichia jadinii DSM 2361, Pichia minuta DSM 7018, Kluyveromyces lactis DSM 4394, Pseudomonas putida DSM 50026, Alcaligenes sp. DSM 30002, Arthrobacter nicotianae DSM 20123 as well as Issatchenkia orientalis DSM 70077 and Rhodococcus erythropolis DSM 311 were characterized by the sensor technique by injection of 30 different substrates and substrate mixtures. Th,e obtained data wich are based on the determination of respiratory rate of microorganisms are similar to physiological characteristics obtained with conventional methods. In comparison to these conventional methods the sensor technique works much more rapid and permits quantification of the data. Therefore, the described technique provides an alternative method for the characterization of microorganisms. Key words: biosensor
Introduction Detailed physiological characterization is one of the important prerequisites for the genetical and molecular biological handling of a microorganism. The actual characterization technique of microorganisms is very time consuming work which needs precise standard conditions. In recent years many microbial sensors were developed (Karube 1989, Riedel et al. 1989c; Karube and Suzuki 1990; Racek 1995; Wittmann et al. 1997). In these sensors microorganisms are in direct contact Corresponding author: G. Kunze
with a transducer, which converts the biochemical signal into a quantifiable electrical response signal. This principle can be used for sensitive detemlination of a large spectrum of substances to monitor pollution. Each microorganism is able to recognize a specific set of substances which is exploited for the determination of complex variables, such as the sum of biodegradable compounds and mutagenic compounds in waste water (Neujahr 1984; Karube and Endo 1991; Riedel et al. 1989c, 1992; Riedel 1994a, b). Wild types as well as genetically engineered organisms can be used as microorganisms for such sensors (Riedel et al. 1989b). The biosensor technique, which uses in most cases a combination of microorganisms and an amperometric oxygen electrode, offers an elegant possibility for the characterization of microorganisms (Riedel et al. 1985, 1988 a, 1989 a; Riedel 1996, 1997 a; Karube and Suzuki 1990; Byfield and Abuknesha 1994; Wood and Gruber 1996). The signal reflecting the changes of the respiratory rate must arise without limitation by substrate diffusion. A very low "microbe loading" of the biosensor is a prerequisite for such a kinetically controlled respiration electrode (Riedel et al. 1988b; Riedel 1991). In the present paper we describe the production and use of 11 biosensors containing various microorganisms. The data obtained characterize these 11 microorgamsms.
Materials and methods Microorganisms strains and culture conditions. Issatchenkia orientalis DSM 70077, Candida boidinii DSM 70034, C. lactis-condensi DSM 70635, Pichia jadinii DSM 2361, P. minuta DSM 7018, Kluyveromyces lacMicrobiol. Res. 152 (1997) 3
233
tis DSM 4394, Rhodococcus erythropolis DSM 311, Pseudomonas putida DSM 50026, Alcaligenes sp. DSM 30002 and Arthrobacter nicotianae DSM 20 123 were obtained from the "German Collection of Microorganisms and Cell Cultures" Braunschweig, Gennany. The strain Arxula adeninivorans LS3 (Basionym: Trichosporon adeninovorans) isolated from wood hydrolysates in Siberia (Russia) was taken from the yeast collection of the "Institute of Plant and Crop Plant Research" Gatersleben, Germany (Gienow et al. 1990). The yeast strains were grown under nonselective and aerobic conditions using yeast-extract-peptone-medium (YEPD) with 1% glucose as carbon source at 30 DC for 24 h. All bacterial strains were cultured in PM medium (0.5% peptone, 0.3% meat extract) or a especial Arthrobacter medium (1 % casein peptone - tryptic digest, 0.5% yeast extract, 1.5% NaCl, 0.5% glucose) under aerobic conditions at 24 DC for 24 h. Immobilization. Cells were harvested from 50 ml culture suspension by centrifugation and the sediment was resuspended with 0.1 M phosphate buffer (pH 6.8) to get a biomass concentration of 0.2-1.0 g wet weight! m1 (corresponding to approximately 4 times the dry weight: 0.05 - 0.25 mg dry weight!ml). An aliquot of a 5% polyvinylalcohol (PVA) solution was added to the microbial suspension and mixed thoroughly. This suspension was dropped onto a capillary membrane Rotroc (pore size 0.6llm; Oxyphen GmbH Dresden, Germany) in a volume of 20 fll to cover an area of about 6 mm diameter. These microbial membranes could be kept at 4 DC and were used between one and two weeks. The data of measurements varied between individual membranes by ±5%. Microbial sensor. The membran with immobilized microorganisms is placed on the teflon membrane of an oxygen electrode (Au cathode with a diameter of 5 mm; Dr. Bruno Lange GmbH Berlin, Germany) and fixed in place using either a bored cap or "O"-ling. Subsequently, the sensor was placed in a measuring cell containing 2.5 ml 100 mM phosphate buffer (pH 6.8, 37 DC). The change in CUlTent was followed at - 600 mV against Ag/AgCI (Riedel et al. 1985, Riedel 1994a, b, 1997a). The measurement of the biosensor response to an added substrate followed with endpoint determination as well as with kinetic measurements (the first derivative of the cUlTent/time curve) by a Sensor BOD-system ARAS (Dr. Lange GmbH Berlin, Germany) (Riedel 1993). The latter method was used for the determinations reported below. It allowed response times of 1 min. A recovery time of approximately 5 min was required between measurements. 234
Microbiol. Res. 152 (1997) 3
Results and discussion A. adeninivorans LS3, C. boidinii DSM 70034, C. lactis-condensi DSM 70635, P. jadinii DSM 2361, P. minuta DSM 7018, Kl. lactis DSM 4394, Ps. putida DSM 50026, Alc. sp. DSM 30002, Arth. nicotianae DSM 20123, I. orientalis DSM 70077 and Rho. erythropolis DSM 311 were characterized by the sensor technique. A cell mass of 6.8 mg dry weight per cm2 was necessary for immobilization on the capillary membrane to achieve a basis level of CUlTent (3 flA). In contrast to A. adeninivorans, 1. orientalis and Rho. el},thropoiis the immobilization was difficult for the other 8 microorganisms tested. Cultures of C. lactis-condensi, C. boidinii and Ps. putida achieved only low cell densities in the stationary growth phase and had to be highly concentrated for the immobilization. The concentration of Art. nicotianae, P. jadinii, P. minuta, Kf. lactis and Alc. sp. cells by centrifugation was also difficult. In most cases the required cell concentration could not be achieved. Therefore, these sensors were incubated with culture medium for some hours. Under these conditions the cells fixed on the membrane of the sensor divide. This procedure was calTied out until a basis CUlTent level (3 flA) was achieved similar to those of A. adeninivorans, I. orientalis and Rho. erythropolis. The sensors with various microorganisms were studied by injection of 30 different substrates and mixtures. Each microorganism showed differential sensitivities to the tested substrates. With glucose as standard substrate highest signals were measured with P. jadinii, C. lactiscondensi and Kl. lactis sensors whereas C. boidinii, P. minuta, Alc. sp., Art nicotianae, Ps. putida and I. Olientalis sensors gave only weak signals (Tab. 1). These results showed that the oxygen consumption varied among the microorganisms. Differences could be detected between the used substrates. The sensor containing Art. nicotianae reacted only with 10 of the 30 substrates and mixtures. In contrast, the sensor containing P. minuta recorded signals with all 30 substrates. However, these signals were relatively low between 1 and 84 nAimin. The best results were obtained with the A. adeninivorans, C. lactis-condensi, P. jadinii and Kl. lactis sensors. These reacted with most substrates and the signals were relatively high. Furthermore, the spectrum of substrate signals registered by the microbial sensors was compared with physiological data from the seven yeast species obtained with conventional techniques (Barnett et af. 1990, Riedel et af. 1997b). Many results were similar, but some results diverged (Tab. 2). For example in contrast to our technique the conventional technique revealed no usage of sucrose and maltose by C. boidinii (Barnett et af. 1990). The reasons for these differences are the metho-
a::(S.
V1
W
N
w
~
-.)
'D 'D
N
-
.... Ul
:n
(D
:;>.:I
?:
cr"
.... 0
3 32 0 37 I
10 7 32 4 12 5
21 28 18 11 23 7 4
citric acid acetic acid methyl alcohol ethyl alcohol glycerol
alanine glycine glutamic acid lysine methionine tryptophan
butyric acid capron acid Na capryl acid Na caproic acid Na lauryl acid Na propionic acid oil
1
0 3
61 291 581 458 88 40 3
96 102 19 87 81 27
5 180 0 160 7
134 64 114 2 48 0 57 20 55 24
LS3
Substrate concentrations were 1 mM.
5 5
43 8 3 0 4 0 17 0 15 7
glucose fructose 'galactose ribose xylose sorbitol sucrose lactose maltose glucosamin
phenol benzoate
70077
vorans
I. orientalis A. adenini-
DSM-Nr
microorganism
2 3
10 11 23 17 57 8 7
13 45 133 42 13 1 1 1 7
10 4 6 8 6 2
0 11 3 6 19
41 22 5 1 7 0 43 4 51 2
70034
C. boidinii
78 41 29 16 18 25
0 97 0 93 10
195 59 4 1 5 3 231 7 34 0
70635
C. laetiseondensi
2 1
0 0
0 0 1 1 1 0 2
143 60 185 175 228 49 3
26 12 3
11
0 0 0 0 0 0
35 10
20 5 9 0 0 0
4 6 2 2 1 0 21 17 31 22 20 28
23 34 21 69 8 7
4 13
34 125 232 256 83 128 9
3 12
28 76 84 57 43 38 2
4 5
15 31
72 85 134 34 52 4 7
0 281 0 19 89
0 35 0 1 184 1 63 0 121 2
3 78 28 39 8
0 250 0 152 160
0 0 0 7 30
0 2 0 28 6
5
11
19 78 35 45 15 35 2
98 204 0 0 2 0 9 0 2 I
55 7 12 4 0 9 0 37 0 12
189 49 187 3 3 0 168 299 25 0
53 7 3 1 7 6 6 6
195 105 0 0 4 0 313 57 66 10
61 0 0 0 0 0 2 2 4 0
72 26 17 3 10 0 52 7 20 10
7018
5 5
346 446 551 495 316 216 11
24 9 51 2 13 0
311
30002
4394
K. lactis
R. erythropolis
P. minuta
Ale. sp.
P. jadinii
2361
Art. nieotianae 20123
50026
Ps. putida
Table 1. Selectivities of sensors containing I. orientalis, A. adeninivorans, C. lactis-eondensi, C. boidinii, Ps. putida, Arth. nieotianae, P. jadinii, P. min uta, Kl. laetis, Ale. sp. and R. erythropolis to various substrates and substrate mixtures (nA/min)l.
Table 2. Comparison between the sensor data and the results obtained by the conventional technique and used for the taxonomic classification of A. adeninivorans, C. lactis-condensi, C. bo idin ii, P jadinii, P minuta, Kl. lac tis and I. orientalis. A. adeninivorans
C. lactiscondensi
C. boidinii
P jadinii
P minuta
K. lactis
I. orientalis
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
conv. sens.
+ + + + + D + +
+ + +,+ + + + +
+
+
+
+
+
+ +
+ +
+,+ + + +,-
citric acid acetic acid methyl alcohol ethyl alcohol
D
+,+
+
+
+
Abbreviations:
conventional data (conv.)
microorganism
glucos~
galactose ribose xylose sucrose lactose maltose glucosarnin
+
+ +,D
W
+
+ +,+,+,+ +,+
+,-
+ +,+,+,+ +,+ +,-
+ +
+,+,+
+ +
+
positive response negative response variable response delayed positive response weak positive response
Microbiol. Res. 152 (1997) 3
+ +
+
+
+ +
+,+ + +
-D + +,+
+ + +,+,+ +.
+ -D
+,+
+ -D
+,-
+
+ +,-
+ +,-
+,+
-W
+,+
+
+
+
+,-
sensor data (sens.) + +,-
dical principles. Whereas the conventional technique measured utilisation of substrates using standard conditions, the biosensor technique registered changes in the oxygen consumption during a very short time. Effects like adaptation of cells to the substrate induction are not taken into consideration by the biosensor technique. The advantage of our technique is the sensitivity and the quantitative registration of the signals which enables recording of very low signals. Based on these results the sensor technique is a suitable method for the rapid characterization of microorganisms and allows gather information about the substrate spectrum. The quantitative data enables comparisons of different strains, mutants of transformants of one and the same species or investigations of transport effects as well as the activation of cell metabolism by the substrate. The comparison of obtained data among the microorganisms showed that especially the A. adeninivorans sensor is relatively simple to prepare. It reacted with most of the tested substrates. Additionally this yeast is a thermoresistant and osmotolerant yeast. It can be cultured up to 48°C in media containing up to 20% NaCI (Wartmann et ai. 1995; Kunze and Kunze 1996). All these properties favour this yeast for the microbial sensor technique for the determination of complex parameters (biochemical oxygen demand [BOD], mutagenicity and toxicity - Riedel 1996). Investigations in this field are in progress. 236
+
+D +D
+ +,+,+,+,+,+ +,-
>10 nAirnin 1-10 nA/min o nAirnin
Acknowledgements We are grateful to Dr. 1. Philipps, Prof. F. Schauer and Prof. K. MUntz for helpful discussions and critical reading of the manuscript. We also thank R. Franz and C. Riedel for excellent technical assistance.
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