DE10034578B4 - Biosensors, process for their preparation and their use - Google Patents

Abstract

Biosensor consisting of microorganisms as biocomponent and a physical transducer, characterized in that the biocomponent is in mycelial form.

Description

The The present invention relates to novel microbial biosensors mycelium-based, process for their preparation and their use. According to the invention, microorganism mycelium becomes thin compact layers on a solid medium produced and preferably immobilized on polymers. These solid nutrient media will be in combination with physical transducers as biosensors for the Environmental analysis, biotechnology, food analysis and medical Diagnostics, in particular for the determination of BOD and determination of disaccharides, applied. Such sensors are used in various fields of application, especially used in environmental control

Cell-based microbial biosensors are known for the determination of a large number of organic and inorganic compounds, such as sugars, amino acids, peptides, aromatics, haloaromatics, urea, phosphate, etc. (see Riedel K. et al .: Microbial Sensors: Fundamentals and Application for Process Control, J. Chem., Biotechnol., 44, 85-106 (1989), Riedel, K. et al .: Microbial Sensors Based on Respiratory Measurements, Chem.-Ing.-Techn., 64, 518-528 (1989). Wittmann, C. et al .: Microbial and enzyme sensors for environmental monitoring In: Handbook of biosensors and electronic noses (Ed .: Kress-Rogers, E.) CRC Press, pp. 299-332 (1997)) , Their use in the wastewater control is also widespread, for the determination of the "Biochemical oxygen demand" (BOD), your statement about the load of the respective wastewater allows ( DD 253 045 A1 . DD 282 472 A5 . DE 43 01 087 A1 , Riedel K. vom Wasser 81, 243-256 [1993]).

Microbial sensor components used for these sensors are microorganism species with the broadest possible substrate spectrum, such as Trichosporon cutaneum (Hikuma, M. et al., European Journal, Appl. Microbiol. Biotechnol., 8: 289-297 [1979]), Klebisella (1979). EP 0 543 407 A1 ), Arxula adeninivorans ( DE 196 20 250 A1 ) and combinations of complementary microorganism species ( DE 431 4981 A1 ).

Of the The invention was based on the object of providing further biosensors, the special performance of microorganisms for Biosensors can harness and to develop corresponding processes for their production.

Surprised it was found possible is also mycelia of microorganisms as a microbial component to use the biosensors. The mycelium presents under these conditions a compact layer.

So far Microorganisms were used only in the form of cells in biosensors. For one However, a whole series of microorganisms exist different growth forms. One speaks also of Pleiomorphismus. Depending on the cultivation or environmental conditions form, for example, yeasts and streptomycetes in addition to the cells (single-celled growth form) also mycelia, which also be referred to as mushroom braid (Schlegel, H. G. [1981]: Allgemeine Microbiology. 5th edition Thieme, Stuttgart).

The object is achieved according to the claims. Particularly preferred is a biosensor with the yeast Arxula adeninivorans as mycel forming biocomponent. The object is achieved in particular by providing biosensors in which the mycelium obtained by surface growth on a solid medium in the form of a compact 2-100 micron thick plate and this is used directly in biosensors. Further solidification is possible in a preferred embodiment by immobilization with inorganic and organic polymers in analogy to known techniques ( DE 197 36510 A1 Polymers within the meaning of the invention are polyurethanes, polyvinyl alcohols and the like

In a further preferred embodiment, the mycelia are fixed on a permeable carrier membrane, preferably a dialysis membrane, and optionally further with an immobilizing agent, preferably polymers in analogy to DE 197 36510 A1 stabilized. As a dialysis membrane, a nuclear spore membrane is preferably used (Riedel, K. et al., Anal. Lett. 31: 1-12 [1998]). It serves as a carrier on which yeast cells and mycelia are immobilized.

The biosensors according to the invention are diverse usable, in particular for the determination of the biological oxygen demand (BSB) and numerous substrates, such. B. of monosaccharides, Disaccharides, polysaccharides, alcohols, amino acids or other organic compounds.

The morphological morphology is changed by culturing in a conventional culture dium according to Riedel et al. (1998), Anal. Lett. 31: 1-12). In contrast to the known cultivation method, however, the cultivation is carried out at a higher temperature,> 42 ° C, which leads to the formation of mycelia in A. adeninivorans, preferably strain LS3.

Preferably is at a temperature of 45 ° C cultured. There is no morphological change in the sensor more.

following the invention is representative Examples explained. For this, the yeast A. adeninivorans, which is particularly distinguished by its broad Substrate spectrum, as well as their high temperature stability and salt tolerance is suitable as a microbiological BOD sensor (Kunze, I. & Kunze G. Antonie van Leeuwenhoek 65, 29-34 [1994], Wartmann T. et al. Antonie van Leeuwenhoek 68, 215-223 [1995]), used. This yeast is able, with the help of its environmental Dimorphism to change their cell morphology. So A. adeninivorans grows at one Temperature up to 41 ° C as cell culture, at 42 ° C as pseudomycel and at higher Temperatures as mycelium.

Of the Use of morphological forms in biosensors is not yet known. According to the invention were For the first time, mycelia, preferably of A. adeninivorans, are preferred Strain LS3, used. As can be seen from the examples, the Microbiological sensors with mycelium as a microbial sensor component Superior to yeast cells. The mycelial form is now characterized by greater stability (eg. Thermal stability, Osmostabilität) and in comparison to the cell shape by increased metabolic activity.

There These mycelia also in their physiological parameters of Distinguish yeast cells, such superiority was unpredictable. The measurement is carried out at a temperature of 15 ° C-50 ° C, wherein the measuring liquid optionally up to 30% salt, in particular saline.

One Use of mycelia in biosensors is due to their broad Substrate spectrum and the large temperature stability and salt tolerance therefore preferable to the use of yeast cells.

embodiments

example 1

For the preparation of A. adeninivorans LS3 as a microbial sensor component a 36-hour Cultivation in YEPD medium (0.3% peptone, 0.3% malt extract, 0.3% Yeast extract, 1% glucose) at 45 ° C. The after cultivation at 45 ° C (Medium: 0.5% yeast extract; 0.5% peptone; 1% glucose) obtained mycelium of A. adeninivorans LS3 is centrifuged off, resuspended, immobilized and preparing the microbial sensors in a known manner.

Around preferably Obtaining high sensor signals requires an optimal amount of mycelia be immobilized on the dialysis membrane. It is important to take into account that with increasing mycelium concentration of oxygen consumption and thus also the measuring signal and the stability of the sensor increases become. At the same time, too high mycelial concentrations influence this Sensor signal negative, as this reduces oxygen and Substrate diffusion leads.

Dialysis membranes were loaded with 2.5-30 mg mycelia / cm ² and tested for their suitability for BOD measurement. After a 12-hour activation of the sensors with 1.88 mM glucose, immobilization of 6.8 mg mycelium / cm ² achieves the optimum basic current (Io) of 4500 nA for the BOD measurement.

At the same time, the SensorBSB values of 0-8 mM glucose were determined with the loaded dialysis membranes. The best readings were obtained with the 6.8 mg mycelium / cm ² loaded membranes. Here, a linearity of the SensorBSB-signal up to 524 mg O _2/1 will be achieved with a r ² of 0.9681. Both the quantification limit of 7.73 mg O ₂ / l and the detection limit of 2.61 mg O ₂ / l are significantly more favorable for measurements with such a mycelium sensor than for the cell sensor ( 1 , Tab. 1). Tab. 1: Detection and quantification limit of Arxula mycelium and cell sensors. Mycelium sensor Cell sensor Detection limit (mg O ₂ / l) 2.61 3.05 Limit of quantification (mg O ₂ / l) 7.73 8.38

Example 2

The stability and storage capacity of A. adeninivorans mycelial or cell sensors was determined by glucose standard (1.88 mM). For this purpose, dialysis membranes with 6.8 mg mycelia / cm ² or 6.8 mg cells / cm ^{2 were} tested for their suitability for BOD measurement over a period of 110 days. Both the background current (Io) and the sensor signals achieved with glucose were determined. For both sensor types (mycelial and cell sensor) the standard deviation of both parameters was less than 10% within this period.

Also the storage stability the membrane loaded with mycelia or cells was determined. At storage temperatures of 4 ° C the membranes loaded with mycelium could be stored for up to 6 months, without it to change the sensor parameter came. In contrast, the cell sensor is only Storable for 2 months.

Example 3

With the mycelium sensor, 27 different substrates were measured and the received signals (ΔI) compared with those of the cell sensor. In this case, higher sensor signals are achieved with 17 substrates with the mycelium sensor. This applies in particular to mono-, di- and oligosaccharides (glucose, galactose, xylose, lactose, maltose, sucrose, raffinose), but also citric acid, acetic acid, glucosamine, glycerol and the amino acids asparagine, lysine, arginine, histidine, phenylalanine and proline , Only 8 of the 27 substrates (fructose, ethanol, glutamine, alanine, leucine, methionine, threonine, tryptophan) can be better measured with the cell sensor (Table 2). Tab. 2: Comparison of the sensor signals of 27 substrates obtained with an A. adeninivorans cell or mycelial sensor. For the production of the sensors, the cells were cultured at 37 ° C (cell sensor) or 45 ° C (mycelium sensor). The substrate concentration of the test samples was always 1 mM. .DELTA.I (nA) Mycelium sensor / sensor cell Cell sensor Mycelium sensor glucose 233 338 1.45 fructose 60 32 0.53 galactose 211 310 1.47 xylose 62 75 1.22 lactose 25 36 1.44 maltose 65 246 3.79 sucrose 32 50 1.56 raffinose 0 32 citric acid 6 27 4.50 acetic acid 370 520 1.41 methanol 0 0 0.00 ethanol 899 653 0.73 asparagine 177 202 1.14 glutamine 85 76 0.89 lysine 145 209 1.44 arginine 141 176 1.25 histidine 143 197 1.38 alanine 138 80 0.58 Leuci 182 155 0.85 phenylalanine 56 95 1.70 proline 58 105 1.81 methionine 130 47 0.36 threonine 130 90 0.69 tryptophan 52 23 0.44 tyrosine 179 185 1.03 glucosamine 51 81 1.59 glycerin 91 224 2.46

Example 4

In order to be able to use the mycelium sensor to measure the SensorBSB, the measured values of a sample should vary only slightly with one another. To test this, a substrate mixture was used, each containing 1 mM glucose, glutamine, glycine, histidine, lysine, asparagine, valine, tryptophan, methionine, proline, alanine, aspartic acid, tyrosine, methanol, fructose, sucrose, maltose, serine, Contains threonine and isoleucine. The BOD ₅ value of this mixture is 138 mg / l. The 6-times measurement of the sample with the mycelium and the cell sensor shows that the measured values obtained with the mycelium sensor differ only slightly from one another, which is reflected in the very low standard deviation. In addition, the averaged SensorBSB value of 144 mg / L obtained with the mycelial sensor comes very close to the BOD ₅ value, whereas the Sensor B B value of the cell sensor deviates more strongly from the BOD ₅ value (Table 3). Tab. 3: Mean value, standard deviation and VK value of the substrate mixture measured in each case with the mycelium and cell sensor 6 ×. Sensor BOD (mg / l) BOD ₅ (mg / l) Mycelium sensor Cell sensor Average 144 263 138 standard deviation 9 29 20 VK value (%) 6.25 11.03 14.4

Example 5

The suitability of the mycelium sensor for measuring NaCl-containing samples is described in Example 5. For this purpose, the sensor was prepared as described and the influence of increasing concentrations of NaCl on the sensor signals (ΔI) measured at 37 ° C. was tested. In contrast to the cell sensor, the signals (ΔI) of the mycelium sensor change only slightly with the addition of NaCl. This effect (physical effect) is attributable above all to the reduction of the dissolved oxygen concentration present in the measurement solution. However, in contrast to the cell sensor, the mycelium sensor has no additional inhibition or activation of the sensor up to a NaCl concentration of 30% in the sample, which is of great advantage for the BOD measurement of saline samples ( 2 ).

To further investigate the physiological effect of NaCl, NaCl was first added to the measurement liquid, causing a decrease in the sensor signal (ΔI). 1 mM glucose was added and the change of the current was measured until the NaCl concentration of 1.5% in the measuring cell (corresponding to 30% in the test sample) was reached, the sensor signal (ΔI ) with the mycelium sensor is relatively constant, ie up to this concentration only the physical effect of NaCl on the biosensor has to be taken into account in the BOD measurement In contrast, the cell sensor has a NaCl concentration of 0.5% in the measuring cell (corresponds to 10% NaCl in the test sample) to the fall of the measured sensor signal (.DELTA.I), ie in addition to the physical effect of the physiological effect of NaCl on the cell sensor must be considered in the BOD measurement ( 3 ).

The dependence of the substrate specificity of the mycelium sensor on the NaCl concentration was tested with glucose, acetic acid and maltose. For this purpose, these substrates were added in a concentration of 1 mM with increasing salt concentrations in the measuring cell and the sensor signal (.DELTA.I _{substrate effetk + salt effetk} - .DELTA.I _{salt effect} ) determined. As a control, the sensor signal without substrate was determined with increasing NaCl concentration. By subtracting the control curve, the physiological effect of the salt on the corresponding substrate can be determined.

The glucose, acetic acid and maltose signals are up to a NaCl concentration of 30% in the test sample only by the physical and not or only slightly influenced by the physiological effect ( 4 ).

Example 6

The A. adeninivorans sensors loaded with 6.8 mg mycelia / cm ² or 6.8 mg cells / cm ² were tested for their suitability for SensorBSB determination. Since effluents usually form substrate mixtures, a synthetic wastewater, yeast extract, was used for these comparisons. Here, the SensorBSB values determined with the mycelium sensor correlate directly with the BOD ₅ value (m = 1.168). In contrast, the values obtained with the cell sensor (m = 0.75) achieve a lower correlation between the two parameters ( 5 ).

Example 7

The suitability of the mycelium sensor for SensorBSB measurement was tested on the example of wastewater samples with a high disaccharide content, taken on different days from the inlet and outlet of a sewage treatment plant. For this purpose, the sensor was prepared as described in Example 1, and the measured values obtained at 37 ° C. were compared with those of the A. adeninivorans cell sensor. At the same time, the BOD ₅ value of all samples was determined (Table 4). Table 4: Comparison of the measured sensorBBB values of various wastewater samples with the BOD ₅ value using an Arxula cell sensor or an Arxula mycelium sensor. Samples no. BS B ₅ SensorBSB (mg / l) SensorBSB / BSB ₅ Cell sensor Mycelium sensor Cell sensor Mycelium sensor enema outlet enema outlet enema outlet enema outlet enema outlet MW 2503 82.6 43.2 50.3 23.8 121.2 34.0 0.6 0.5 1.5 0.8 MW 0104 59.0 38.6 42.3 16.2 40.7 15.0 0.7 0.4 0.7 0.4 MW 2907 116 33.7 51.8 16.7 124 20.5 0.4 0.5 1.1 0.6 MW 0209 52.3 46.4 27.7 16.8 54.2 27.7 0.5 0.4 1.0 0.6 MW 0909 49.5 34.3 32.3 10.0 42.8 14.0 0.6 0.3 0.9 0.4 MW 1609 19.6 32.9 27.0 9.3 22.5 28.2 1.4 0.3 1.2 0.9 MW 2309 28.2 30.2 36.8 1.0 26.3 29.5 1.3 0.1 0.9 1.0

The measured values of Tab. 4 prove that with a permitted deviation of the SensorBSB values from the BOD ₅ value 65% of the measured values achieved by the mycelium sensor from the inlet and outlet of the sewage treatment plant correspond to the BOD ₅ value. In contrast, only a degree of agreement of 14% is achieved with the cell sensor.

Example 8 (Immobilization)

For the preparation of A. adeninivorans LS3 as a microbial sensor component is a 36-hour cultivation in YEPD medium (0.3% peptone, 0.3% malt extract, 0.3% yeast extract, 1% glucose) at 45 ° C. 10 μl of this culture are then added dropwise to a dialysis membrane on YEPD agar and incubated at 45 ° C. for several hours. A mycelial colony now forms on the dialysis membrane which is stabilized with PVA or PU in a known manner ( DE 197 36510 A1 ). The thus loaded dialysis membrane can be used immediately as a microbial sensor component.

Example 9 (loading density)

Around to obtain reproducible readings, must have an optimal cell count be immobilized on the dialysis membrane. The more applies Cells are immobilized, the higher the oxygen consumption and thus the measuring signal. At the same time increases with the loading density the stability of the sensor. This process works however, contrary to the diffusion rate, which is proportional to strength the immobilized cell layer is. This means that one too high loading density reduces the diffusion of oxygen and substrate, which leads to a decrease of the background current (Io) and thus to smaller measuring signals leads.

Around to ensure a constant mycelial growth on a dialysis membrane, have to z. B. dripped 10 .mu.l Mycelial cultures over a defined area are struck out (diameter: 0.4 cm). By use different culture times and the use of two dialysis membrane types with different pore size (0.6 μm, 10 μm), can be optimize the loading procedure.

Tab. 5: Loading of the dialysis membrane with mycelia of A. adeninivorans LS3. For this purpose, the strain LS3 was cultured for 36 hours at 45 ° C in YEPD medium, 10 .mu.l of this mycelium culture on dialysis membranes with 0.6 micron and 10 micron pore size, which were placed on YEPD agar. Everything was incubated for 6, 10, 12 and 24 hours at 45 ° C, the loading of the membrane (mg dry weight / cm ² ) calculated according to the formula m / π · d ² , the mycelia immobilized with PU and the base current (Io) as well as the last sensor signal the activation measurement (.DELTA.I) determined. For activation, 100 mM glucose was used. Incubation period (h) Loading (mg TG / cm ² ) Io (nA) IA (nA / min) 0.6 μm membranes 6 4.7 5,321.1 139.9 10 5.2 3,962.7 250.5 12 6.8 5,442.0 113.0 20 10.5 91.0 4.0 24 13.1 - - 10 μm membranes 6 4.6 4,070.8 46.6 10 9.3 2,836.5 228.8 12 13.8 3,656.0 57.0 24 6.5

To 6 hours Incubation of both types of membranes at 45 ° C reach the mycelial colonies a diameter about 0.4 cm. While but the mycelium mass at the 0.6 microns Dialysis membrane is between 1.6 and 4.0 mg, this is the 10 micron membranes only between 1.6 and 3.3 mg. This is the increase in thickness the mycelial colony in pendency of the pore size of the dialysis membrane. While the further cultivation will be mycelium masses of over 10 mg reached.

If the mycelium masses grown on both types of membranes are compared with regard to their achievable activation signals (ΔI), which should always be between 150 and 450 nA, these values can only be determined with mycelium masses between 2.1 and 8.2 mg, ie 4.2 and 15.5 mg TG / cm ² can be achieved. Such values are reached after 10 hours incubation at 45 ° C. If longer incubation times are used for the loadings, there is a further increase in the mycelium mass, which at the same time results in a decrease in the activation signal (ΔI).

Optimum loading of the 0.6 μm and 10 μm dialysis membranes with A. adeninivorans mycelia is thus achieved after 10 hours of incubation at 45 ° C. ( 1 ).

At the same time, the loaded 0.6 μm and 10 μm dialysis membranes were used to determine the sensor BOD values of 0-8 mM glucose. The best readings were obtained with the 6.8 mg mycelium / cm ² loaded membranes of both types (0.6 μm, 10 μm). Both the quantification limit (4.46 mg O ₂ / l - 0.6 μm membrane, 5.8 mg O ₂ / l - 10 μm membrane) and the detection limit (1.5 mg O ₂ / l - 0.6 μm Membrane, 2.0 mg O ₂ / l-10 μm membrane) are significantly more favorable in measurements with such prepared mycelium sensors than in the conventionally prepared cell sensors ( 2 ). Tab. 6: Detection and determination limit of mycelium and cell sensors, which were prepared with the newly developed procedure or conventionally. Procedure with 0.6 μm dialysis membrane Procedure with 10 μm dialysis membrane conventionally prepared mycelium sensor conventionally prepared cell sensor Detection limit (mg O ₂ / l) 1.5 2.0 2.6 3.0 Limit of quantitation 4.5 5.8 7.7 8.4

Example 10 (stability, storage capacity)

The stability and storage capacity of both sensors was determined using glucose standard (1.88 mM glucose). For this purpose, the two dialysis membranes (0.6 μm, 10 μm) were loaded with 6.8 mg mycelia / cm ² and tested for their suitability for BOD measurement over a period of 110 days. Both the background current (Io) and the sensor signals achieved with glucose were determined. At both Sen The standard deviation of these measurements within 110 days was less than 10%.

Also the storage capacity both loaded membrane types was determined. At storage temperatures of 4 ° C The membranes were storable for up to 6 months without causing any changes the sensor parameter came. Thus, both loaded membrane types have similar stabilities and storage capacities like the conventionally prepared one Mycelium sensor on. In contrast, the conventionally prepared Cell sensor can be stored for only 2 months.

Example 11 (substrate specificity)

The immobilized on dialysis membranes with 0.6 or 10 microns pore diameter via PU Mycelia were used as microbial sensor components in a known manner and prepared. With both sensor types different substrates were measured and the signals obtained with a conventionally prepared mycelium sensor compared.

Of the Comparison of SensorBSB values of 29 different substrates, measured with all three sensor types shows that their substrate specificity is very similar.

Table 3: Comparison of the substrate specificity of A. adeninivorans LS3 mycelium sensors prepared according to the method newly described in this patent and in the conventional manner. For the preparation of the sensors dialysis membranes were used with 0.6 microns and 10 microns pore diameter for the immobilization of Arxula mycelia (cultured at 45 ° C) or cells (cultured at 30 ° C). The measurement temperature was 37 ° C and the substrate concentration of the measurement samples 1 mM. ΔI (nA) Mycelium sensor / sensor cell Cell sensor Mycelium sensor glucose 233 338 1.45 fructose 60 32 0.53 galactose 211 310 1.47 xylose 62 75 1.22 lactose 25 36 1.44 maltose 65 246 3.79 sucrose 32 50 1.56 raffinose 0 32 citric acid 6 27 4.50 acetic acid 370 520 1.41 methanol 0 0 0.00 ethanol 899 653 0.73 asparagine 177 202 1.14 glutamine 85 76 0.89 lysine 145 209 1.44 arginine 141 176 1.25 histidine 143 197 1.38 alanine 138 80 0.58 leucine 182 155 0.85 phenylalanine 56 95 1.70 proline 58 105 1.81 methionine 130 47 0.36 threonine 130 90 0.69 tryptophan 52 23 0.44 tyrosine 179 185 1.03 glucosamine 51 81 1.59 glycerin 91 224 2.46

Example 12 (parameters that the sensor influence)

The Suitability of the three sensor types for the measurement of NaCl-containing samples describes Example 12. For this they were prepared as described and the influence of increasing concentrations of NaCl to those measured at 37 ° C Sensor signals (ΔI) tested.

Legend too 1 to 5 and 1 and 2

1 : Calibration curve of the A. adeninivorans mycelial sensor. For the experiments dialysis membranes were used with 6.8 mg mycelia / cm ² , which were immobilized with PVA in a conventional manner. The measuring time was 70 seconds. The glucose concentration is the final concentration in the test sample.

2 : Influence of NaCl on the sensor signals (ΔI) of the cell (⎕) or mycelium sensor

3 : Dependence of the glucose signal (ΔI) on the NaCl concentration of the test sample using a cell (⎕) or mycelium sensor

bzw. Maltose

Die dargestellte Kurve ist die Differenzkurve aus Substratkurve – Salzkurve. Die NaCl-Konzentration ist als Konzentration in der Meßprobe angegeben. 4 : Influence of NaCl on the sensor signal (ΔI) using 1 mM glucose (⎕), acetic acid

or maltose

The curve shown is the difference curve from substrate curve - salt curve. The NaCl concentration is given as the concentration in the test sample.

bei der Verwendung von Hefeextrakt als Substrat. 5 : Correlation of SensorBSB to the BOD ₅ value of the cell (⎕) or mycelial sensor

when using yeast extract as a substrate.

1 : Dependence of the activation signal (ΔI) on the biomass of the mycelia immobilized on the dialysis membranes.

2 : Calibration curves of A. adeninivorans LS3 mycelium sensors prepared according to (A) conventional and the manner according to the invention. For this purpose, dialysis membranes with (B) 0.6 μm and (C) 10 μm pore diameter were used. The measuring temperature was 37 ° C.

Claims (15)

Biosensor consisting of microorganisms as biocomponent and a physical transducer, characterized in that the biocomponent is in mycelial form. Biosensor according to claim 1, characterized in that that the mycelium solidifies with inorganic and organic polymers is. Biosensor according to claim 1, characterized in that that mycelium with foil-forming inorganic and organic Overcoated polymers is. Biosensor according to any one of claims 1-3, characterized by that the mycel-forming organism is the yeast Arxula adeninivorans is. Biosensor according to one of claims 1-4, characterized in that that the mycelium is in thin compact layers on a nutrient on the nutrient medium permeable support membrane fixed. Biosensor according to one of claims 1-4, characterized in that that the mycelium is fixed to a dialysis membrane containing the mycelia preferably protects the measuring side. Biosensor according to claim 6, characterized in that the dialysis membrane is a nuclear spore membrane. Process for the preparation of microbial biosensors according to one of the claims 1 to 7, characterized in that a mycelial braid in thin compact Layers by surface growth on solid nutrient media Temperatures> 42 ° C generated which are used directly in the biosensors. Method according to claim 8, characterized in that that the mycelium is placed on a nutrient on the nutrient medium permeable support membrane generated and this support membrane is used with the mycelium in the biosensors. A method according to claim 8 or 9, characterized in that this carrier membrane a dialysis membrane is immobilized on the yeast cells and mycelia become. Method according to one of claims 8 to 10, characterized in that the mycelium solidifies by immobilizing agent becomes. Method according to claim 11, characterized in that as immobilizing agents polymers, in particular PVA and PU be used. Use of biosensors according to claims 1-7 for BOD determination. Use of biosensors according to claims 1-7 for determination of substrates and substrate mixtures. Use according to claim 14 for the determination of saccharides, alcohols and / or organic Acids.