J. KREUTZMANN, J. WEBER, A. PLANTIKOW

For evaluation of water quality both chemico-physical and biological assays are used. In the latter test organisms (e.g. fishes, algae, photobacteriae, daphniae) indicate toxic effects of dissolved substances. A large number of organisms is used, because there is no "ideal" biotest for water control. Yeasts have been used, to some extend, as well as test organisms for various investigations (1), but the sensitivity of simple yeast growth assays was too low for environmental investigations (2). A better pollutant sensitivity of yeast cells was obtained by a mutation line with increased cell permeability (3). An other way to better senstivity is the use of a biological performance parameter as an indicator for toxic influences of pollutants. It is a wellknown fact in animal and plant breeding, that organisms unilaterally selected for a special performance react to suboptimal life conditions with a high performance decrease. That is also valid for the fermentation activity of selected yeast strains.

In the company NORDUM Institut für Umwelt und Analytik GmbH & Co. KG was developed a biotest for water pollution on this base. It was selected a single-cell-line from an industrial used strain of the yeast Saccharomyces cerevisiae with high fermentation performance (2). This yeast is an eukaryotic monocellular organism with the detoxification system Cytochrom P 450, corresponding to that of higher eukaryots. The yeast cells of a single-cell –line are genetically identical and therefore react uniformly. The cell-line is characterized by a good pollutant sensitivity and by a high fermentation activity at low pH values.

Variations of the new yeast test are used as:

1. fermentation biotest - method for water pollution control in laboratories
2. yeast sensor - small gel bodies for the detection of pollutants in water samples, simplifying the fermentation biotest
3. educational training - as a set for pupil experiments to demonstrate the action of pollutants in the environment
4. sediment contact test - method for detection of pollutants in sediments and sludges.

All items base on the same test principle (4): yeast cells and a nutrition medium are added to the sample (water, sediment) and incubated for a period of 18 - 20 hrs at 25° - 28°C. During this time yeast cells multiply by 5 - 6 generations (from one cell grow 50 cells).

Pollutants reduce the vitality, reproduction rate and thereby the fermentation potency of the cells. The fermentation is subsequently induced by a thermal shock (40°C). The fermentation activity is determined after 2 - 3 hrs by measuring the volume of formed carbon dioxide. The results are compared with an unpolluted water sample (drinking water).

- development of a simple biosensor

- availability of test results within 24 hrs

- simple handling

- risk-free procedure

- no additional laboratory equipment

- activity over 6 months storage

- no special requirements for keeping test organisms

- detection of ecologically relevant pollutant concentrations

- application for turbid resp. coulored water samples

The yeast sensor (Fig. 1) is an open, cylindrical hollow gel body of 16 x 6 mm (height x diameter), whose inner wall is coated with immobilized yeast cells. The yeast cells are fixed in a gel layer. Gel and gel carriers are permeable for all materials dissolved in the water sample. The yeast sensor is durable with storage temperatures from 2° to 4°C. Temperatures up to 30°C can be tolerated for one day.

The nutrition medium (in powder or tablet form) supplies all necessary nutrients for the yeast cells and adjusts a pH of around 3.5 in the sample. The acidic medium represses sample borne microorganisms, possible competitors to the yeast cells.

After an incubation period the fermentation in the gel bodies is induced by increasing the temperature to 40°C. The carbon dioxide gas formed fills the bodies and they float up to the water surface (Fig. 2). Pollutants delay the floating up.

20 ml water sample is filled into a 50 ml tube and 0.8 g nutrition medium are added and dissolved (Fig. 2). Then three yeast sensors are put into the tube after filling with the solution with a pipette. The sensors sink downwards.

Drinking water is used as a reference sample added with five sensors. The test tubes are incubated for 18 - 20 hrs at 25° - 28°C. Then the test tubes are warmed up to 40°C. When four sensors float up in the reference sample (after 2 - 3 hrs) the classification can be started (Fig. 3). If the sensors do not float up this means they are no longer active.

At least two sensors have floated up within one additional hour (=one hour delay) to the reference sample.

At least two sensors have floated up within two hours delay to the reference sample.

Non or only one sensor has floated up after three hours delay.

The sensitivity of yeast sensor wass tested against concentration series of 45 pollutants, e.g. biocides, pesticides, heavy metals, softening agents and organic solvents. The data are given in Table 1.

The yeast sensor has an increased sensitivity against the toxicants of group I. The sensitivities against toxic pollutants of the groups II and III are in the range of other test organisms. The pollutants of group IV have a moderate toxicity against yeast sensors, in most cases lower than to other test organisms. One reason is the detoxification by the cytochrom P 450 system. Especially pesticides with low warm-blooded animal toxicity (e.g. DDT) are detoxificated by this system and therefore moderately indicated by yeast sensors.

A high sensitivity against pure solutions of chemicals is an important base of a biotest. But this feature can not allone characterize the suitability for practical water and sewage control. Industrial and municipal sewages, especially sewages from dumps, may contain very different organic and anorganic toxicants beside proteins, carbohydrates, surfactants, salts a.o.. This complex composition can cause synergistic and antagonistic effects to the test organism. Additionally sewages contain a lot of different microorganisms, potential competitors and prädators of the test organism. Therefore many microbial assays demand a previous sample sterilisation against enemy organisms. But this procedure may change the chemical composition. The testing of natural samples is an advantage of the yeast sensor. Its immobilised cells are protected against an uptake by prädators and competitors are inhibited by an acidic pH in the samples.

Intending an evaluation of the suitability of yeast sensors for practical control, more than 180 water samples were tested with yeast sensors. They were as well as tested with photobacteriae and fishes and additionally chemically analysed by standard methodes (5). One sample only was classified by yeast sensors as "false positive", that means: no pollutants were found by chemical analysis. Fishes and photobacteriae each indicated five "false positive" classifications. Yeast sensors detected all polluted samples (no "false negative"), photobacteriae did not detect toxic salt loads and fishes did not detect any nitrite loads.

Table 1. Pollutant detection limits of yeast sensors
(the given pollutant concentrations initiate classification I)

The "false positive" classifications by tests with fishes and photobacteriae were caused by high nutrient loads. The most well-known biotests are not able to detect a pollution by harmful substances in the presence of high nutrient concentrations, because they are inhibited by them alone.Especially high ammonium concentrations are frequently indicated as toxic. Yeast cells tolerate high nutrient loads . That is an important advantage especially for municipal sewage control. They are frequently overloaded with organic nutrients causing an oxygen shortage in the course of the bacterial decomposition process. Subsequently toxic substances as nitrite (NO₂^-) and hydrogen sulfide (H₂S) can be generated under anaerobic conditions. These biotic formed toxicants prevent normally an indication of further pollutants in nutrient loaded sewages. For yeast sensor application H₂S can be eliminated simply by blowing air through the sample and NO₂^- is eliminated by addition of some drops of amidosulfuric acid. After these procedures it is possible to detect further pollutants by yeast sensors in the presence of nutrient loads.

The application of the yeast sensor is possible for a wide range of samples, inclusive turbid, coulored, acidic and basic (pH 2 – 10) without any additions for neutralisation, and microbial settled samples without any additional steps.

1. Marquardt H., Schaefer S. G., 1994. Lehrbuch der Toxikologie. BI-Wiss. Verlag, Mannheim.

3. Terziyska A., Waltschewa L., Venkov P., 2000. A new sensitive test based on yeast cells for studying environmental pollution. Environmental Pollution, 109 : 43-52.

5. Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlamm-untersuchung Wiley – VC Verlag GmbH & Co. KgaA, Bd. II, III, IV und VI (2003).