Or[g na| Papers

Test Instructions for Measuring the Microbial Metabolic Activity in Water Samples
Ursula Obst DVGW und Stadtwerke Mainz AG, Rheinallee 41, D-6500 Mainz 1, Federal Republic of Germany Tcstvorschriften zur Bestimmung der mikrobiellen Stoffwechselaktivit.~it in W~issern Zusammenfassung. Es werden Analysenvorschriften zur Messung von acht verschiedenen Enzymaktivit/iten beschrieben. Die Messung dieser Enzymaktivitfiten des mikrobiellen Stoffwechsels in Wasserproben erfolgt nach der Zugabe von entsprechenden chromogenen Substraten und deren Umsatz. Die der Enzymaktivit/it proportionale Farbstoffkonzentration nach der Reaktion wird photometrisch bestimmt. Bei bekanntem Molverh/iltnis von Farbstoff zu ursprfinglichem Substrat und linearem Reaktionsverlauf wird der prozentuale Substratumsatz pro Liter Probevolumen und Stunde Inkubationszeit berechnet. Summary. Test instructions for measuring eight different enzymatic activities are described. These enzymatic activities of the microbial metabolism in water can be measured after an addition of chromogenic substrates and their turnover. After the reaction the concentration of the chromophoric group proportional to the enzymatic activity is determined photometrically. I f the molar value of the chromophore in the original substrate is known and the reaction is linear, the enzymatic turnover can be calculated in percentual degree of conversion per liter and hour.
are: small expenditure of reagents, short incubation time, and no necessity of large-scale equipment. The respective enzymatically separatdd chromophore is quantified with a calibration curve, which has been set up for the photometric measurement under test conditions. The percentual substrate turnover in the mixture can be calculated if the concentration of covalently bounded chromophore in the originally used substrate is known. With a linear rate of catabolism during incubation a comparison of samples of different origin and of different activity parameter can be achieved. The substrate turnover (ST) in liters -~ and hours - 1 is calculated by the general equation: ST -

E F 1000 x - 100% Sxt V

E = spectral absorption measure of the final product at wavelength x F = photometric calibration factor of final product in tool 1- ~ S = chromophoric matter in added substrate in mol 1- ~ V = used testing volume in ml t = incubation time in h. We abstain from converting into enzyme units (U), as commercially available pure enzymes used for calibration with known activity often have a non-microbial origin and, furthermore, in-vitro conditions can hardly be compared with in-vivo conditions of the testing dispositions discussed here. With the biochemical methods described on the following pages it is possible to determine enzyme activities concerning the catabolism of: polymer substrates, di- and monomer fragments resulting from it, and finally energydelivering metabolic activities.

1. Introduction
The methods used up to now for determining the biological quality of water either do not allow any statement on catabolism or are too expense and time consuming for application in a standard laboratory. Physiological activity of biocenoses in water, i.e. self-purification, can be quantified by direct measurements of microbial metabolic degradation. Such methods have been described for analyses of sediments and sludge [1]. Recently, these methods could be adapted to an analysis of surface- and groundwater. First results showed that they proved a success in testing waste water-influence on surface water, describing the quality of water, analysing groundwater and biological treatment [2].

3. Activity Tests of Catabolism of Water-Insoluble and Polymer Substrates 3.1. Analysis of Esterase Activity 3.1.1. Theory. Fluorescein diacetate as a water-insoluble substrate can be splitted in the same degree by extracellular esterases, proteases, and lipases as well as by bacteria, fungi, protozoa and some algae. Thus, enzymatic hydrolysis of fluorescein diacetate (FDA) is well suited for a broad detection of heterotrophic activity [3]. Fluoresccin emerges as one of the final products of the enzymatic reaction and is determined photometrically. Enzymatic FDA-hydrolysis is largely oxygen-independent and can be measured under aerobic and anaerobic conditions.

2. Basic Principles
The enzymatic activity to be tested is determined on the basis of its specific activity after adding and degrading a corresponding chromogenic substrate. The advantages
Fresenius Z Anal Chem (1985) 321:166-168 9 Springer-Verlag 1985

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3.1.2. Material. Test tubes. Shaking machine. Spectrophotometer. FDA-reagent = 20 mg of fluorescein diacetate (Serva, No. 21 575) are brought into solution with 10 ml of acetone (reagent-grade) and stored at - 1 8 ~C. Incubation times for surface- and groundwater are the same in this method. The next step is to centrifuge for 10 rain at 20,000 rpm (or respectively longer at lower speed) and to determine the optical density at 595 nm against the blank reading. For samples with a strong inherent colour the optical density of the sample without substrate has to be substracted. To convert the optical density into the concentration of the dye Azure (final product) a corresponding amount of Azure-B-chloride dissolved in distilled water is used for a concentration series under testing conditions and for setting-up a calibration curve. The standard deviation of the protease-test is about _ 4%, of the amylase-test about _+ 9% from the average.

3.1.3. Method. 10 ml of the fresh sample are pipetted into a

test tube and 40 ~tl of FDA-reagent are added. The blank reading for the photometrical measurement consists of 10 ml distilled water and 40 ~tl FDA-reagent. An additional blank reading of the sample is prepared, consisting of 10 ml sample without any FDA-reagent, if the sample shows a strong inherent colour. The batches are mixed well, closed and shaken on a shaking machine; the incubation time for samples from surface water is 5 h, from groundwater 24 h. Photometric measurement is carried out at 490 nm against the respective blank reading. Turbid samples are centrifuged before measurement. For the conversion of the optical density into concentration of fluorescein, an aliquot of the FDAreagent is chemically hydrolysed at a p H value of about 11, or thermically at about 100~ With the help of the thus splitted fluorescein a concentration series is set up under test conditions for the calibration curve. The standard deviation of the test is _+ 12% from the average.

4. Activity Tests of Catabolism of Water-Soluble Di- and Monomer Substrates After an extracellular degradation of polymeric compounds by microorganisms the thus formed soluble fragments are taken up into the cells; there they undergo a further decomposition by intracellular enzyme systems and are utilised energetically. Main components of these fragments are esters of the phosphorus metabolism, peptides and oligosaccharides. The turnover of these three substrate groups is measured by the same method [5].

3.2. Analysis of Protease and Amylase Activity 3.2.1. Theory. Polymer substrates with an extracellular
catabolism generally establish the beginning of microbial metabolic chains. Proteins and polysaccharides like cellulose and starch are the mainly degraded compounds. Enzymes which hydrolyse peptide bondings of polymeric proteins belong to the protease group. The substrate specifity of these proteases may be high as well as low; microbial proteases generally show a lower specifity than those ofeucaryonts. Two different enzymes are responsible for the catabolism of polymeric starch: endo-(=~-)amylase cracks 1/4-glucanebondings within the starch molecule, exo-(=/%)amylase cracks the respective bondings at the end of the molecule. The same analytical principle is the basis for the following activity test for proteases and amylases: the respective substrate is applied, covalently bound with an azure group [4]. This is the reason for a joint description of the analyses. We are sorry to say that with the specimens used a conversion into a percentual substrate turnover cannot be done at the present time as the producer does not give any data on the percentage of dye in the original substrate. Centrifuge tubes. Shaking machine. Centrifuge with refrigeration (20,000 rpm). Spectrophotometer. Hide Powder Azure (Calbiochem, No. 37716). Amylopectin Azure (Calbiochem, No. 172678). Quench-solution = 1 M phosphoric acid + concentrated formaldehyde solution, mixture 1:1. Calibrating substance = Azure-Bchloride (Sigma, No. A-1029).

4.1. Analysis of Phosphatase, Aminopeptidase and Glucosidase Activity Theory. Phosphomonoesters are converted by phosphatases, enzymes belonging to the esterases group. Phosphatases show a relatively low substrate specifity and can be taken as a widely-scattered activity parameter, the more so as enzymatic separation of ester-bound phosphate residues represent a common reaction in central metabolism. Peptides are attacked by aminopeptidases which cleave off a specific amino acid from the terminal end of a peptide chain. In the following we describe a model activity test for alanine-aminopeptidase. Disaccharides as metabolites of polysaccharides are hydrolyzed by enzymes of the glucosidases group. According to their respective substrate they are specified into ~- and/~-glucosidases. E.g., c~-glucosidases degrade maltose and sucrose and further convert soluble fragments of the starch-metabolism;/%glucosidases degrade cellobiose into/~-glucose-units and take part in the cellulose metabolism. Phosphatases, aminopeptidases, and glucosidases are detected by nitrophenyl- or nitroanilide derivatives of their substrates. All activity tests can be done in a joint process. 4.1.2. Material. Test tubes. Water bath (30~ Spectrophotometer. 0.14 M Solution of sodium chloride (insterile, preservable for a week at cool storage). 1 M Solution of sodium carbonate. 10% Trichloroacetic acid. Substrate solution = l mg of 4-nitrophenyl-phosphate (Merck, No. 6850), L-alanine-4-nitroanilide-hydrochloride (Merck, No. 1014), p-nitrophenyl-e-o-glucopyranoside (Serva, No. 30716) or 4-nitrophenyl-/%D-glucopyranoside (Merck, No. 6793) is dissolved in 1 ml of 14 M sodium chloride. The substrate solutions are prepared freshly before use. Calibrating substances = 4-nitrdphenol (Merck, No. 6798), 4-nitroaniline (Merck, No. 6760).
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4.1.1.

3.2.2. Material.

3.2.3. Method. 30 mg of the respective substrate are weighed into a centrifuge tube for each sample and the blank reading. This initial step can be done some time before use; the tubes with substrate can be stored at - 18 ~ 5 ml of the fresh sample are brought into the substrate-filled tubes; the blank reading for the photometrical measurement consists of 5 ml distilled water and substrate. The tubes are shaken on a shaking machine for 2 4 h at room temperature; after incubation 2.5 ml of quench-solution are added to each tube.

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4.1.3. Method. 1 ml of the corresponding substrate solution
is added to 1 ml of the fresh sample. The blank reading for the photometrical measurement consists of 1 ml of 0.14 M sodium chloride and 1 ml of substrate solution. Samples with a strong inherent colour need a special blank reading consisting of 1 ml of the sample and 1 ml of 0.14 M sodium chloride. The batches are mixed well, closed, and incubated in a water bath (30~ Incubation time for samples of surface water is 6 h, for samples of groundwater 24 h. After incubation 1 ml of 1 M sodium carbonate is added to each tube containing nitrophenyl phosphate or nitrophenyl glucopyranoside; to each tube containing alaninenitroanilide 1 ml of 10% trichloroacetic acid is added. Turbid samples are centrifuged before photometric measurement. The optical density of the test mixtures is determined at 405 nm against the respective blank reading. To convert the optical density into the concentration of the final product (nitrophenol, resp. nitroaniline), 30 mg nitrophenol are dissolved in 100 ml 1 M sodium carbonate, resp. 10 mg nitroaniline in 100 ml 10% trichloroacetic acid. These solutions are used for concentration series under testing conditions for setting-up calibration curves. The standard deviation of the tests is about _+ 10% from the average. 115 ml 0.1 M phosphate buffer pH 8.5, divided in small portions and stored at - 1 8 ~ until use. INT-solution = 200 mg 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliumchloride (Serva, No. 26840) are dissolved in 100 ml distilled water and stored cool and fightprotected. Quench-solution = 1 M phosphoric acid + concentrated formaldehyde solution, 1 : 1 mixture.

4.2.3. Method. 100 ml of the fresh sample are filtered through

a membrane filter. The rolled-up membrane filter is put into a centrifuge tube together with 5 ml of ETS-B-solution and treated with ultrasonics for 10 min. After that the tubes are centrifuged for 10 min at 20,000 rpm (or respectively longer at a lower speed). To 1 ml of the supernatant including the released enzyme system 3 ml of the ETS-substrate-solution and 1 ml of the INT-solution are added. The blank reading for the photometrical measurement consists of 1 ml of the ETS-B-solution, 3 ml of the ETS-substrate-solution and 1 ml of the INT-solution. After thorough mixing the test solutions are incubated in a water bath (37~ for 1 h. Incubation times for surface- and groundwater are the same. After incubation 1 ml of the quench-solution is added to each tube and the optical density is measured at 490 nm against the blank reading. For converting the optical density into the concentration of the final product (iodonitrophenylformazane) no special calibration curve is needed. The molar extinction coefficient eg9Onm is known [6] with a value of 15.9 mM -1 cm -1. The standard deviation of the test is + 17% from the average.

4.2. Analysis of the Activity of Energy-Producing Metabolic Reactions (Electron-Transport System) 4.2.1. Theory. The main pathways of heterotrophic metabolism lead to energy-producing enzymatically catalysed electron transport and to a following energyconserving oxidative phosphorylisation (ATP, energy charge). Testing the activity of the energy charge costs a lot of work and equipment. Therefore we confined ourselves to determine the activity of this metabolic chain to N A D H and NADPH-dependent dehydrogenases of the electrontransport system. When adding the cosubstrates N A D H and NADPH, the conditions for electron transport are optimised; thus the test sensitivity is increased approximatively tenfold [6, 7]. The activity of the electron-transport system can be determined under aerobic as well as anaerobic conditions. Generally, it is higher in the anaerobic range, as this testing mode preferably records the reducing part of the reaction. A cell-lysis, carried out before the analysis, is needed, as the enzymes of the electron-transport system are membrane-bound. 4.2.2. Material. Membrane filters (0.2-0.45 ~ma pore diameter) and filtration device. Centrifuge tubes. Ultrasonic bath. Centrifuge with refrigeration (20,000 rpm). Test tubes. Water bath (37~ Spectrophotometer. 0.1 M phosphate buffer, pH 8.5. ETS-B-solution = 9.25 mg magnesium sulphate, 750 mg polyvinylpyrrolidone (Merck, No. 7443) and I g Triton X 100 (Merck, No. 11869) are dissolved in 500 ml 0.1 M phosphate buffer pH 8.5 and stored cool. ETS-substrate-solution = 25 mg NADPH2 (Boehringer, No. 107824), 145 mg NADH2 (Boehringer, No. 127345) and 0.23 g Triton X 100 (Merck, No. 11869) are dissolved in

5. Conclusions

The enzymatic activity tests for surface- and groundwater described in this paper are suitable for use in a standard laboratory as a supplement of chemical and biological analyses; they do not call for extensive effort, nor for expensive equipment. Applications are proposed for all water samples whose microbial metabolism is of any interest. First experiences in practical application of these methods have already been made [2] and prove their utility in water analysis. It is possible to extend the list of enzymatic tests if new chromogenic substrates can be obtained or similar tests are developed by biochemical research.

Acknowledgements. We would like to thank the Umweltbundesamt

Berlin for its financial support and numerous collaborators of the Stadtwerke Mainz AG for their help with our work.

References

1. Obst U, Schmitz W (1983) Vom Wasser 61 : 187-198 2. Obst U (1984) Vom Wasser 63: in press 3. Schntirer J, Rosswall Th (1982) Appl Environ Microbiol 43:1256-1261 4. Meyer-Reil L-A (1983) Marine Biol 77:247-256 5. Teuber M, Brodisch K (1977) Eur J Appl Microbiol 4:185-- 194 6. Owens TG, King FD (1975) Marine Biol 30:27--36 7. Zimmermann AP (1975) Verh Int Verein Limnol 19:1518-1523 Received September 5, 1984

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