A new rapid method for determining sludge activity

War. Res. Vol. 20, No. 12, pp. 1529-1534, 1986

0043-1354/86 $3.00+0.00

Printcd in Great Britain

Pergamon Journals Lid

A NEW RAPID METHOD FOR D E T E R M I N I N G SLUDGE ACTIVITY JOZSEF OLAH and PE'rm~ P R ~ c z Centre for Water Resources Development, 1095 Budapest, Kvassay J.u.l., Hungary (RecewedJuly 1985)

Abstraet---One of the most reliable methods for characterizing an activated sludge is by measurement of its activity. The measuring principle of the method developed is based upon the determination of glucose activity of the activated sludge. The activated sludge sample is continuously aerated in a reactor and glucose standard solution is given to it. The glucose uptake in the activated sludge system is measured by a glucose selective electrode. The sludge activity is calculated on the basis of the reaction time required for the 50% decomposition of the glucose. Only the change of dissolved oxygen concentration of the sample interferes the measurement. This interference is eliminated by the measurement of dissolved oxygen. The electrical response of glucose electrode is corrected with that of the oxygen electrode. The method is selective, the reproducibility of it is better than 10%. Key words---activated sludge, activity, glucose-electrode, glucose uptake, instrumentation

INTRODUCTION In the course of the operation of sewage treatment plant, it has been increasingly recognized that due to the fluctuations of load and pH, toxicity of industrial wastes etc. it is necessary to control and regulate the activated sludge biological systems. To fulfil this need the activity of the system must be measured. The knowledge of sludge activity provides on opportunity to estimate the biological capacity of the waste water treatment plants. F o r instance, in the ease of introducing toxic wastes to the plant the harmful effect on activated sludge could be reduced by decreasing the amount of toxic input, on the basis of the functional relationship between the amount of toxic waste and sludge activity. In this way the treatment plant can be protected from the total toxification. The activity of activated sludge can be determined on the basis of assimilation and dissimilation processes as well as by enzyme activity measurement. Bardtke and Thomanetz (1976) and Raebel and Schiierf (1980) developed measuring methods for the determination of the dezoxyribonucleic acid (DNA) content of activated sludges. Roe and Bhagat (1982) propose a method based on adenosine triphosphate ( A ' I T ) measurement. Von Sekulov and Bardtke (1970) developed an analytical method by which respiration activity can be measured on the basis of dissimilation processes. Pagga (1980) gives an account of the respirometric method of activity measurement and Farkas (1981) describes a respirigraphic method for acetate. The method recommended by Sridhar and Pillai (1973) and that of Thiel and Hattingh (1966) are based on enzyme activity, protease activity, measurements. The technique in which chromogen substrate (i-leucine-p-nitranilide) is used for the

measurement of protease activity was developed by Vankov/t et al. (1980). Jones and Prasad (1969) and Dickson Liu (1983) described the measurement of dehydrogenase activity (NADH). A simple electrochemical method was worked out for the measurement of catalase activity by Obst and Frank (1983). The technique introduced by Brodisch et al. (1979) is based on the measurement of ,,-glucosidase and L-alanine aminopeptidase activities. The aforementioned methods of activity measurement are complicated, their automatization has not been solved yet, and therefore they cannot be used for the purpose of continuous operation control. THEORY

Elaboration o f a new method for activity measurement

The activity of an activated sludge may be characterized as the specific rate of glucose assimilation. This can be determined by adding a measured quantity of glucose to the system and registering the changes in glucose concentration with time. The glucose activity of activated sludge means the quantity of glucose decomposed by unit weight or volume of activated sludge during unit period of time. mg~o~ g~[ss h - ' or mg~0~ l~-~s~ h -1 . Though it is possible to follow the changes of glucose content in activated sludge system by the measurement of 14C-labelled glucose, this method (Larson and Schaeffer, 1982) due to its complexity is primarily of theoretical importance. The simple glucose activity measuring method developed by the authors is based on the addition of a known concentration and quantity of glucose solution to the activated sludge suspension simultaneously with continuous aeration, and the measurement of the changes of glucose concentration in time by using a glucose-selective membrane electrode.

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Fig. i. (a) Calibration of the glucose-selective membrane electrode for glucose. Concentration of glucose

added: 0, 10, 20, 30, 40, 50mgl -I. Temperature; 20°C. (b) Calibration curve of the glucose-selective membrane electrode for glucose

Measuring principle of the glucose-selective membrane electrode The basic sensor of the glucose-selective electrode is an amperometric dissolved oxygen sensor, the teflon membrane of which is coated with immobilized glucose-oxidase enzyme. During measurement the glucose is oxidized to gluconic acid by the enzyme according to the following reaction: glucose + 02 + H20 ~ o ~ o,~d.s~ gluconic acid + H202.

(1)

The oxygen necessary for the glucose oxidation is removed from the solution by the enzyme, which causes the oxygen concentration of the solution to diminish proportionately to the glucose concentration ([glucose]) in the immediately vicinity of the electrode membrane. If the oxygen concentration in the solution is DOs and on the surface of the electrode membrane is DOM, then DOs - DOM = f ([glucose]).

(2)

Equation (2) shows that DO M measured by the glucose electrode is inversely proportional to glucose concentration at constant DO s value. MEASURING M E T H O D

From the operating principle of the glucose-selective electrode it follows that the electrode is sensitive both to the concentration changes of glucose and dissolved oxygen. The

electrode current can be described, as 1 = SDo'DOs - S~ [glucose] + K (3) where I = electrode current (nA) Soo = DO sensitivity of the electrode ( n A / m g . l ~ ) = glucose sensitivity of the electrode (nA/mg' 1 ~ ) ~ = constant dependent on the formation of electrode enzyme (nA). It can be seen that equation (3) contains two other independent variables Soo and DOs, other than glucose concentration. It means that theoretically there are two possibilities for the measurement of glucose: DO s is kept at a constant value during the measurement, so SDo' DOs = constant DO s is continuously measured and the momentary values of SnO,. DOs are calculated on the basis of oxygen sensitivity of the glucose electrode. Considering that DOs, in most cases, cannot be kept at a constant value in activated sludge systems, generally the second method can only be used. In using this measuring method, in addition to the glucose sensitivity of the electrode, its sensitivity to DO, as well as the DO concentration of each sample must be known. For this reason, apart from glucose concentration, the DO concentration of the sludge should also be measured. The calibration curve for glucose of the glucose electrode plotted by means of using standard solutions of constant DO concentration is shown in Fig. l(a) and (b). The

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Determination of sludge activity

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When the percentage of glucose decomposition that was programmed into the microcomputer is reached, the drain valve is opened by the control unit and the sample examined flows out of the reactor space. After the completion of the reaction cycle the microcomputer computes the value of glucose activity on the basis of reaction time necessary for reaching the programmed glucose decomposition percentage. The result is given in digital form by the printer. The air-compressor maintained the dissolved oxygen level in the activated sludge is operate during the whole measuring cycle.

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RESULTS AND DISCUSSION

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Characterization of glucose decomposition curves

The glucose decomposition curves of activated sludges of different origin and identical concentration (So = 2.50 g 1-]) are shown in Fig. 4. The figure shows Disolved oxygen (DO)concentration (mg1-1) Fig. 2. Calibration c u r v e s of glucose-selective membrane that the rate of glucose decomposition is different in electrode for dissolved oxygen (DO), glucose concentration the various sludges. (mgl-~): 050; x40; Fq30; 020; ®10. The difference between the activities of the sludges is even more apparent on the basis of Table 1, where dependence of the electrode on DO, determined by changing the activity values of various sludges are expressed in the values of DO at constant glucose concentrations is seen terms of the 100 and 50 percentage of decomposition in Fig. 2. of the glucose dose (50 mg 1-]). The data show that The figures show that the calibration curve of the glucose electrode (Radelkis OP-GI-7113)is linear in both cases and the activity value related to total glucose content (At) was the highest in the activated sludge originating its sensitivity to dissolved oxygen SDo= 24 nA/mg IriS. from the winery (Villainy) ( 1 0 0 m g l - l h - l ) . Lower Operation of the activity-measuring apparatus Figure 3 shows the construction of the automatic glucose activity values (80 and 66 mg 1-] h-~) were obtained activity-measuring analyzer. Its operation is the following: in the case of sludges taken from sewage treatment the control unit closes the drain valve of the reactor, plants (V~ic, South Pest). opens the valves through which activated sludge and glucose In the case of the sludge originated fron~ South are fed in. After reaching of the level-indicator, both valves Pest, activity was measured also with the addition of close, terminating the inflow of activated sludge and glucose. The signals of the glucose and dissolved oxygen different o-chlorophenol doses. It was found that at electrodes are made available for the microcomputer by 10 mg 1-1 o-chlorophenol concentration there was no polarizing blocks and mV meters. decrease in activity, while at the dose of 30 mg 10

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o-chlorophenol, activity decreased by about 30% (46mg l-l.h-1). It is considered necessary to mention that the investigations in connection with the calibration of the glucose electrode--performed with increasing concentrations of o-chlorophenol and cyanide solutions--have unequivocally verified that glucoseoxidase is not deactivated up to about 150mg chlorphenoll -I and 10mg cyanide 1-1 concentrations, i.e. the activity decreases cannot be explained by the diminishing activity of the electrode enzyme. The significant differences between the glucose oxidase activity of sludge and glucose probe can be explained that the process of immobilization provides a certain protection for the electrode enzyme against inhibitors (Ma and Hassan, 1982). Activity values calculated after 50% decomposition of added glucose are, of course, greater values than those related to the time of total glucose

decomposition. This can be explained by the fact that the change of concentration in time is not linear but approximately hyperbolic. Activities pertaining to 50% decomposition levels are of importance particularly from the viewpoint of measuring technique, i.e. partly because of the reduction of measuring time and partly because of the increased accuracy of end point indication. Considering that the toxicity of chemicals is often characterized by the 50% glucose decomposition, the authors accepted the activity values (As0) belong to 50% decomposition. As mentioned before, the slopes of glucose decomposition curves are approximately hyperbolic. This is in accordance with the findings of Larson and Schaeffer (1982) and Vivilin (1982) who used a hyperbolic curve for the representation of glucose oxidation on the basis of four measuring points. The data registered continuously by our apparatus, how-

Table 1. Glucose activity values of different activated sludges expressed in terms of different decomposition percentages of added glucose A(mg~

Glucose activity values 1- i h- i ); B(mg~oK gfi~ss h ])

After complete decomposition (At) Activated sludge studied

South Pest (sewage treatment plant) Activated sludge II. activated sludge+ 10rag o -chlorophenol I 1. activated sludge + 30 mg o-chlorophenol V~c (sewage treatment plant) Activated sludge Villgmy (winery; sewage treatment plant) Activated sludge

After 50% decomposition (A~o)

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66

26

136

54

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83

33

46

18

48

19

80

32

115

46

100

40

187

75

Note: 50 mg glucose was added to 1 1. sewage sludge. Activated sludge concentration was 2.50gl -I in each case.

Determination of sludge activity

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Fig. 5. Acetate, glucose and prol~ase activities in the function of activated sludge concentrations. x Acetate activity; • protease activity: 0 glucose activity.

It is seen that there is a linear relationship between each of the three activities and the activated sludge concentration (the relationship is closely linear up to 3.4 g 1- t activated sludge concentration, beyond that, however, each activity curve shows a slight downward bend). It means that any of the three different Technical specifications of the measuring method activities can be used to characterize the sludge The technical specifications of the measuring activity. The method to be applied for activity meamethod were established on the basis of data obsurement should be selected on the basis of practical tained from seven measurements performed in paralconsiderations (simplicity, rapidity etc.), but in the lels with five communal and five industrial sewage case of a given sludge its activity must be determined sludges each, glucose decomposition being carried by the same method. out in each instance up to 50%. Reproducibility of In Table 2 the individual measuring techniques are the method was better than 10% in each sample. The compared with each other. The table shows that measuring range of the method is between glucose, the respirographic acetate and protease ac0-200 mgsl~o~ g ~ ¢ h - l • tivities of different sludges as well as the activities The method is selective. Disturbance that might be measured following cyanide dosage. On the effect of caused by occasional temperature changes can be the same quantity of cyanide the percentage value of eliminated by thermostating the reactor. the activity decreases of various sludges using various Comparison of different activities activity measuring methods, were different. This may The relationships of activated sludge concentration be explained that those enzymes of bacterium species vs glucose, respirographic acetate and protease activ- which accomplish the decomposition of various substrates become deactivated in different degrees. ity are shown in Fig. 5.

ever, suggest a more complex oxidation process. Steps can be observed in the curves of decomposition presented in Fig. 4. The appearance of the steps can be explained by adsorption between glucose and activated sludge.

Table 2. Comparisonof different methods used for determiningsludgeactivity Glucose Respirographic Proteaseenzyme activity(At) acetate activity activity Activated sludgestudied (mg~.g~.ss h-I) (mg~m~=g~ssh-I) (mg~s=~g~ssh-') South Pest Activated sludge 82 134 38 I I. activatedlodge+ 2 mg cyanide 47 72 18 Szentendre Activated sludge 31 65 18 1 1. activatedsludge+ 2 mg cyanide 22 41 10 V~c Activated sludge 53 41 26 1I. activatedsludge+ 2rag cyanide 41 31 18

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JOZSEF OLAH and PETER PRINCZ CONCLUSIONS

On the basis of the measurements the following conclusions can be drawn: The glucose electrode is sensitive both to glucose and dissolved oxygen. When the dissolved oxygen concentration fluctuates, glucose values measured with the electrode should be converted to values corresponding to identical dissolved oxygen level. When activity is measured with glucose electrode in an activated sludge system, dissolved oxygen content should also be measured continuously. There is a linear relationship among glucose, respirographic acetate and protease activities at concentrations ranging up to 3.4 g 1-~ activated sludge, but the changes in activity of a given sludge with time can only be determined by the same measuring method. On the glucose decomposition curve, adsorption steps appear. Despite these stages, the glucose decomposition curve can be replaced by a hyperbola. The simplicity of the method and instrument developed, make the supervision and control of sewage treatment plants possible. REFERENCES

Bardtke D. and Thomanetz E. (1976) Untersuchung zur Erfassung der Biomasse von Belebtschliimmen dutch quantitative Analyse der Desoxyribonukleinsfiure (DNS). GWF Wass. Abwass. 117, 451-453. Brodisch K., Teuber M. and Hegemann W. (1979) Enzymaktivitfiten als Kennwerte des bclebten Schlammes. GWF Wass. Abwass. 120, 524-527. Dickson Liu (1983) Resazurin reduction method for acti-

vated sludge process control. Envir. Sci. Technol. 17, 407-411. Farkas P. A. (1981) The respirography in biological treatment plant control. Wat. Sci. Technol. 13, 125-131. Jones P. H. and Prasad D. (1969) The use of tetrazolium salts as a measure of sludge activity. J. Wat. Pollut. Control Fed. 41, R441-R449. Larson R. J. and Schaeffer S. L. (1982) A rapid method for determining the toxicity of chemicals to activated sludge. Wat. Res. 16, 675~80. Ma T. S. and Hassan S. S. M. (1982) Organic Analysis Using Ion-selective Electrodes, pp. 123-132. Academic Press, London. Obst V. U. and Frank H. K. (1983) Ein einfaches Verfahren zur Bestimmung der Katalase-Aktivit/it in Schlamm, Sediment und Wasser. Z. WassForsch 16, 138-139. Pagga U. (1980) Respirometrischer Abbau und Toxizit/itstest mit Belebtschlamm zur Priifung von Substanzen und Abwiissern. Vom Wasser 55, 313-325. Raebel M. and Schlierf H. (1980) Ermittlung der aktiven Biomass im Bclebtschlamm dutch Bcstimmung der Desoxyribonucleins~iure (DNS). Vom Wasser 54, 293-505. Roe P. C. and Bhagat S. K. (982) Adenosine triphosphate as a control parameter for activated sludge process. J. War. Pollut. Control Fed. 54, 244-254. Sekulov I. yon and Bardtke D. (1970) Untersuchungen zur schnellen Bestimmung der Aktivitfit von Belebtschl~mmen. GWF Wass. dbwass, ll, 18-20. Sridhar M. K. C. and Pillai S. C. (1973) Protease activity in sewage sludges and effluents. War. Waste Treat. August. 35-42. Thiel P. G. and Hattingh W. H. (1966) Determination of hydrolitic enzyme activities in anaerobic digesting sludge. Wat. Res. 1, 191-196. Vankovfi, S., Kasafirek E., Kupec J. and Mlfidek M. (1980) Bcurteilung der Hydrolyseaktivit/it von Belebtschlamm mittels spezifischer chromogener Substrate. Acta Hydrochim. hydrobiol. 10, 15-22. Vavilin V. A. (1982) Models and design of aerobic biological treatment processes. Acta Hydrochim. hydrobiol. 10, 211-242.