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Characterization of textile wastewater and other industrial wastewaters by respirometric and titration biosensors
~ Pergamon
War. Sci. Tech Vol. 40, No. I, pp, 161-168, 1999 c 1999IAWQ Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0273-1223/99520.00 + 0.00
PH: S0273-1223(99)00376-5
CHARACTERIZATION OF TEXTILE WASTEWATER AND OTHER INDUSTRIAL WASTEWATERS BY RESPIROMETRIC AND TITRATION BIOSENSORS A. Rozzi *, E. Ficara*, C. M. Cellamare** and G. Bortone** *DIIAR, Politecnico di Milano. Piazza L da Vinci 32. 20133 Milano. Italy uENEA. Via Martiri di Monte Sole 4, 40129 Bologna, Italy
ABSTRACT Many industrial effluents, such as textile and tannery wastewater, contain slowly biodegradable, refractory or even toxic compounds at variable concentrations which may interfere with the efficient operation of biological wastewater treatment plants, in particular with the nitrification stage. Agro-industrial effluents may occasionally contain sanitising agents which are by definition biocides. Two different biosensors, based on respirometry (oxygen uptake rate, OUR measurements) and on basic titration respectively, were used to measure degradation rates of industrial wastewater samples by autotrophic bacteria (ammonia oxidizers). Specific sanitisers such as sodium hypochlorite and benzalconium chloride were used to evaluate and compare the nitrifying activity measured by the two different instruments. 0 1999 IAWQ Published by Elsevier Science Ltd. All rights reserved.
KEYWORDS Respirometry; titration; biosensors; inhibition; nitrification; industrial wastewaters. INTRODUCTION Automatic process control is increasingly used in biological industrial wastewater treatment since it makes possible the on-line optimization of operating conditions even as regards to variable loading rates and accidental or deliberate addition of toxicants in the influent to the biological treatment plant. Among the instruments which may be used to monitor biotechnological processes, such as aerobic and anaerobic wastewater treatments, biosensors are particularly useful because they make possible to measure the activity of the biomass within the reactors as well as substrate or product concentrations. The respiration rate is frequently used in wastewater treatment plants (WWTPs) to evaluate the potential toxicity and organic load of the incoming streams on active biomass. Biosensors measuring the respiration rate use oxygen probes. The determination is based either on oxygen measurement after interruption of the aeration ("closed respirometry") or on the oxygen depletion curve in a continuously aerated system ("open respirometry"). Examples of the former biosensor may be found in Cech et al. (1984), Drtil et al. (1993), Spanjers and Klapwijk (1990), Verschuere et al. (1995) and in Surmacz-Gorska et al. (1996) while the 161
162
A. ROZZI et al.
RODTOX (Rapid Oxygen Demand & TOXicity) biosensor (Vanrolleghem et al., 1990) is an example of the latter. Titration biosensors measure the activity of an acidifying (Ramadori et al., 1980) or alkalising (Massone et al., 1996) bacterial consortia by titration at constant pH. Among this class of biosensors, the ANITA (Ammonium Nitrification Analyser) is a titration system which monitors the ammonium oxidation kinetics by determining theO rate of alkali titrant required to keep the pH at a given set point value (Massone et al., 1995; Massone, 1997). Nitrification is an oxidation reaction which produces two moles of protons per mole of oxidized ammonium. The biosensor determines the acid products released during the first step of the above conversion:
NH: +~02 ~ NO'2 +H 20+2W 2
(1)
Moreover, comparing the maximum degradation rate before and after the addition of a certain amount of a chemical, the inhibition effect of the latter may be estimated (Maffei, 1995; Massone, 1997; Gemaey, 1997). The use ofbiosensors based either on dissolved oxygen (DO) depletion (closed respirometry) or on titration, in order to monitor inhibition of nitrification by industrial (textile) wastewaters and by solutions containing sanitizing agents, is addressed in this paper. MATERIALS AND METHODS Respirometric biosensor The instrument is made of two parallel moving bed continuous flow completely mixed reactors, equipped with oxygen sensors (Danfoss mod.2500) connected to a computer that acts as controller, data acquisition -and processing unit (Fig. I). The working volume of each reactor is 5.850 I. The control program is implemented on Lab View (National Instrument Environment). Air is provided by a compressor to the reactors through a sparging porous diffuser. Aeration is stopped when the oxygen concentration reaches a set point upper value (6 mg/l). After air is shut-off, DO begins to decrease, and oxygen uptake rate (OUR) can be determined from the slope of the curve. Calculation of OUR is made as soon as the curve is sufficiently linear or when oxygen concentration reaches a lower set point value (2 mgt!) (Randall et al., 1991). The moving bed was made of polyurethane macroreticulated foam (120 PPD which is colonized by fixed biomass. The biofilm reactor was selected to simplify the operation of the biosensor, by avoiding secondary clarifiers and recycling pumps, and by allowing it to operate at low HRTs and, therefore, to obtain rapid responses in terms of OUR changes. The experimental scheme is similar to the one reported in the ISO Method 11733, but information about biodegradability and potential toxicity of the tested stream is derived by OUR profiles (including EC 50 estimation) rather than measuring effiuent COD (Bortone,et al., 1997). Although the biosensor was designed to evaluate the degree of treatability related to carbonaceous pollutants and to ammonia in a wastewater, the main goal in this study was to determine the potential inhibitionon nitrifiers only. For the evaluation of the treatability of specific industrial wastewaters into a given WWTP, the two reactors are, first, inoculated with activated sludge drawn from that plant and acclimated to the composite wastewater fed to the same plant. Then, the two reactors of the respirometer are fed at the same constant flow rate with the same influent as the full scale WWTP. The industrial wastewater to be tested (the candidate toxicant) is analysed for COD and NH 4 • Extra feed, made of this effiuent is then added to one of the two reactors at a given load, while the same additional amounts of COD and ~ are provided, as acetate and ammonium, to the second one in order to keep constant the total organic and nitrogen loads in both the moving beds. At least three increasing dosages are used. The test is completed by ammonia addition to both reactors (Fig. 2). From the analysis of the differential responses, the percentage of biodegradable COD, the inhibition to respiration, inhibition to nitrification and process kinetics can be obtained. The first step in Fig. 2 allows estimation of the amount of biodegradable COD in the test stream. By increasing the dosage of COD in both reactors, it is possible to evaluate whether the OUR increase in the
Characterization of textile wastewater
163
reactor fed on the industrial wastewater is proportional (absence of respiration inhibition) or less than proportional (inhibition) to the OUR increment in the blank reactor. The same procedure can be used with increasing dosages of ammonia to detect nitrification inhibition. During the experimental comparison with the titrimetric biosensor, spike dosages of ammonia and inhibitory compounds were carried out at the beginning of each test, to quickly reach steady state conditions.
-------~
I
BLANK REACTOR
I I
I I
I '--------'
·"-d
........ Figure I. Scheme of the differential respirometer.
CD +(COl>'-NID) +NH3* + (COl>'-NID)
I
I I
Tm,. + (COl>'-NID) +NID· + (COl>'-NID)
......
I
- Indualrial __
- Synthetic mnUure (COD+N.
Figure 2. Graphic example of the test procedure.
The titration biosensor A standardized procedure is being developed at the Politecnico di Milano which allows determination of the inhibition effects of specific toxicants or toxic wastewaters on nitrifying bacteria found in wastewater treatment plants. The activated sludge to be tested is introduced into the aerated beaker, i.e. the biosensor reactor and the equilibrium pH of the system is first reached. Then, a given amount of ammonium (usuaIly NlLtCl), of the order of some mg NH4-NIl, a concentration which is appreciably higher than the half saturation constant of the Michaelis-Menten equation, is added. As a consequence a nitrification activity
A. ROZZlel al.
164
starts which is practically constant (substrate saturated conditions and zero order reaction rate with respect to the substrate). This nitrification rate (v nn ) , which corresponds to the activity in non-inhibiting conditions (blank test), is measured using the above mentioned titration technique as: V n it =
v*N* 14/2 (ppm NH 4-N/min)
(2)
fyp lcal pr ofile of the added titr ant du rtng .. to. lclty t•• t
Og
-~
01 07
01 t! 0
§
I
~
O' 0
>
0 2 01
500
1000
. 500
2000
tim.
2~00
>000
3500
. 000
t• •
Figure 3. Example of inhibition test by the ANITA biosensor.
where v (ml/min) is the flow rate of alkali titrant calculated by a 10 points centered moving window, N (meq/ml) is the titrant normality, 14 (g/mole) is the nitrogen molecular weight and 2 is the ratio between the mole of acid and mole of nitrogen in equation I. Then, the candidate toxicant is added and the new nitrification rate is measured again. The ratio between the nitrification rate related to the potential inhibition over the nitri fication rate related to the blank is the percentage of activity. In Fig. 3 the experimental results of a test are illustrated as an example. The titrant used was NaOH 0.05 N. The sludge volume was 1.0 or 2.0 liters depending on the sludge concentration. Wastewaters The wastewater used to feed the differential respirometer during the colonization period was the influent to the Livescia (Como) WWTP, mainly made of wastewater discharged by local textile printing industries diluted with domestic sewage. Segregated and mixed effluents were sampled from textile mills to be used for the inhibition tests. In Table I, the main characteristics of the textile effluents tested are reported. Table I. The main characteristics of the tested effluents Effluent Effluent A Effluent B
N-TKN ml 60.17 148.01
The sludge used to inoculate the di fferential respirometer and to run the activity tests with the AN ITA biosensor was drawn from the aeration tank of the Livescia WWTP. Two sanitising agents normally used in agro-industries, namely sodium hypochlorite and benzalconium chloride (Effluent C and B) were also tested for acute toxicity on nitrifiers. RESULTS AND DISCUSSION Acute inhibition by several potential toxicants (both textile effluents and sanitising agents) was assayed and different results were obtained according to the nature of the toxicant.
Characterization of textile wastewater
165
Tests on the titrimetric biosensor Two tests related to two textile wastewaters are reported. Effluent A contained no inhibitors to the nitrifiers and, therefore, a straight line titration was obtained (i.e. constant activity) before and after the addition of 33-50-55 % volume of effluent (diagram not reported). Effluent B (Figure 4) contained inhibitors, as could be shown by the respirometric tests, but, during the titration test, other interfering reactions took place, mainly oxidation of organics by heterotrophic bacteria and acidification of the mixed liquor by C02 release, and inconsistent results were obtained. In fact, the activity after the addition of the effluent was higher than the activity of the blank, even after addition of allylthiourea, a specific inhibitor of ammonia oxidizers. The toxic effect of a typical disinfectant (NaCIO) and of a cationic surfactant (benzalconium chloride) has been evaluated according to the procedure already described. For both toxicants the tests have been run at different sludge concentrations. In fact, as already reported by King and Dutka (1986) , toxicity may depend upon the latter parameter, the lower the biomass concentration the higher the inhib iting effect of the same toxicant concentration. In Figures 5 and 6 the activity of ammonium oxidizing bacteria in the presence of a certain toxicant concentration is plotted versus the relative toxicant concentration for the three activated sludge concentrations tested . The activated sludge concentration of the aeration tank at the wastewater treatment plant was around 3.3 g YS/I, therefore, close to the highest sludge concentration tested. Toxicity tests with much lower sludge concentrations than those actually used in the biological process were performed in order to compare the titrimetric and respirometric results, the latter being performed in presence of about 1.7 g YS/I. According to the titration results, the toxic effect of the cationic surfactant seems more dependent upon biomass concentration than that of NaCIO. 1.6
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20
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60
50
70
80
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Figure 4. Titration test with effluent B.
ToxIcIty of NaCrO 120 100
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ra·
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NaCIO.c1 (ppm)
Figure 5. Inhibition effect ofNaCIO.
14
18
18
A. ROZZI et ill.
166
Respirometric results Two tests related to two textile wastewaters arc reported. The respirometric test on Effluent A (Fig. 7) showed no inhibition either to heterotrophs or to nitrifiers and is consistent with the titrirnetric results.
- - - - - ---- --
-
l
Toxicity of bcnzalconium chlorid e 120
•
100 ~
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co
80
60
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40
20
l_
•
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•
~~
•
0 0
300
200
600
400
•
700
Bonza lcon lum chlorldo (ppm)
Figure 6. Inhibition effect of benzalconiurn chloride.
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-
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lest reactor
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·1
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10
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16
Figure 7. Respirometric test on Effluent A.
As can be seen, an OUR increase was recorded after each ammonia addition at different Effluent A dosages. On the basis of the test, Effluent A biodegradability was estimated around 48%. The respirometric test carried out on Effluent 8 showed a slight inhibition of nitrificrs (Fig. 8 a, b, c). Effluent 8 biodegradability was estimated around 78')10. This figure confirms that the high concentration of biodegradable organic substances might have interfered with the titrirnctric test, which did not show any nitrificr inhibition. In Figure 9, rcsults of the sodium hypochlorite test are reported. The first dosage (09 mg NuCIO-el/!) did not cause any decrease in OUR (Fig. 9a). The second dosage (1.8 mg NaCIO-elll) caused a 75% OUR decrease (Fig. 9b), while the third dosage almost IOO"!., (Fig. ge). After this latter dosage, another ammonia addition was carried out into the reactor. No increase in OUR was observed, demonstrating the acute and irreversible inhibition of nitrifiers at such high NaCIO concentration. The E<. \0 was then cstimatcd to be around J.5 mg ClIl.
Characterizatio n o f te xtile wastewa ter 100 ,
167
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Figure 8. Effluent B respirometric test.
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The experimental procedure used to compare the respirometric biosensor to the titrimetric biosensor did not allow differentiation of the OUR due to heterotrophic activity on the benzalconium chloride and its inhibitory effect on the nitrifiers activity, therefor e it was not possible to evaluate any nitrifier inhibition.
A. ROZZI et al.
168
CONCLUSIONS The biosensors described in this paper have been demonstrated as very powerful tools for the characterization of industrial and agro-industrial wastewaters. The respirometer biosensor recorded a higher toxic effect of both the disinfectants and of almost all the wastewaters tested during this work. This higher sensitivity might be due, as previously indicated, to the effect of the inhibitory compoundlbiomass ratio in the titrimetric biosensors. The tests have shown that the toxicity level is strongly dependent on the sludge concentration. Nevertheless, inhibitory effects were well shown at all the biomass concentrations. By this point of view, further standardization trials are necessary.
It is very important that both tests are carried out at the same sludge concentration and, possibly, at the same inhibitory compound/biomass ratio as in the full scale treatment plant where the waste is treated. Nevertheless, it can be concluded that both biosensors may be used for rapid and simple inhibitory screening tests of industrial products and wastewater. ACKNOWLEDGEMENTS This research has been partly funded by the EU Contract ENV4-CT95-0064 "Integrated recycling and emission abatement in textile industry" and by the Italian Ministry of University and Scientific Research MURST Contract 40% "Trattamento, utilizzazione e monitoraggio di reflui e residui di attivita agroalimentari", The authors thank the Company AUSTEP for placing a ANITA biosensor at the disposal of the Department lIAR of the Politecnico di Milano and also thank CIDA for funding the development of the different ial respirometer and related tests. REFERENCES Bertone, G., Gemelli, S., Tilche , A., Bianchi, R., and Bergna, G. (1997). A new approach to the evaluat ion of the biological treatab ility of industrial wastewater for the implementation of the "waste design concept ". Wat. Sci. Tech., 36(2-3), 81-90 . Cech, I . S., Chudoba, I., and Grau, P. (1994). Determination of kinetic constants of activated sludge microorganisms. Wat. Sci. Tech., 17, 259-272. Drtil, M., Nemeth, P. and Bodtk, I. (1993) . Kinetic constants of nitrification. Wat. Res., 27(1), 35-39 . Gernaey , K. (1997) . Development of Sensors for On-line Monitoring of Nitrification in Activated Sludge. Ph. D. Thes is, Univer siteit Gent, Belgium. King, E. F. and Dukta, B. J. (1986). Toxicity Testing using Microorganisms. Vol. I, Eds. Bitton G. and Dukta 8., CRC Press, Florida . Kong, Z., Vanrolleghem, P.A. and Verstraete, W. (1993) . An activated sludge-based biosensor for rapid IC50 estimat ion and online toxicity monitonng. Biosens. and Bioelect., 8, 49-58. Maffei, D. (1995) . Biosensore per 10Misura dell'Attivlla Nitrificante e dell'Ammoniaca in Soluzione . M.Sc. Thesis, Politecnico di Milano, Italy. Massone, A. G., Gernaey, K., Rozz i, A., Willems, P. and Verstraete , W. (1995) . Biosensor. for nitrogen control in wastewaters . Wat. Sci. Technol., 34(1-2), 213·220. Massone, A. G. (1997). I Sensori a Titolazione. Sviluppo di una Nuova Tecnologia per il Controllo degli Impianti dt Depurazione. Ph.D. thesis, Politecnico di MIlano, Italy. Massone, A., Antonelli, M. and Rozzi, A. (1996) . The Denicon: a Novel Biosensor to Control Denitrification in Biological Wastewater Treatment Plants. Med. Fac, Landbouww. Univ. Gent, 61/4a, 1709. Ramadori, R., Rozzi, A. and Tandoi, V. (1980) . An automated system for monitoring the kinetics of biological oxidation of ammonia. Wat. Res., 14, 1555-1557 . Randall , E. W., Wilkinson, A. and Ekama, G. A. (1991) An instrument for the direct determination of oxygen utilisation rate. Water SA. 17(1), 11-17. Spanjers, H. and Klapwijk, A. (1990). On-line meter for respiration rate and short-term biochemical oxygen demand in the control of the activated sludge process. In: Advances in Water Pollution Control. (Ed . Briggs R.), Pergamon Press, London, 67·77. Surmacz-Gorska, J., Gernaey, K., Demuynck, C., Vanrolleghem, P. and Verstraete, W. (1996) . Nitrification monitoring in activated sludge by oxygen uptake rate (OUR) measurements. War. Res., 30(5), 1228-1236. Vanrolleghem. P.A., Dries, D. and Verstraete, W. (1990) . RODTOX: Biosensor for rapid determination of the biochemical oxygen demand and on-line monitoring of the toxicity of wastewaters. In: Proceedings of the Fifth European Congress on Biotechnology. Copenhagen, Denmark , July 8·13 1990. Vol. I, 161-164. Vershuere, L., Gernaey, K., and Verstraete, W. (1995) . De NITROX : een snelle en gevoelige on-line toxiciteitsmeter voor water en afvalwater. Water, 14, 163-168.
War. Sci. Tech Vol. 40, No. I, pp, 161-168, 1999 c 1999IAWQ Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0273-1223/99520.00 + 0.00
PH: S0273-1223(99)00376-5
CHARACTERIZATION OF TEXTILE WASTEWATER AND OTHER INDUSTRIAL WASTEWATERS BY RESPIROMETRIC AND TITRATION BIOSENSORS A. Rozzi *, E. Ficara*, C. M. Cellamare** and G. Bortone** *DIIAR, Politecnico di Milano. Piazza L da Vinci 32. 20133 Milano. Italy uENEA. Via Martiri di Monte Sole 4, 40129 Bologna, Italy
ABSTRACT Many industrial effluents, such as textile and tannery wastewater, contain slowly biodegradable, refractory or even toxic compounds at variable concentrations which may interfere with the efficient operation of biological wastewater treatment plants, in particular with the nitrification stage. Agro-industrial effluents may occasionally contain sanitising agents which are by definition biocides. Two different biosensors, based on respirometry (oxygen uptake rate, OUR measurements) and on basic titration respectively, were used to measure degradation rates of industrial wastewater samples by autotrophic bacteria (ammonia oxidizers). Specific sanitisers such as sodium hypochlorite and benzalconium chloride were used to evaluate and compare the nitrifying activity measured by the two different instruments. 0 1999 IAWQ Published by Elsevier Science Ltd. All rights reserved.
KEYWORDS Respirometry; titration; biosensors; inhibition; nitrification; industrial wastewaters. INTRODUCTION Automatic process control is increasingly used in biological industrial wastewater treatment since it makes possible the on-line optimization of operating conditions even as regards to variable loading rates and accidental or deliberate addition of toxicants in the influent to the biological treatment plant. Among the instruments which may be used to monitor biotechnological processes, such as aerobic and anaerobic wastewater treatments, biosensors are particularly useful because they make possible to measure the activity of the biomass within the reactors as well as substrate or product concentrations. The respiration rate is frequently used in wastewater treatment plants (WWTPs) to evaluate the potential toxicity and organic load of the incoming streams on active biomass. Biosensors measuring the respiration rate use oxygen probes. The determination is based either on oxygen measurement after interruption of the aeration ("closed respirometry") or on the oxygen depletion curve in a continuously aerated system ("open respirometry"). Examples of the former biosensor may be found in Cech et al. (1984), Drtil et al. (1993), Spanjers and Klapwijk (1990), Verschuere et al. (1995) and in Surmacz-Gorska et al. (1996) while the 161
162
A. ROZZI et al.
RODTOX (Rapid Oxygen Demand & TOXicity) biosensor (Vanrolleghem et al., 1990) is an example of the latter. Titration biosensors measure the activity of an acidifying (Ramadori et al., 1980) or alkalising (Massone et al., 1996) bacterial consortia by titration at constant pH. Among this class of biosensors, the ANITA (Ammonium Nitrification Analyser) is a titration system which monitors the ammonium oxidation kinetics by determining theO rate of alkali titrant required to keep the pH at a given set point value (Massone et al., 1995; Massone, 1997). Nitrification is an oxidation reaction which produces two moles of protons per mole of oxidized ammonium. The biosensor determines the acid products released during the first step of the above conversion:
NH: +~02 ~ NO'2 +H 20+2W 2
(1)
Moreover, comparing the maximum degradation rate before and after the addition of a certain amount of a chemical, the inhibition effect of the latter may be estimated (Maffei, 1995; Massone, 1997; Gemaey, 1997). The use ofbiosensors based either on dissolved oxygen (DO) depletion (closed respirometry) or on titration, in order to monitor inhibition of nitrification by industrial (textile) wastewaters and by solutions containing sanitizing agents, is addressed in this paper. MATERIALS AND METHODS Respirometric biosensor The instrument is made of two parallel moving bed continuous flow completely mixed reactors, equipped with oxygen sensors (Danfoss mod.2500) connected to a computer that acts as controller, data acquisition -and processing unit (Fig. I). The working volume of each reactor is 5.850 I. The control program is implemented on Lab View (National Instrument Environment). Air is provided by a compressor to the reactors through a sparging porous diffuser. Aeration is stopped when the oxygen concentration reaches a set point upper value (6 mg/l). After air is shut-off, DO begins to decrease, and oxygen uptake rate (OUR) can be determined from the slope of the curve. Calculation of OUR is made as soon as the curve is sufficiently linear or when oxygen concentration reaches a lower set point value (2 mgt!) (Randall et al., 1991). The moving bed was made of polyurethane macroreticulated foam (120 PPD which is colonized by fixed biomass. The biofilm reactor was selected to simplify the operation of the biosensor, by avoiding secondary clarifiers and recycling pumps, and by allowing it to operate at low HRTs and, therefore, to obtain rapid responses in terms of OUR changes. The experimental scheme is similar to the one reported in the ISO Method 11733, but information about biodegradability and potential toxicity of the tested stream is derived by OUR profiles (including EC 50 estimation) rather than measuring effiuent COD (Bortone,et al., 1997). Although the biosensor was designed to evaluate the degree of treatability related to carbonaceous pollutants and to ammonia in a wastewater, the main goal in this study was to determine the potential inhibitionon nitrifiers only. For the evaluation of the treatability of specific industrial wastewaters into a given WWTP, the two reactors are, first, inoculated with activated sludge drawn from that plant and acclimated to the composite wastewater fed to the same plant. Then, the two reactors of the respirometer are fed at the same constant flow rate with the same influent as the full scale WWTP. The industrial wastewater to be tested (the candidate toxicant) is analysed for COD and NH 4 • Extra feed, made of this effiuent is then added to one of the two reactors at a given load, while the same additional amounts of COD and ~ are provided, as acetate and ammonium, to the second one in order to keep constant the total organic and nitrogen loads in both the moving beds. At least three increasing dosages are used. The test is completed by ammonia addition to both reactors (Fig. 2). From the analysis of the differential responses, the percentage of biodegradable COD, the inhibition to respiration, inhibition to nitrification and process kinetics can be obtained. The first step in Fig. 2 allows estimation of the amount of biodegradable COD in the test stream. By increasing the dosage of COD in both reactors, it is possible to evaluate whether the OUR increase in the
Characterization of textile wastewater
163
reactor fed on the industrial wastewater is proportional (absence of respiration inhibition) or less than proportional (inhibition) to the OUR increment in the blank reactor. The same procedure can be used with increasing dosages of ammonia to detect nitrification inhibition. During the experimental comparison with the titrimetric biosensor, spike dosages of ammonia and inhibitory compounds were carried out at the beginning of each test, to quickly reach steady state conditions.
-------~
I
BLANK REACTOR
I I
I I
I '--------'
·"-d
........ Figure I. Scheme of the differential respirometer.
CD +(COl>'-NID) +NH3* + (COl>'-NID)
I
I I
Tm,. + (COl>'-NID) +NID· + (COl>'-NID)
......
I
- Indualrial __
- Synthetic mnUure (COD+N.
Figure 2. Graphic example of the test procedure.
The titration biosensor A standardized procedure is being developed at the Politecnico di Milano which allows determination of the inhibition effects of specific toxicants or toxic wastewaters on nitrifying bacteria found in wastewater treatment plants. The activated sludge to be tested is introduced into the aerated beaker, i.e. the biosensor reactor and the equilibrium pH of the system is first reached. Then, a given amount of ammonium (usuaIly NlLtCl), of the order of some mg NH4-NIl, a concentration which is appreciably higher than the half saturation constant of the Michaelis-Menten equation, is added. As a consequence a nitrification activity
A. ROZZlel al.
164
starts which is practically constant (substrate saturated conditions and zero order reaction rate with respect to the substrate). This nitrification rate (v nn ) , which corresponds to the activity in non-inhibiting conditions (blank test), is measured using the above mentioned titration technique as: V n it =
v*N* 14/2 (ppm NH 4-N/min)
(2)
fyp lcal pr ofile of the added titr ant du rtng .. to. lclty t•• t
Og
-~
01 07
01 t! 0
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~
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>
0 2 01
500
1000
. 500
2000
tim.
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. 000
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Figure 3. Example of inhibition test by the ANITA biosensor.
where v (ml/min) is the flow rate of alkali titrant calculated by a 10 points centered moving window, N (meq/ml) is the titrant normality, 14 (g/mole) is the nitrogen molecular weight and 2 is the ratio between the mole of acid and mole of nitrogen in equation I. Then, the candidate toxicant is added and the new nitrification rate is measured again. The ratio between the nitrification rate related to the potential inhibition over the nitri fication rate related to the blank is the percentage of activity. In Fig. 3 the experimental results of a test are illustrated as an example. The titrant used was NaOH 0.05 N. The sludge volume was 1.0 or 2.0 liters depending on the sludge concentration. Wastewaters The wastewater used to feed the differential respirometer during the colonization period was the influent to the Livescia (Como) WWTP, mainly made of wastewater discharged by local textile printing industries diluted with domestic sewage. Segregated and mixed effluents were sampled from textile mills to be used for the inhibition tests. In Table I, the main characteristics of the textile effluents tested are reported. Table I. The main characteristics of the tested effluents Effluent Effluent A Effluent B
N-TKN ml 60.17 148.01
The sludge used to inoculate the di fferential respirometer and to run the activity tests with the AN ITA biosensor was drawn from the aeration tank of the Livescia WWTP. Two sanitising agents normally used in agro-industries, namely sodium hypochlorite and benzalconium chloride (Effluent C and B) were also tested for acute toxicity on nitrifiers. RESULTS AND DISCUSSION Acute inhibition by several potential toxicants (both textile effluents and sanitising agents) was assayed and different results were obtained according to the nature of the toxicant.
Characterization of textile wastewater
165
Tests on the titrimetric biosensor Two tests related to two textile wastewaters are reported. Effluent A contained no inhibitors to the nitrifiers and, therefore, a straight line titration was obtained (i.e. constant activity) before and after the addition of 33-50-55 % volume of effluent (diagram not reported). Effluent B (Figure 4) contained inhibitors, as could be shown by the respirometric tests, but, during the titration test, other interfering reactions took place, mainly oxidation of organics by heterotrophic bacteria and acidification of the mixed liquor by C02 release, and inconsistent results were obtained. In fact, the activity after the addition of the effluent was higher than the activity of the blank, even after addition of allylthiourea, a specific inhibitor of ammonia oxidizers. The toxic effect of a typical disinfectant (NaCIO) and of a cationic surfactant (benzalconium chloride) has been evaluated according to the procedure already described. For both toxicants the tests have been run at different sludge concentrations. In fact, as already reported by King and Dutka (1986) , toxicity may depend upon the latter parameter, the lower the biomass concentration the higher the inhib iting effect of the same toxicant concentration. In Figures 5 and 6 the activity of ammonium oxidizing bacteria in the presence of a certain toxicant concentration is plotted versus the relative toxicant concentration for the three activated sludge concentrations tested . The activated sludge concentration of the aeration tank at the wastewater treatment plant was around 3.3 g YS/I, therefore, close to the highest sludge concentration tested. Toxicity tests with much lower sludge concentrations than those actually used in the biological process were performed in order to compare the titrimetric and respirometric results, the latter being performed in presence of about 1.7 g YS/I. According to the titration results, the toxic effect of the cationic surfactant seems more dependent upon biomass concentration than that of NaCIO. 1.6
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Figure 5. Inhibition effect ofNaCIO.
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18
18
A. ROZZI et ill.
166
Respirometric results Two tests related to two textile wastewaters arc reported. The respirometric test on Effluent A (Fig. 7) showed no inhibition either to heterotrophs or to nitrifiers and is consistent with the titrirnetric results.
- - - - - ---- --
-
l
Toxicity of bcnzalconium chlorid e 120
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300
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As can be seen, an OUR increase was recorded after each ammonia addition at different Effluent A dosages. On the basis of the test, Effluent A biodegradability was estimated around 48%. The respirometric test carried out on Effluent 8 showed a slight inhibition of nitrificrs (Fig. 8 a, b, c). Effluent 8 biodegradability was estimated around 78')10. This figure confirms that the high concentration of biodegradable organic substances might have interfered with the titrirnctric test, which did not show any nitrificr inhibition. In Figure 9, rcsults of the sodium hypochlorite test are reported. The first dosage (09 mg NuCIO-el/!) did not cause any decrease in OUR (Fig. 9a). The second dosage (1.8 mg NaCIO-elll) caused a 75% OUR decrease (Fig. 9b), while the third dosage almost IOO"!., (Fig. ge). After this latter dosage, another ammonia addition was carried out into the reactor. No increase in OUR was observed, demonstrating the acute and irreversible inhibition of nitrifiers at such high NaCIO concentration. The E<. \0 was then cstimatcd to be around J.5 mg ClIl.
Characterizatio n o f te xtile wastewa ter 100 ,
167
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The experimental procedure used to compare the respirometric biosensor to the titrimetric biosensor did not allow differentiation of the OUR due to heterotrophic activity on the benzalconium chloride and its inhibitory effect on the nitrifiers activity, therefor e it was not possible to evaluate any nitrifier inhibition.
A. ROZZI et al.
168
CONCLUSIONS The biosensors described in this paper have been demonstrated as very powerful tools for the characterization of industrial and agro-industrial wastewaters. The respirometer biosensor recorded a higher toxic effect of both the disinfectants and of almost all the wastewaters tested during this work. This higher sensitivity might be due, as previously indicated, to the effect of the inhibitory compoundlbiomass ratio in the titrimetric biosensors. The tests have shown that the toxicity level is strongly dependent on the sludge concentration. Nevertheless, inhibitory effects were well shown at all the biomass concentrations. By this point of view, further standardization trials are necessary.
It is very important that both tests are carried out at the same sludge concentration and, possibly, at the same inhibitory compound/biomass ratio as in the full scale treatment plant where the waste is treated. Nevertheless, it can be concluded that both biosensors may be used for rapid and simple inhibitory screening tests of industrial products and wastewater. ACKNOWLEDGEMENTS This research has been partly funded by the EU Contract ENV4-CT95-0064 "Integrated recycling and emission abatement in textile industry" and by the Italian Ministry of University and Scientific Research MURST Contract 40% "Trattamento, utilizzazione e monitoraggio di reflui e residui di attivita agroalimentari", The authors thank the Company AUSTEP for placing a ANITA biosensor at the disposal of the Department lIAR of the Politecnico di Milano and also thank CIDA for funding the development of the different ial respirometer and related tests. REFERENCES Bertone, G., Gemelli, S., Tilche , A., Bianchi, R., and Bergna, G. (1997). A new approach to the evaluat ion of the biological treatab ility of industrial wastewater for the implementation of the "waste design concept ". Wat. Sci. Tech., 36(2-3), 81-90 . Cech, I . S., Chudoba, I., and Grau, P. (1994). Determination of kinetic constants of activated sludge microorganisms. Wat. Sci. Tech., 17, 259-272. Drtil, M., Nemeth, P. and Bodtk, I. (1993) . Kinetic constants of nitrification. Wat. Res., 27(1), 35-39 . Gernaey , K. (1997) . Development of Sensors for On-line Monitoring of Nitrification in Activated Sludge. Ph. D. Thes is, Univer siteit Gent, Belgium. King, E. F. and Dukta, B. J. (1986). Toxicity Testing using Microorganisms. Vol. I, Eds. Bitton G. and Dukta 8., CRC Press, Florida . Kong, Z., Vanrolleghem, P.A. and Verstraete, W. (1993) . An activated sludge-based biosensor for rapid IC50 estimat ion and online toxicity monitonng. Biosens. and Bioelect., 8, 49-58. Maffei, D. (1995) . Biosensore per 10Misura dell'Attivlla Nitrificante e dell'Ammoniaca in Soluzione . M.Sc. Thesis, Politecnico di Milano, Italy. Massone, A. G., Gernaey, K., Rozz i, A., Willems, P. and Verstraete , W. (1995) . Biosensor. for nitrogen control in wastewaters . Wat. Sci. Technol., 34(1-2), 213·220. Massone, A. G. (1997). I Sensori a Titolazione. Sviluppo di una Nuova Tecnologia per il Controllo degli Impianti dt Depurazione. Ph.D. thesis, Politecnico di MIlano, Italy. Massone, A., Antonelli, M. and Rozzi, A. (1996) . The Denicon: a Novel Biosensor to Control Denitrification in Biological Wastewater Treatment Plants. Med. Fac, Landbouww. Univ. Gent, 61/4a, 1709. Ramadori, R., Rozzi, A. and Tandoi, V. (1980) . An automated system for monitoring the kinetics of biological oxidation of ammonia. Wat. Res., 14, 1555-1557 . Randall , E. W., Wilkinson, A. and Ekama, G. A. (1991) An instrument for the direct determination of oxygen utilisation rate. Water SA. 17(1), 11-17. Spanjers, H. and Klapwijk, A. (1990). On-line meter for respiration rate and short-term biochemical oxygen demand in the control of the activated sludge process. In: Advances in Water Pollution Control. (Ed . Briggs R.), Pergamon Press, London, 67·77. Surmacz-Gorska, J., Gernaey, K., Demuynck, C., Vanrolleghem, P. and Verstraete, W. (1996) . Nitrification monitoring in activated sludge by oxygen uptake rate (OUR) measurements. War. Res., 30(5), 1228-1236. Vanrolleghem. P.A., Dries, D. and Verstraete, W. (1990) . RODTOX: Biosensor for rapid determination of the biochemical oxygen demand and on-line monitoring of the toxicity of wastewaters. In: Proceedings of the Fifth European Congress on Biotechnology. Copenhagen, Denmark , July 8·13 1990. Vol. I, 161-164. Vershuere, L., Gernaey, K., and Verstraete, W. (1995) . De NITROX : een snelle en gevoelige on-line toxiciteitsmeter voor water en afvalwater. Water, 14, 163-168.