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Characterization of a phenol degrading mixed population by enzyme assay
PII: S0043-1354(99)00248-1
Wat. Res. Vol. 34, No. 4, pp. 1127±1134, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
CHARACTERIZATION OF A PHENOL DEGRADING MIXED POPULATION BY ENZYME ASSAY MULUGETA KIBRET*, WALTER SOMITSCH and KARL-HEINZ ROBRA Institute of Environmental Biotechnology, Technical University Graz, Petersgasse 12/I, A-8010 Graz, Austria (First received 1 November 1998; accepted in revised form 1 May 1999) AbstractÐThe metabolic response of a mixed population to stepwise increase in phenol concentration was followed with the assay of pyrocatechase activity. The mixed population was grown in a laboratory scale activated sludge plant for 40 days. The inlet phenol concentration (750 mg lÿ1) was then increased stepwise to 1250, 2500 and 4500 mg lÿ1, corresponding to low, moderate and high level increases in phenol loading, respectively. Pre- and post-shock assessments were performed on samples collected from the aeration tank. Pyrocatechase activity increased following each step increase in phenol and the level of increase was dependent on the magnitude of the step increase. Following low level increase in loading, phenol was not detected from the aeration tank. At moderate level loading phenol was temporarily detected in the aeration tank but disappeared after 7 days. At high level loading phenol was accumulated in the aeration tank throughout. Pyrocatechase activity was dependent on the concentration of phenol in the aeration tank. From pyrocatechase activity assay it was possible to demonstrate the metabolic response of the activated sludge to changes in phenol concentration. The accumulation of phenol and decrease in pyrocatechase activity are considered as the stage when the system no longer copes with increase in phenol loading. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐenzyme, mixed microbial population, phenol, pyrocatechase, degradation
INTRODUCTION
Phenol and other phenolic compounds are common constituents of aqueous euents from processes such as polymeric resin production, oil re®ning and cokeing plants. Phenol is both toxic and lethal to ®sh at relatively low concentrations e.g. 5±25 mg lÿ1 and imparts objectionable tastes to drinking water at far lower concentrations (Hill and Robinson, 1975). Euents containing such compounds are usually treated in activated sludge processes, where the organic constituents of the wastewater are removed oxidatively by a mixed population of microorganisms, primarily heterotrophic bacteria (Richards et al., 1984; Nybroe et al., 1992). The activated sludge is subjected to periodic ¯uctuations in the inlet substrate concentration, in¯uent ¯ow rate, aeration rate, pH and temperature. Many operational problems are associated with such ¯uctuations since these factors play an important role in the establishment and maintenance of the microbial communities in the activated sludge (Rozich et al., 1983). The methods, which have traditionally been used to investigate the activity of activated sludge, *Author to whom all correspondence should be addressed. Tel: +43-316-873/8310; fax: +43-316-518-636; e-mail: [email protected]
include mixed liquor suspended solids, mixed liquor volatile suspended solids, biochemical oxygen demand, chemical oxygen demand and dissolved organic carbon. Most of these methods although useful in practice, do not give much insight into the factors that control the proper biological process (Peters et al., 1975; Ros et al., 1988). It has been found for the activated sludge process, that the activity of certain oxido-reductive enzymes follows closely the physico-chemical indicators commonly used in monitoring treatment works (Richards et al., 1984). In a study on a pilot scale plant, Nybroe et al. (1992) showed that enzyme activities re¯ect microbial activities in wastewater. These ®ndings gave impetus to the use of enzymatic methods to characterize the activity of activated sludge. Enzyme activities have received particular attention for a number of reasons: (1) these activities play a key role in the hydrolysis and mineralization of organic wastes, (2) the characterization of enzyme activities can provide insight into the biochemical factors controlling the treatability of xenobiotic chemicals, (3) enzymes can be added exogenously to treatment processes to improve overall treatment eciencies and (4) patterns of enzyme activities can be useful for the identi®cation of microbial populations (Boczar et al., 1992).
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To design the best control strategies for wastewater treatment plants, it is necessary to study the performance of the system in relation to varying conditions. In this context, it is desirable to examine the response of an activated sludge to shock loads because similar situations are encountered in the ®eld. There are few earlier investigations on the response of activated sludge to shock loading (Rozich and Gaudy, 1985; Allsop et al., 1993; Watanabe et al., 1996b). In this study the activity of activated sludge from a laboratory scale treatment plant, which was subjected to stepwise increases in the inlet phenol concentrations, was characterized by the assay of pyrocatechase activity. This enzyme catalyzes a reaction in the initial steps of aerobic phenol degradation where phenol is ®rst oxidized to catechol that is further oxidized in the meta and ortho pathways. In the ortho pathway, pyrocatechase, i.e. catechol 1,2-dioxygenase (EC. 1.13.11.1) catalyses the cleavage of the aromatic ring of catechol to form cis,cis-muconic acid by the insertion of two atoms of molecular oxygen. In the meta pathway, metapyrocatechase, i.e. catechol 2,3-dioxygenase (EC. 1.13.11.2) catalyzes conversion of catechol to a-hydroxymuconic semialdehyde (Stanier and Ornston, 1973). MATERIALS AND METHODS
Overall design The activated sludge was subjected to stepwise increase in phenol loading rate, by increasing the phenol concentration in the feed stepwise from 750 mg lÿ1 to 1500, 2500 and 4500 mg lÿ1 at a constant ¯ow rate of 960 ml dÿ1 and at a dilution rate of 0.282 dayÿ1. The phenol-loading rate was increased to 1440, 2400 and 4320 mg lÿ1 dÿ1, respectively. Performance assessments were done in the pre- and post-shock conditions for each step increase. Microorganism and cultivation A mixed population of organisms was used as a source of inoculum. It was originally obtained from soil contaminated with phenol. It has repeatedly been used for phenol biodegradation studies. For use in this experiment, 500 ml of mineral salts medium supplemented with 0.5 g lÿ1 phenol was inoculated with 40 ml of homogenized culture and incubated in rotary shaker at 278C with a speed of 180 rpm. Growth of culture was monitored spectrophotometrically by measuring optical density at 578 nm with a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland). Phenol disappearance in the culture was also measured. Actively growing culture was transferred into a reactor containing the same medium. Initially the culture was grown in batch and continuous ¯ow was started. The medium contained the following constituents (mg lÿ1): phenol variable (750±4500); MgSO47H2O, 9.4; CaSO42H2O, 4.7; Na2HPO42H2O, 752; KH2PO4, 63.92; NH4Cl, 18.8 and trace minerals solution 0.47 ml. The trace minerals solution contained the following components (mg lÿ1): Na2EDTA, 2500; ZnSO47H2O, 100; MnCl26H2O, 30; H3BO3, 300; CoCl26H2O, 200; CuCl22H2O, 10; NiCl22H2O, 20; Na2Mo42H2O, 900; Na2SeO3, 20 and FeSO47H2O, 1000. The pH of the medium was adjusted to 7.2 with NaOH. All components of the medium except phenol were autoclaved at 1218C.
Phenol was ®lter sterilized separately and added to other components after autoclaving. Apparatus A laboratory scale bioreactor was employed for the cultivation of organisms (German Standard Methods, DIN 38412, 1994). The aeration tank of the reactor had a total volume of 6 l (with active volume of 3.4 l) and the settlement tank had a volume of 2.6 l (Fig. 1). The ¯ow rate of the medium to the reactor was maintained at 1 l dÿ1 with Watson Marlow peristaltic pump Model 313 (Watson Marlow, Falmouth, U.K.). The temperature of the reactor was maintained at 308C with a circulating water bath (Lauda K4RD, MessgeraÈte-Werk Lauda, Vienna, Austria). Air and agitation were provided from a compressed air source at a ¯ow rate of 130 l hÿ1. A portion of the sludge from the settlement tank was recirculated into the aeration tank using an air pump at a ¯ow rate of 130 ml minÿ1. Initially the culture was grown in mineral salts medium supplemented with 750 mg lÿ1phenol for 40 days. After phenol was completely degraded (phenol concentration less than 5 mg lÿ1), the phenol concentration in the feed was increased to 1500, 1250 and 4500 mg lÿ1. Analytical methods The dissolved oxygen concentration in the aeration tank was directly measured with Microprocessor oximeter OXI 96 (WTW, Weilheim, Germany). The pH was measured with Orion pH meter model 240 (Orion Research Incorporation, Boston, USA). For the determination of dissolved organic carbon (DOC) and phenol, samples from the aeration tank were ®ltered under pressure through Magna Nylon membrane ®lters of 50-mm diameter, having a pore size of 0.22 mm (Miro Separations, Westboro, USA). Phenol was measured spectrophotometrically by the modi®ed 4-aminoantipyrin method of Martin indicated elsewhere (Yang and Humphrey, 1975). To 50 ml of a ®ltered sample from the aeration tank, 0.5 ml of 1% (w/v) potassium ferricyanide and 2.5 ml of 1% (w/v) 4-aminoantipyrine were added. The absorbancy of the resulting mixture was read at 505 nm with a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland). Phenol concentration was determined with a calibration curve made from known phenol standard. The dry weight was measured by ®ltering aeration tank sample through a dried, tarred, 0.22 mm nylon ®lter and drying at 1058C for a minimum of 24 h. The dissolved organic carbon (DOC) concentration in the aeration tank was measured with ASTRO Model 2001 system
Fig. 1. Schematic diagram of the activated sludge plant. A is the medium reservoir, B the thermotrap, C the pump, D the aeration tank, E the settlement tank, F the euent receiver and G the temperature control. Solid lines indicate the direction of ¯ow of medium and dashed lines indicate the direction of ¯ow of sludge.
Characterization of a mixed population 2 computer controlled laboratory total organic carbon analyzer (Texas Instruments, USA). Enzyme assays Enzyme assays were performed at room temperature using quartz cuvettes of 1 cm path length, in a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland) in time drive mode so that a progress curve could be obtained for calculation of enzyme activity. To harvest cells, samples from the aeration tank were centrifuged at 5000 rpm for 20 min in a Sorvall RC-5B refrigerated centrifuge (Du Pont Instruments, Hamburg, Germany). The pellet was washed twice with 0.1 M phosphate buer (pH 7.5) and resuspended in the same buer (Hegeman, 1966). Subsequently the cell pellet was disrupted by sonication for seven min using SONOPULS HD sonicator (BANDELINE electronics, Berlin, Germany). The cell debris was removed by centrifugation of the homogenized cell suspension at 20,000 rpm for 25 min. The supernatant was immediately used for enzyme assays. Pyrocatechase, i.e. catechol 1,2-dioxygenase (E.C.1.13.11.1) assay Pyrocatechase activity was assayed spectrophotometrically based on the measurement of the rate of cis,cismuconic acid formation from catechol at 260 nm. The reaction mixture contained 200 ml of 0.1 mM catechol, 200 ml of 500 mM EDTA, 100 ml crude cell-free extract and 0.1 M of phosphate buer (pH 7.5) to the ®nal volume of 3.0 ml (Murray et al., 1972). The reaction was initiated by addition of the crude extract. The protein concentration of the crude extract was measured following the method of Lowry et al. (1951) using crystalline bovine serum albumin as a standard. Calculation of enzyme activity Enzyme activities were determined from the initial gradients of the absorption curves. Molar extinction coecient (E ) of 16,900 for cis,cis-muconic acid was used to calculate initial velocity of the enzyme reaction (Gibson,
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1975). All chemicals were analytical grade obtained from Merck, Boehringer Mannheim and Sigma. RESULTS
Change in the speci®c metabolic activities of the activated sludge i.e. pyrocatechase activity during step change in phenol concentration is shown on Figs 2±4. When the inlet phenol concentration was increased from 750 to 1500 mg lÿ1, phenol was almost completely removed by the activated sludge. Phenol concentration in the aeration tank was below the detection limit through out the entire period. Pyrocatechase activity increased shortly 1 day after the change in phenol concentration and dropped sharply the next day and stayed constant thereafter. A concomitant momentary decrease in the aeration tank dissolved oxygen (from 5.7 to 5.3 mg lÿ1) was observed 1 day after the step increase (Fig. 2). Following intermediate step increase in inlet phenol concentration, i.e. from 1500 to 2500 mg lÿ1, the aeration tank phenol concentration increased temporarily and then decreased to zero. Seven days were required to remove phenol completely from the aeration tank, with a peak on the third day after the step increase [Fig. 3(B)]. Pyrocatechase activity increased after the step change in phenol concentration and a concurrent drop in dissolved oxygen concentration in the aeration tank (from 5.2 to 3.7 mg lÿ1) after 1 day was observed due to the step increase [Fig. 3(A)]. With increase in inlet phenol concentration from 2500 to 4500 mg lÿ1, phenol was not completely removed from the aeration tank, its concentration
Fig. 2. Enzyme activity and dissolved oxygen concentration pro®les of the activated sludge following a low level increase in phenol concentration from 750 to 1500 mg lÿ1.
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Fig. 3. Enzyme activity and dissolved oxygen (A), phenol and DOC (B) concentration pro®les of the activated sludge following a moderate level increase in phenol concentration from 1500 to 2500 mg lÿ1.
rather increased with time [Fig. 4(B)]. Pyrocatechase activity increased after the step increase in phenol concentration but declined after 4 days following the step increase. The dissolved oxygen concentration in the aeration tank dropped sharply from 2.6 to 0.5 mg lÿ1 1 day after the step increase in phenol, then to zero 4 days after the step change [Fig. 4(A)]. Pyrocatechase activity was dependent on the phenol concentration in the aeration tank. As shown on Fig. 3, pyrocatechase activity increased with increase in phenol concentration in the aeration tank up to 158 mg lÿ1 and dropped below this concentration. Pyrocatechase activity also decreased when the phenol concentration in the aeration tank was above 160 mg lÿ1. (Fig. 4).
Dissolved organic carbon (DOC) was accumulated even when the phenol concentration was below the detection limit, during the low-level loading followed by an increase in DOC concomitant with the increase in phenol concentration at the moderate and high level loadings. Following moderate level increase in loading, DOC was temporarily accumulated but declined on the second day [Fig. 3(B)]. At high level increase however, the DOC concentration in the aeration tank increased steadily [Fig. 4(B)]. Overall increase in the dry weight concentration of 0.16 g lÿ1 and 1.13 g lÿ1 were observed following step increase in phenol concentration from 750 to 1500 mg lÿ1 and 1500 to 2500 mg lÿ1, respectively. However, a momentary increase in dry weight con-
Characterization of a mixed population
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Fig. 4. Enzyme activity and dissolved oxygen (A), phenol and DOC (B) concentration pro®les of the activated sludge following a high level increase in phenol concentration from 2500 to 4500 mg lÿ1.
centration of 0.38 g lÿ1 followed by a decline after day 4 was observed for high level increase in phenol concentration. The speci®c phenol utilization rates were estimated from the amount of phenol degraded per day and from the dry weight. The speci®c phenol utilization rate generally increased after the step increase in phenol concentration and decreased with time thereafter. For instance, the speci®c phenol utilization rate before increase in inlet phenol from 1500 to 2500 mg lÿ1 was 1.65 g lÿ1 dÿ1 and increased to 2.064 g lÿ1 dÿ1 soon after the step increase. However it came down to 1.75, 1.6 and 1.2 g lÿ1 dÿ1 on days 2, 3, and 7 after the step increase, respectively [Fig. 5(B)]. Similarly the speci®c phenol utilization rate increased from a preshock value of 1.25 g lÿ1 dÿ1 to 2.06 g lÿ1 dÿ1 after
step increase in phenol from 2500 to 4500 g lÿ1, then it decreased to 1.85 g lÿ1 after 7 days [Fig. 5(A)]. DISCUSSION
Step increases in phenol loading into the activated sludge plant were accompanied by a temporary increase in pyrocatechase activity. A study of the literature reveals that both pure and mixed microbial cultures experience physiological adaptation which involves adjustments in the relative amounts of the primary macromolecules, the level of enzymes and the concentration of intracellular metabolites in response to change in environmental conditions. This is due to available reaction potential (ARP) which is de®ned as the ability of a mi-
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Fig. 5. Speci®c phenol utilization rate of the activated sludge following increase in phenol concentration from 2500 to 4500 mg lÿ1 (A) and from 1500 to 2500 mg lÿ1 (B).
crobial culture to rapidly increase in growth and substrate removal rates during a transient response (Daigger and Grady, 1982). Comparison of pre-shock and the maximum pyrocatechase activity after the step increase in phenol concentration indicates dierences in the level of response to changes in phenol loading. The level of response was dependent on the magnitude of the step increase. There was only increase in pyrocatechase activity of 0.024 units 1 day after the change in the inlet phenol concentration from 750 to 1500 mg lÿ1. Intermediate step change in phenol loading caused moderate level response with increase of 0.034 units of pyrocatechase after day 3. At high level loading, pyrocatechase activity reached its peak (1.069 units) on day 4 after the shock load. Obviously, the in vitro enzyme assay
re¯ects the enzymatic response to the in vitro test conditions. However, one of the aims of the work is to develop a quick method of measuring sludge activity that is of practical signi®cance. For this purpose simulation of reactor conditions in the laboratory would be impractical. As far as we use the same standardized assay condition, the in vitro test should re¯ect changes in enzyme activities with changes in phenol concentration in the feed. Majorities of the microorganisms that degrade phenol use the ortho cleavage pathway. For instance, of the 11 phenol oxidizing Pseudomonas strains which Feist and Hegeman (1969) examined, eight strains degrade phenol through the ortho cleavage pathway. Indeed, we have measured the metapyrocatechase activity in this speci®c mixed population however we found no signi®cant ac-
Characterization of a mixed population
tivity. After repeated measurement of metapyrocatechase activity in this speci®c mixed population, we obtained activity between 0.002 to 0.009 mmol (mg protein)ÿ1(min)ÿ1 and in the subsequent experiments the activity came down to zero. In contrast, it was possible to measure pyrocatechase activity between 0.07 to 0.15 mmol (mg protein)ÿ1 (min)ÿ1. The initial metapyrocatechase activity was low, moreover it came down to zero in due time. Therefore only the activity of ortho cleavage pathway enzyme was further measured in the investigation. The meta pathway enzyme requires a special precaution to prevent its inactivation and maintain its stability during sonication (Nozaki et al., 1963). One of the measures taken is to suspend the crude extract in phosphate buer supplemented with 10% acetone. Given that the metapyrocatechase activity detected is low, even none at some point during the investigation, and given that no special precautionary measure was taken, the possibility of disturbance of the pyrocatechase activity by the meta cleavage pathway enzyme would be minimum. Therefore the use of H2O2 will have little signi®cance to avoid interference of pyrocatechase activity by meta cleavage enzyme. At low step increase, phenol was almost completely degraded by the activated sludge with its concentration below the detection limit. Phenol was temporarily detected in the aeration tank and its concentration dropped to below the detection limit after 7 days following moderate level step increase in loading but was detected throughout the whole experiment in the case of high level increase. Similar results have been reported previously (Yang and Humphrey, 1975; Rozich et al., 1983; Allsop et al., 1993). It has been interpreted as cases in which the enzyme levels induced before the increase in loading were sucient to accommodate the higher loading rate up to certain limits. The system could not cope with phenol loading of 4500 mg lÿ1 as manifested by the continuous accumulation of phenol and DOC in the aeration tank and a decline in enzyme activity after day 4 as opposed to the low level and moderate level step changes. This is supposed to be the beginning of system failure. Watanabe et al. (1996b) reported a similar phenomenon with phenol oxygenating activity. The other physical indicator of system failure was the accumulation of red to red brown color in the settlement tank after this increase in phenol concentration. Contrary to low and medium phenol concentrations, steady phenol build-up in the reactor began even in the presence of dissolved oxygen. Therefore, it is possible to say that the organisms reached their limits and the drop in enzyme activity was due to phenol inhibition. In the case of moderate and high-level changes in phenol loading, the pyrocatechase activity increased with phenol concentration in the aeration tank up to a maximum of 178 mg lÿ1 and decreases in
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enzyme activities were observed below this concentration. This was in agreement with the ®ndings of Watanabe et al. (1996a) in which the phenol oxygenating activity increased with increase in phenol concentration up to certain concentration followed by a gradual decline. The concentration of dissolved oxygen in the aeration tank decreased after the step increase corresponding to increase in enzyme activity. Initial oxygen consumption rates were dependent on the level of phenol loading with 0.4 mg lÿ1 oxygen per day after the low level increase in phenol from 750 to 1500 mg lÿ1 followed by 1.5 mg lÿ1 dÿ1 during intermediate increase in phenol from 1500 to 2500 mg lÿ1 and 2.1 mg lÿ1 dÿ1 during the high level increase in phenol from 2500 to 4500 mg lÿ1. From the carbon content of phenol (approximately 77 wt%) it is clear that the DOC accumulated after the low-level step increase was nonphenol DOC. During intermediate level increase non-phenol DOC was temporarily accumulated. At high level increases however phenol DOC was progressively accumulated in the aeration tank. Accumulation of non-phenol DOC in a continuous culture subjected to step increase in the feed phenol concentration, with a signi®cant delay before phenol leakage has been reported by Allsop et al. (1993) and con®rmed by Watanabe et al. (1996b). The origin of this DOC was assumed to be a nucleotide containing intermediate, possibly acetylCoA. CONCLUDING REMARKS
With pyrocatechase activity assay, it was possible to demonstrate the metabolic response of the activated sludge to changes in the phenol loading rates. An increase in pyrocatechase activity was always found as a result of increase in phenol loading. The degree of increase in activity was dependent on the degree of changes in loading rate. In this experiment pyrocatechase activity increased with increase phenol concentration in the aeration tank up to 160 mg lÿ1 and decrease below and above this range of concentration. At high level increase in phenol concentration, the accumulation of DOC and apparent increase in phenol concentration are considered to be signs of the process breakdown.
REFERENCES
Allsop P. J., Chisti Y., Moo-Young M. and Sullivan G. R. (1993) Dynamics of phenol degradation by Pseudomonas putida. Biotechnol. Bioeng. 41, 572±580. Boczar B. A., Begley W. M. and Larson R. J. (1992) Characterisation of enzyme activity in activated sludge using rapid analysis for speci®c hydrolases. Water Environ. Res. 64, 792±797. Daigger G. T. and Grady J. R. C. P. L. (1982) The dynamics of microbial growth on soluble substrates: a unifying theory. Water Res. 16, 365±382.
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Feist C. F. and Hegeman G. D. (1969) Phenol and benzoate metabolism by Pseudomonas putida: regulation of tangential pathways. J. Bacteriol. 100, 869±877. German standard methods for the examination of water, waste water and sludge; Bioassay (group L) (1994). Surfactant biodegradation and elimination test for simulation of municipal wastewater treatment plants. DIN 38412 part 26. Hegeman G. D. (1966) Synthesis of enzymes of the mandalate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91, 1140±1154. Hill G. and Robinson C. W. (1975) Substrate inhibition kinetics: phenol degradation by Pseudomonas putida. Biotechnol. Bioeng. 17, 1599±1615. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265±275. Murray K., Duggleby C. J., Sala-Trepat J. M. and Williams P. A. (1972) The metabolism of benzoate and methylbenzoate via the meta cleavage pathway by Pseudomonas arvilla mt-2. Eur. J. Biochem. 28, 301±310. Gibson D. T. (1975) In Assay of enzymes of aromatic metabolism, eds J. R. Norris and D. W. Ribbons, Methods in Microbiology, Vol. 6A. Academic Press, London, pp. 462±477. Nozaki N., Kagamiyama H. and Hayaishi O. (1963) Metapyrocatechase. I. Puri®cation, crystalization and some properties. Biochem. Z. 333, 582±590. Nybroe N., Jùrgensen P. E. and Henze M. (1992) Enzymatic activities in wastewater and activated sludge. Water Res. 26, 579±584.
Peters G. M., Hamaide-Deplus M., Monaux V. and Nyns E. J. (1975) Quantitative determination of the population biological activity of biological sludge used in the activated sludge wastewater treatment process. Water Res. 9, 901±911. Richards S. R., Hastwell C. and Davies M. (1984) The comparative examination of 14 activated sludge plants using enzymatic techniques. Water Pollut. Contr. 38, 300±313. Ros M., Dular M. and Fakas P. A. (1988) Measurement of respiration of activated sludge. Water Res. 22, 1405± 1411. Rozich A. F. and Gaudy A. F. (1985) Response of phenol-acclimated activated sludge process to quantitative shock loading. J. Water Pollut. Contr. Feder. 57, 795± 804. Rozich A. F., Gaudy A. F. and D'Adamo P. D. (1983) Predictive model for treatment of phenolic wastes by activated sludge. Water Res. 17, 1453±1466. Stanier R. Y. and Ornston L. N. (1973) The b-ketoadipate pathway. Adv. Microbial. Physiol. 9, 89±151. Watanabe K., Hino S., Onodera K., Kajie S. and Takahashi N. (1996a) Diversity in kinetics of bacterial phenol-oxygenating activity. J. Ferm. Bioeng. 81, 560± 563. Watanabe K., Hino S. and Takahashi N. (1996b) Response of activated sludge to an increase in phenol loading. J. Ferm. Bioeng. 82, 522±524. Yang R. D. and Humphrey A. E. (1975) Dynamics and steady state studies of phenol biodegradation in pure and mixed cultures. Biotechnol. Bioeng. 17, 1211±1235.
Wat. Res. Vol. 34, No. 4, pp. 1127±1134, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
CHARACTERIZATION OF A PHENOL DEGRADING MIXED POPULATION BY ENZYME ASSAY MULUGETA KIBRET*, WALTER SOMITSCH and KARL-HEINZ ROBRA Institute of Environmental Biotechnology, Technical University Graz, Petersgasse 12/I, A-8010 Graz, Austria (First received 1 November 1998; accepted in revised form 1 May 1999) AbstractÐThe metabolic response of a mixed population to stepwise increase in phenol concentration was followed with the assay of pyrocatechase activity. The mixed population was grown in a laboratory scale activated sludge plant for 40 days. The inlet phenol concentration (750 mg lÿ1) was then increased stepwise to 1250, 2500 and 4500 mg lÿ1, corresponding to low, moderate and high level increases in phenol loading, respectively. Pre- and post-shock assessments were performed on samples collected from the aeration tank. Pyrocatechase activity increased following each step increase in phenol and the level of increase was dependent on the magnitude of the step increase. Following low level increase in loading, phenol was not detected from the aeration tank. At moderate level loading phenol was temporarily detected in the aeration tank but disappeared after 7 days. At high level loading phenol was accumulated in the aeration tank throughout. Pyrocatechase activity was dependent on the concentration of phenol in the aeration tank. From pyrocatechase activity assay it was possible to demonstrate the metabolic response of the activated sludge to changes in phenol concentration. The accumulation of phenol and decrease in pyrocatechase activity are considered as the stage when the system no longer copes with increase in phenol loading. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐenzyme, mixed microbial population, phenol, pyrocatechase, degradation
INTRODUCTION
Phenol and other phenolic compounds are common constituents of aqueous euents from processes such as polymeric resin production, oil re®ning and cokeing plants. Phenol is both toxic and lethal to ®sh at relatively low concentrations e.g. 5±25 mg lÿ1 and imparts objectionable tastes to drinking water at far lower concentrations (Hill and Robinson, 1975). Euents containing such compounds are usually treated in activated sludge processes, where the organic constituents of the wastewater are removed oxidatively by a mixed population of microorganisms, primarily heterotrophic bacteria (Richards et al., 1984; Nybroe et al., 1992). The activated sludge is subjected to periodic ¯uctuations in the inlet substrate concentration, in¯uent ¯ow rate, aeration rate, pH and temperature. Many operational problems are associated with such ¯uctuations since these factors play an important role in the establishment and maintenance of the microbial communities in the activated sludge (Rozich et al., 1983). The methods, which have traditionally been used to investigate the activity of activated sludge, *Author to whom all correspondence should be addressed. Tel: +43-316-873/8310; fax: +43-316-518-636; e-mail: [email protected]
include mixed liquor suspended solids, mixed liquor volatile suspended solids, biochemical oxygen demand, chemical oxygen demand and dissolved organic carbon. Most of these methods although useful in practice, do not give much insight into the factors that control the proper biological process (Peters et al., 1975; Ros et al., 1988). It has been found for the activated sludge process, that the activity of certain oxido-reductive enzymes follows closely the physico-chemical indicators commonly used in monitoring treatment works (Richards et al., 1984). In a study on a pilot scale plant, Nybroe et al. (1992) showed that enzyme activities re¯ect microbial activities in wastewater. These ®ndings gave impetus to the use of enzymatic methods to characterize the activity of activated sludge. Enzyme activities have received particular attention for a number of reasons: (1) these activities play a key role in the hydrolysis and mineralization of organic wastes, (2) the characterization of enzyme activities can provide insight into the biochemical factors controlling the treatability of xenobiotic chemicals, (3) enzymes can be added exogenously to treatment processes to improve overall treatment eciencies and (4) patterns of enzyme activities can be useful for the identi®cation of microbial populations (Boczar et al., 1992).
1127
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To design the best control strategies for wastewater treatment plants, it is necessary to study the performance of the system in relation to varying conditions. In this context, it is desirable to examine the response of an activated sludge to shock loads because similar situations are encountered in the ®eld. There are few earlier investigations on the response of activated sludge to shock loading (Rozich and Gaudy, 1985; Allsop et al., 1993; Watanabe et al., 1996b). In this study the activity of activated sludge from a laboratory scale treatment plant, which was subjected to stepwise increases in the inlet phenol concentrations, was characterized by the assay of pyrocatechase activity. This enzyme catalyzes a reaction in the initial steps of aerobic phenol degradation where phenol is ®rst oxidized to catechol that is further oxidized in the meta and ortho pathways. In the ortho pathway, pyrocatechase, i.e. catechol 1,2-dioxygenase (EC. 1.13.11.1) catalyses the cleavage of the aromatic ring of catechol to form cis,cis-muconic acid by the insertion of two atoms of molecular oxygen. In the meta pathway, metapyrocatechase, i.e. catechol 2,3-dioxygenase (EC. 1.13.11.2) catalyzes conversion of catechol to a-hydroxymuconic semialdehyde (Stanier and Ornston, 1973). MATERIALS AND METHODS
Overall design The activated sludge was subjected to stepwise increase in phenol loading rate, by increasing the phenol concentration in the feed stepwise from 750 mg lÿ1 to 1500, 2500 and 4500 mg lÿ1 at a constant ¯ow rate of 960 ml dÿ1 and at a dilution rate of 0.282 dayÿ1. The phenol-loading rate was increased to 1440, 2400 and 4320 mg lÿ1 dÿ1, respectively. Performance assessments were done in the pre- and post-shock conditions for each step increase. Microorganism and cultivation A mixed population of organisms was used as a source of inoculum. It was originally obtained from soil contaminated with phenol. It has repeatedly been used for phenol biodegradation studies. For use in this experiment, 500 ml of mineral salts medium supplemented with 0.5 g lÿ1 phenol was inoculated with 40 ml of homogenized culture and incubated in rotary shaker at 278C with a speed of 180 rpm. Growth of culture was monitored spectrophotometrically by measuring optical density at 578 nm with a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland). Phenol disappearance in the culture was also measured. Actively growing culture was transferred into a reactor containing the same medium. Initially the culture was grown in batch and continuous ¯ow was started. The medium contained the following constituents (mg lÿ1): phenol variable (750±4500); MgSO47H2O, 9.4; CaSO42H2O, 4.7; Na2HPO42H2O, 752; KH2PO4, 63.92; NH4Cl, 18.8 and trace minerals solution 0.47 ml. The trace minerals solution contained the following components (mg lÿ1): Na2EDTA, 2500; ZnSO47H2O, 100; MnCl26H2O, 30; H3BO3, 300; CoCl26H2O, 200; CuCl22H2O, 10; NiCl22H2O, 20; Na2Mo42H2O, 900; Na2SeO3, 20 and FeSO47H2O, 1000. The pH of the medium was adjusted to 7.2 with NaOH. All components of the medium except phenol were autoclaved at 1218C.
Phenol was ®lter sterilized separately and added to other components after autoclaving. Apparatus A laboratory scale bioreactor was employed for the cultivation of organisms (German Standard Methods, DIN 38412, 1994). The aeration tank of the reactor had a total volume of 6 l (with active volume of 3.4 l) and the settlement tank had a volume of 2.6 l (Fig. 1). The ¯ow rate of the medium to the reactor was maintained at 1 l dÿ1 with Watson Marlow peristaltic pump Model 313 (Watson Marlow, Falmouth, U.K.). The temperature of the reactor was maintained at 308C with a circulating water bath (Lauda K4RD, MessgeraÈte-Werk Lauda, Vienna, Austria). Air and agitation were provided from a compressed air source at a ¯ow rate of 130 l hÿ1. A portion of the sludge from the settlement tank was recirculated into the aeration tank using an air pump at a ¯ow rate of 130 ml minÿ1. Initially the culture was grown in mineral salts medium supplemented with 750 mg lÿ1phenol for 40 days. After phenol was completely degraded (phenol concentration less than 5 mg lÿ1), the phenol concentration in the feed was increased to 1500, 1250 and 4500 mg lÿ1. Analytical methods The dissolved oxygen concentration in the aeration tank was directly measured with Microprocessor oximeter OXI 96 (WTW, Weilheim, Germany). The pH was measured with Orion pH meter model 240 (Orion Research Incorporation, Boston, USA). For the determination of dissolved organic carbon (DOC) and phenol, samples from the aeration tank were ®ltered under pressure through Magna Nylon membrane ®lters of 50-mm diameter, having a pore size of 0.22 mm (Miro Separations, Westboro, USA). Phenol was measured spectrophotometrically by the modi®ed 4-aminoantipyrin method of Martin indicated elsewhere (Yang and Humphrey, 1975). To 50 ml of a ®ltered sample from the aeration tank, 0.5 ml of 1% (w/v) potassium ferricyanide and 2.5 ml of 1% (w/v) 4-aminoantipyrine were added. The absorbancy of the resulting mixture was read at 505 nm with a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland). Phenol concentration was determined with a calibration curve made from known phenol standard. The dry weight was measured by ®ltering aeration tank sample through a dried, tarred, 0.22 mm nylon ®lter and drying at 1058C for a minimum of 24 h. The dissolved organic carbon (DOC) concentration in the aeration tank was measured with ASTRO Model 2001 system
Fig. 1. Schematic diagram of the activated sludge plant. A is the medium reservoir, B the thermotrap, C the pump, D the aeration tank, E the settlement tank, F the euent receiver and G the temperature control. Solid lines indicate the direction of ¯ow of medium and dashed lines indicate the direction of ¯ow of sludge.
Characterization of a mixed population 2 computer controlled laboratory total organic carbon analyzer (Texas Instruments, USA). Enzyme assays Enzyme assays were performed at room temperature using quartz cuvettes of 1 cm path length, in a UVIKON 940 spectrophotometer (Kontron Inst. AG, Zurich, Switzerland) in time drive mode so that a progress curve could be obtained for calculation of enzyme activity. To harvest cells, samples from the aeration tank were centrifuged at 5000 rpm for 20 min in a Sorvall RC-5B refrigerated centrifuge (Du Pont Instruments, Hamburg, Germany). The pellet was washed twice with 0.1 M phosphate buer (pH 7.5) and resuspended in the same buer (Hegeman, 1966). Subsequently the cell pellet was disrupted by sonication for seven min using SONOPULS HD sonicator (BANDELINE electronics, Berlin, Germany). The cell debris was removed by centrifugation of the homogenized cell suspension at 20,000 rpm for 25 min. The supernatant was immediately used for enzyme assays. Pyrocatechase, i.e. catechol 1,2-dioxygenase (E.C.1.13.11.1) assay Pyrocatechase activity was assayed spectrophotometrically based on the measurement of the rate of cis,cismuconic acid formation from catechol at 260 nm. The reaction mixture contained 200 ml of 0.1 mM catechol, 200 ml of 500 mM EDTA, 100 ml crude cell-free extract and 0.1 M of phosphate buer (pH 7.5) to the ®nal volume of 3.0 ml (Murray et al., 1972). The reaction was initiated by addition of the crude extract. The protein concentration of the crude extract was measured following the method of Lowry et al. (1951) using crystalline bovine serum albumin as a standard. Calculation of enzyme activity Enzyme activities were determined from the initial gradients of the absorption curves. Molar extinction coecient (E ) of 16,900 for cis,cis-muconic acid was used to calculate initial velocity of the enzyme reaction (Gibson,
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1975). All chemicals were analytical grade obtained from Merck, Boehringer Mannheim and Sigma. RESULTS
Change in the speci®c metabolic activities of the activated sludge i.e. pyrocatechase activity during step change in phenol concentration is shown on Figs 2±4. When the inlet phenol concentration was increased from 750 to 1500 mg lÿ1, phenol was almost completely removed by the activated sludge. Phenol concentration in the aeration tank was below the detection limit through out the entire period. Pyrocatechase activity increased shortly 1 day after the change in phenol concentration and dropped sharply the next day and stayed constant thereafter. A concomitant momentary decrease in the aeration tank dissolved oxygen (from 5.7 to 5.3 mg lÿ1) was observed 1 day after the step increase (Fig. 2). Following intermediate step increase in inlet phenol concentration, i.e. from 1500 to 2500 mg lÿ1, the aeration tank phenol concentration increased temporarily and then decreased to zero. Seven days were required to remove phenol completely from the aeration tank, with a peak on the third day after the step increase [Fig. 3(B)]. Pyrocatechase activity increased after the step change in phenol concentration and a concurrent drop in dissolved oxygen concentration in the aeration tank (from 5.2 to 3.7 mg lÿ1) after 1 day was observed due to the step increase [Fig. 3(A)]. With increase in inlet phenol concentration from 2500 to 4500 mg lÿ1, phenol was not completely removed from the aeration tank, its concentration
Fig. 2. Enzyme activity and dissolved oxygen concentration pro®les of the activated sludge following a low level increase in phenol concentration from 750 to 1500 mg lÿ1.
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Fig. 3. Enzyme activity and dissolved oxygen (A), phenol and DOC (B) concentration pro®les of the activated sludge following a moderate level increase in phenol concentration from 1500 to 2500 mg lÿ1.
rather increased with time [Fig. 4(B)]. Pyrocatechase activity increased after the step increase in phenol concentration but declined after 4 days following the step increase. The dissolved oxygen concentration in the aeration tank dropped sharply from 2.6 to 0.5 mg lÿ1 1 day after the step increase in phenol, then to zero 4 days after the step change [Fig. 4(A)]. Pyrocatechase activity was dependent on the phenol concentration in the aeration tank. As shown on Fig. 3, pyrocatechase activity increased with increase in phenol concentration in the aeration tank up to 158 mg lÿ1 and dropped below this concentration. Pyrocatechase activity also decreased when the phenol concentration in the aeration tank was above 160 mg lÿ1. (Fig. 4).
Dissolved organic carbon (DOC) was accumulated even when the phenol concentration was below the detection limit, during the low-level loading followed by an increase in DOC concomitant with the increase in phenol concentration at the moderate and high level loadings. Following moderate level increase in loading, DOC was temporarily accumulated but declined on the second day [Fig. 3(B)]. At high level increase however, the DOC concentration in the aeration tank increased steadily [Fig. 4(B)]. Overall increase in the dry weight concentration of 0.16 g lÿ1 and 1.13 g lÿ1 were observed following step increase in phenol concentration from 750 to 1500 mg lÿ1 and 1500 to 2500 mg lÿ1, respectively. However, a momentary increase in dry weight con-
Characterization of a mixed population
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Fig. 4. Enzyme activity and dissolved oxygen (A), phenol and DOC (B) concentration pro®les of the activated sludge following a high level increase in phenol concentration from 2500 to 4500 mg lÿ1.
centration of 0.38 g lÿ1 followed by a decline after day 4 was observed for high level increase in phenol concentration. The speci®c phenol utilization rates were estimated from the amount of phenol degraded per day and from the dry weight. The speci®c phenol utilization rate generally increased after the step increase in phenol concentration and decreased with time thereafter. For instance, the speci®c phenol utilization rate before increase in inlet phenol from 1500 to 2500 mg lÿ1 was 1.65 g lÿ1 dÿ1 and increased to 2.064 g lÿ1 dÿ1 soon after the step increase. However it came down to 1.75, 1.6 and 1.2 g lÿ1 dÿ1 on days 2, 3, and 7 after the step increase, respectively [Fig. 5(B)]. Similarly the speci®c phenol utilization rate increased from a preshock value of 1.25 g lÿ1 dÿ1 to 2.06 g lÿ1 dÿ1 after
step increase in phenol from 2500 to 4500 g lÿ1, then it decreased to 1.85 g lÿ1 after 7 days [Fig. 5(A)]. DISCUSSION
Step increases in phenol loading into the activated sludge plant were accompanied by a temporary increase in pyrocatechase activity. A study of the literature reveals that both pure and mixed microbial cultures experience physiological adaptation which involves adjustments in the relative amounts of the primary macromolecules, the level of enzymes and the concentration of intracellular metabolites in response to change in environmental conditions. This is due to available reaction potential (ARP) which is de®ned as the ability of a mi-
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Fig. 5. Speci®c phenol utilization rate of the activated sludge following increase in phenol concentration from 2500 to 4500 mg lÿ1 (A) and from 1500 to 2500 mg lÿ1 (B).
crobial culture to rapidly increase in growth and substrate removal rates during a transient response (Daigger and Grady, 1982). Comparison of pre-shock and the maximum pyrocatechase activity after the step increase in phenol concentration indicates dierences in the level of response to changes in phenol loading. The level of response was dependent on the magnitude of the step increase. There was only increase in pyrocatechase activity of 0.024 units 1 day after the change in the inlet phenol concentration from 750 to 1500 mg lÿ1. Intermediate step change in phenol loading caused moderate level response with increase of 0.034 units of pyrocatechase after day 3. At high level loading, pyrocatechase activity reached its peak (1.069 units) on day 4 after the shock load. Obviously, the in vitro enzyme assay
re¯ects the enzymatic response to the in vitro test conditions. However, one of the aims of the work is to develop a quick method of measuring sludge activity that is of practical signi®cance. For this purpose simulation of reactor conditions in the laboratory would be impractical. As far as we use the same standardized assay condition, the in vitro test should re¯ect changes in enzyme activities with changes in phenol concentration in the feed. Majorities of the microorganisms that degrade phenol use the ortho cleavage pathway. For instance, of the 11 phenol oxidizing Pseudomonas strains which Feist and Hegeman (1969) examined, eight strains degrade phenol through the ortho cleavage pathway. Indeed, we have measured the metapyrocatechase activity in this speci®c mixed population however we found no signi®cant ac-
Characterization of a mixed population
tivity. After repeated measurement of metapyrocatechase activity in this speci®c mixed population, we obtained activity between 0.002 to 0.009 mmol (mg protein)ÿ1(min)ÿ1 and in the subsequent experiments the activity came down to zero. In contrast, it was possible to measure pyrocatechase activity between 0.07 to 0.15 mmol (mg protein)ÿ1 (min)ÿ1. The initial metapyrocatechase activity was low, moreover it came down to zero in due time. Therefore only the activity of ortho cleavage pathway enzyme was further measured in the investigation. The meta pathway enzyme requires a special precaution to prevent its inactivation and maintain its stability during sonication (Nozaki et al., 1963). One of the measures taken is to suspend the crude extract in phosphate buer supplemented with 10% acetone. Given that the metapyrocatechase activity detected is low, even none at some point during the investigation, and given that no special precautionary measure was taken, the possibility of disturbance of the pyrocatechase activity by the meta cleavage pathway enzyme would be minimum. Therefore the use of H2O2 will have little signi®cance to avoid interference of pyrocatechase activity by meta cleavage enzyme. At low step increase, phenol was almost completely degraded by the activated sludge with its concentration below the detection limit. Phenol was temporarily detected in the aeration tank and its concentration dropped to below the detection limit after 7 days following moderate level step increase in loading but was detected throughout the whole experiment in the case of high level increase. Similar results have been reported previously (Yang and Humphrey, 1975; Rozich et al., 1983; Allsop et al., 1993). It has been interpreted as cases in which the enzyme levels induced before the increase in loading were sucient to accommodate the higher loading rate up to certain limits. The system could not cope with phenol loading of 4500 mg lÿ1 as manifested by the continuous accumulation of phenol and DOC in the aeration tank and a decline in enzyme activity after day 4 as opposed to the low level and moderate level step changes. This is supposed to be the beginning of system failure. Watanabe et al. (1996b) reported a similar phenomenon with phenol oxygenating activity. The other physical indicator of system failure was the accumulation of red to red brown color in the settlement tank after this increase in phenol concentration. Contrary to low and medium phenol concentrations, steady phenol build-up in the reactor began even in the presence of dissolved oxygen. Therefore, it is possible to say that the organisms reached their limits and the drop in enzyme activity was due to phenol inhibition. In the case of moderate and high-level changes in phenol loading, the pyrocatechase activity increased with phenol concentration in the aeration tank up to a maximum of 178 mg lÿ1 and decreases in
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enzyme activities were observed below this concentration. This was in agreement with the ®ndings of Watanabe et al. (1996a) in which the phenol oxygenating activity increased with increase in phenol concentration up to certain concentration followed by a gradual decline. The concentration of dissolved oxygen in the aeration tank decreased after the step increase corresponding to increase in enzyme activity. Initial oxygen consumption rates were dependent on the level of phenol loading with 0.4 mg lÿ1 oxygen per day after the low level increase in phenol from 750 to 1500 mg lÿ1 followed by 1.5 mg lÿ1 dÿ1 during intermediate increase in phenol from 1500 to 2500 mg lÿ1 and 2.1 mg lÿ1 dÿ1 during the high level increase in phenol from 2500 to 4500 mg lÿ1. From the carbon content of phenol (approximately 77 wt%) it is clear that the DOC accumulated after the low-level step increase was nonphenol DOC. During intermediate level increase non-phenol DOC was temporarily accumulated. At high level increases however phenol DOC was progressively accumulated in the aeration tank. Accumulation of non-phenol DOC in a continuous culture subjected to step increase in the feed phenol concentration, with a signi®cant delay before phenol leakage has been reported by Allsop et al. (1993) and con®rmed by Watanabe et al. (1996b). The origin of this DOC was assumed to be a nucleotide containing intermediate, possibly acetylCoA. CONCLUDING REMARKS
With pyrocatechase activity assay, it was possible to demonstrate the metabolic response of the activated sludge to changes in the phenol loading rates. An increase in pyrocatechase activity was always found as a result of increase in phenol loading. The degree of increase in activity was dependent on the degree of changes in loading rate. In this experiment pyrocatechase activity increased with increase phenol concentration in the aeration tank up to 160 mg lÿ1 and decrease below and above this range of concentration. At high level increase in phenol concentration, the accumulation of DOC and apparent increase in phenol concentration are considered to be signs of the process breakdown.
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