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Stability of aerobic benzoate-degrading capability of Pseudomonas aeruginosa 142
Process Biochemistry 40 (2005) 1409–1413 www.elsevier.com/locate/procbio
Stability of aerobic benzoate-degrading capability of Pseudomonas aeruginosa 142 ¨ zer C¸ınar* O Department of Environmental Engineering, Kahramanmaras¸ Su¨tc¸u¨ I˙mam University, Karacasu, Kahramanmaras¸, Turkey 46601 Received 10 October 2003; accepted 11 June 2004
Abstract Pseudomonas aeruginosa 142 was grown on benzoate as sole carbon source in chemostats under fully aerobic condition and tested for the aerobic ability to degrade benzoate at maximum oxygen uptake rate (OUR) in a batch reactor after exposure to anaerobic conditions in the absence of substrate. Aerobically grown P. aeruginosa 142 lost around 75% initial max OUR in 8 h of anaerobic exposure. Laboratory experiments with aerobic ring-cleaving enzymes showed that a small part of the 75% loss of activity was due to the aerobic ring-cleaving enzymes (i.e. C12DO) while the remainder was due to cytochrome oxidases. # 2004 Elsevier Ltd. All rights reserved. Keywords: Benzoate; Biodegradation; Aromatic biodegradative capability; Enzyme stability
1. Introduction Aromatic compounds are important components in petroleum-based fuels, agricultural chemicals, and consumer products. Most aromatic compounds, particularly those containing single benzene rings, are subject to degradation by bacteria at significant rates. Biological processes therefore play an important role in their destruction in wastewater treatment systems. A critical step in aromatic compound biodegradation is cleavage of the benzene ring, because the nature of the ring-cleavage reactions depends on the oxidation state of the environment in which biodegradation is occurring. If molecular oxygen is present, then many of the peripheral and ring-cleavage reactions are oxidative and involve molecular oxygen as a reactant [1–3]. On the other hand, if molecular oxygen is absent, the ring-cleavage reactions are reductive and require a different set of enzymes [4]. Since many bacteria can degrade aromatic compounds under both oxidizing and reducing conditions, they have the ability to synthesize catabolic enzymes for both conditions. This raises the question of how they regulate their aromatic * Tel.: +90-3442512341; fax: +90-3442512342. E-mail address: [email protected]. 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.035
compound degrading enzyme systems when the environment in which they are living cycles between the two oxidation states, such as in biological nitrogen removal (BNiR) systems. This question is becoming increasingly important as more industrial wastewater treatment systems, which frequently receive significant quantities of aromatic compounds, are required to remove nitrogen. Only limited studies have been conducted on the stability of the biodegradative capability for aromatic compounds under alternating aerobic/anoxic conditions. Ma and Love [5] observed that the activities of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase decreased to very low levels when microorganisms were shifted from aerobic to anoxic environments, but that these aerobic aromatic-degrading enzymes were quickly induced when the environment was shifted from anoxic to aerobic conditions. Viliesid and Lilly [6] reported that both the synthesis and activity of catechol 1,2-dioxygenase are regulated by the dissolved oxygen (DO) concentration, decreasing as the DO concentration is decreased. Ma and Love [5] observed similar, but opposite, results for methyl viologen oxidation potential, which represents the activity of benzoyl-CoA reductase (a key enzyme in the anoxic biodegradation of aromatic compounds), suggesting that high DO concentrations inhibit this
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anoxic aromatic-degrading enzyme in vivo. Boll and Fuchs [7] also found that benzoyl-CoA reductase is extremely sensitive to oxygen because in cell-free extracts it is irreversibly inactivated by oxygen with a half-life of about 20 s under saturated DO conditions. Furthermore, this enzyme is located in the cytoplasm. Since the oxygen concentration in the cytoplasm of bacterial cells is proportional to the DO concentration in the medium in which the cells are growing, one would expect benzoyl-CoA reductase also to be rapidly destroyed in whole cells in aerobic environments. In addition, immunoblot analysis of cell-free extracts from Thauera aromatica grown aerobically on benzoic acid showed trace amounts of inactive benzoyl-CoA reductase, causing Heider et al. [8] to speculate that microorganisms might maintain a backup enzyme level for rapid response when they are exposed to shifts in the redox environment. Because of the lack of information on the stability of the biodegradative capability of microorganisms responsible for aromatic compounds and because of the importance of enzyme stability on the fate of aromatic compounds in BNiR systems, extensive research on this subject has been conducted. This study began with simple screening experiments in which pure cultures were grown on benzoate in a totally aerobic chemostat. The culture was harvested from it, held for various times under anoxic conditions in the absence of substrate and the oxygen uptake rate (OUR) and the activity of aerobic aromatic degrading enzymes measured. Benzoate was chosen as the substrate for these studies because it can be degraded aerobically through any of the three central aerobic intermediates [9], depending on the organisms involved and is degraded anoxically through benzoyl-CoA, the major anoxic central intermediate [4]. Consequently, the response of benzoate degrading enzymes is likely to be representative of the response of many aromatic degrading enzyme systems.
2. Materials and methods 2.1. Reactor setup and operation Experiments were performed with microorganisms from the pure culture grown in an aerobic chemostat. The pure culture was Pseudomonas aeruginosa 142, kindly provided by Dr. Robert P. Hausinger of Michigan State University. This bacterium was selected because it was isolated from contaminated soil and thus is environmentally relevant, utilizes a wide variety of aromatic compounds, has a low rate of spontaneous mutation and degrades benzoate by the chromosomally encoded b-ketoadipate pathway [10]. The chemostat was operated with a hydraulic retention time of 48 h on mineral salts media containing benzoate as the carbon and energy source. All inorganic nutrients were provided in excess, making benzoate the growth limiting substance [11]. The feed benzoate concentration was 2000 mg/L as chemical oxygen demand (COD). Oxygen
was provided in excess to the aerobic chemostat, with a residual effluent concentration approaching saturation. The feed to both chemostats contained 0.026 M phosphate buffer, which was adequate to maintain the pH at 7.3 in the aerobic chemostat. 2.2. Experimental design The design of experiments to test the stability of aerobic benzoate-degrading capability of P. aeruginosa 142 is illustrated in Fig. 1. After the aerobic chemostat had been operated for an extended period of time, aliquots of biomass from the chemostat were removed and placed into anoxic continuously mixed batch reactors with no exogenous substrate. Biomass samples from these batch reactors were then removed after 0, 1, 2, 4, and 8 h exposure to measure the ability of cells to take up oxygen (OUR) and the activity of aerobic catabolic aromatic-degrading enzymes within the bacteria (catechol 1,2-dioxygenase (C12DO), catechol 2,3-dioxygenase (C23DO), protocatechuate 3,4-dioxygenase (P34DO), protocatechuate 4,5-dioxygenase (P45DO), and gentisate 1,2-dioxygenase (G12DO)). 2.3. Analytical methods Routine analyses used in operation of the aerobic chemostat were run according to standard methods [12]. Benzoate was measured using high-pressure liquid chromatography (HPLC) [13]. Total organic carbon (TOC) was measured with a Shimadzu TOC-5000 analyzer, equipped with an ASI-5000 auto sampler. The lower detection limit was 0.02 mg/L as C (1.67 mM). 2.4. Aerobic enzyme assays The activities of all the aerobic catabolic enzymes were quantified by measuring changes in absorbance with time in a Beckman DU-640 (Fullerton, CA) UV recording spectrophotometer. The products of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were monitored when catechol was provided as the substrate [14]. They were cis,cismuconic acid (257 nm) and 2-hydroxy-cis,cis-muconic
Fig. 1. Redox conditions imposed during the various phases of the experimental design.
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semialdehyde (375 nm), respectively. The activity of gentisate 1,2-dioxygenase was quantified by measuring the formation of its product, maleylpyruvate, at 337 nm [15]. The activity of protocatechuate 3,4-dioxygenase was quantified by measuring the consumption of protocatechuate at 290 nm [16], whereas the activity of protocatechuate 4,5-dioxygenase was quantified by measuring the generation of 4-carboxy-2-hydroxy muconic semialdehyde at 410 nm [17]. The total protein concentrations in the cells were determined by the bicinchoninic acid–copper reaction [18] and all enzyme activities were normalized to the total protein concentrations to obtain specific enzyme activities. 2.5. Oxygen uptake rate assay Respirometry was used to assess the ability of P. aeruginosa 142 to respire benzoate when added individually to batch respirometers [19]. Cells were removed from a chemostat and placed in a 40 mL, water-jacketed (25 8C), and stirred respirometer with zero head-space. The respirometer contained a port for a dissolved oxygen (DO) microprobe (Instech Laboratories, Plymouth Meeting, PA) and a port for injection of substrate. The DO microprobe was attached to an YSI model 5300 Biological Oxygen Monitor (Yellow Springs, OH), which was interfaced with a microcomputer for automatic data acquisition. The endogenous oxygen uptake was measured for 2–3 min, after which a test substrate was injected and the resultant oxygen uptake was measured for 15–20 min. Linear least squares analysis was used to establish the endogenous oxygen uptake rate (OUR) before the substrate injection. The remaining data were then adjusted by subtracting an amount equivalent to the oxygen that would have been consumed had endogenous metabolism continued, giving a net oxygen consumption curve due to exogenous substrate. The maximum OUR associated with degradation of the injected substrate was then determined by linear least squares analysis of the linear portion of the curve and the results were normalized with respect to the bacterial concentration as measured by absorbance at 600 nm, giving
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a rate with units of mg O2/(L-min-abs). Three respirometers were run simultaneously, providing three replicate measurements of the maximum OUR on a given substrate.
3. Results and discussions The pure culture, P. aeruginosa 142, was grown in an aerobic chemostat with solid retention time (SRT) of 2 days. All inorganic nutrients and electron acceptors were provided in excess, making benzoate the growth limiting substance. The lack of any detectable benzoate in aerobic chemostat indicates that the bacteria were well adapted (the lower detection limit for benzoate was 0.1 mg COD/L). The chemostat performance was also monitored by measuring TOC. The baseline TOC concentration was approximately 15 mg C/L in the chemostat. Overall, the performance of the chemostat was excellent, demonstrating that bacterial cultures can effectively remove high concentrations of benzoate. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their maximum oxygen uptake rate is presented in Fig. 2. As in Fig. 2, assays performed on three different days are presented to demonstrate the reproducibility of the response. The results show the decline in the cells’ ability to uptake oxygen (as both an electron acceptor and a reactant for oxidases) continued throughout the experimental period, until more than 70% of it had been lost within 8 h. There are some potential reasons for the observed results. The loss was not due to starvation per se because there was no loss of activity when the cells were starved under aerobic conditions [11]. This suggests that the loss was associated with exposure to anaerobic conditions in some way. One possibility is that the loss of ability was associated with loss of aerobic ring-cleavage enzymes (e.g. C12DO), while the other is that the terminal cytochrome oxidase was lost during prolonged exposure to anaerobic conditions. The first possibility was investigated by measuring the change in the activity of aerobic ring-
Fig. 2. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their maximum oxygen uptake rate. Percent maximum oxygen uptake rate is presented as a percentage of the rate prior to anaerobic starvation.
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Fig. 3. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their activity of C12DO. The activity of C12DO is presented as a percentage of the rate prior to anaerobic starvation.
cleavage enzymes during anaerobic starvation. Since the different types of microorganisms may express different types of aerobic ring-cleavage enzymes to break the stable benzene ring, aerobic ring-cleavage enzymes involved in the metabolism of benzoate in aerobic chemostat was screened and C12DO was the only aerobic ring-cleavage enzyme expressed as Rudolph and Grady [20] observed the activity of C12DO in P. aeruginosa 142 grown on benzoate aerobically. Thus, to be certain that the enzyme assays were working properly and with sufficient sensitivity, tests were performed with pure cultures known to express each of the key enzymes. These tests confirmed that significant levels of the other aerobic ring-cleavage enzymes were indeed absent from the culture. The effects of anaerobic starvation on the level of C12DO present in the culture are presented in Fig. 3. The results from Figs. 2 and 3 show that the rate of decline on the activity of C12DO was much lower than that on the maximum OUR, suggesting that the second possibility; that is, the terminal cytochrome oxidase could be lost during prolonged exposure to anaerobic conditions, was involved in this study. Thus, it is clear that anaerobic starvation caused both a loss in the ability of the cells to catabolize benzoate aerobically via the mild loss of C12DO activity (see Fig. 3) and a significant decrease in the electron transport capacity of the cells. The effect of oxygen availability on C12DO activity was also investigated by Viliesid and Lilly [6] and they found that the activity of catechol 1,2-dioxygenase present in Pseudomonas putida were dependent on the dissolved oxygen (DO) concentration. The activity of catechol 1,2-dioxygenase is influenced by the DO concentration and the halfsaturation coefficient is fairly large, with values of 1.92 and 3.10 mg/L being reported in Pseudomonas putida [21] and Pseudomonas arvilla [22], respectively [23]. Such values suggest that the activity of catechol 1,2-dioxygenase is likely to be low in systems with limited DO concentrations. The response of the aerobic degradative capacity for benzoate via the measurement of max OUR to anaerobic starvation conditions (see Fig. 2) was summarized in three
important ways. First, much more aerobic degradative capacity was lost, with around 75% being lost in 8 h. Second, loss continued over the entire anoxic period. Both of these observations suggest that the hydraulic retention time (HRT) of the anoxic zone in a biological nutrient removal wastewater treatment plant (WWTP) will influence the aerobic degradative capacity remaining in the biomass entering the aerobic zone. Studies are also under way addressing this question. Third, it was clear that some of the loss of aerobic degradative capacity was associated with the loss of catabolic enzymes; that is, C12DO in this study, and some of that was associated with loss of terminal electron transfer capacity. Ma and Love [5] found that both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase (another ring-cleavage enzyme involved in aerobic benzoate biodegradation) were rapidly lost upon initiation of the anoxic phase in a sequencing batch reactor degrading BTEX compounds under alternating anoxic and aerobic conditions, suggesting that some of the loss of activity observed herein were probably due to loss of catabolic enzymes. Regardless of the mechanism, however, it is clear that the ability of the biomass to carry out aerobic biodegradation of benzoate was seriously compromised by exposure to anoxic conditions. Furthermore, the fact that the aerobic degradative capacity was unaffected by aerobic starvation suggests that the loss under anoxic conditions was due to the lack of oxygen and not to starvation. The fact that aerobic degradative capacity for aromatic compounds is lost under anoxic conditions suggests that new aerobic catabolic enzyme synthesis will be required upon return of biomass in a biological nitrogen removal system to the aerobic zone. Furthermore, Viliesid and Lilly [6] found that both the rate of synthesis and the quantity of catechol 1,2-dioxygenase present in Pseudomonas putida are dependent on the DO concentration. This suggests that the DO concentration maintained in the aerobic zone of a biological nitrogen removal system will influence the success of that system in degrading aromatic compounds.
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Acknowledgements This research was supported by an unrestricted grant from the DuPont Educational Aid Program. I would like to thank Emeritus Prof. Dr. C.P. Leslie Grady, Jr. of Clemson University, Clemson, SC, USA, Assoc. Prof. Dr. Metin Digrak of Kahramanmaras, Turkey, and Dr. John M. Rudolph for valuable contributions.
References [1] Altenschmidt U, Oswald B, Steiner E, Herrmann H, Fuchs G. New aerobic benzoate oxidation pathway via benzoyl-coenzyme and 3 hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp. J Bacteriol 1993;175:4851–8. [2] Powlowski J, Shingler V. Genetics and biochemistry of phenol biodegradation by Pseudomonas sp. CF600. Biodegradation 1994;5:219– 36. [3] Harwood CS, Parales RE. The b-ketoadipate pathway and the biology of self-identity. Ann Rev Microbiol 1996;50:553–90. [4] Harwood CS, Burchhardt G, Herrmann H, Fuchs G. Anaerobic metabolism of aromatic compounds via benzoyl-CoA pathway. FEMS Microbiol Rev 1999;22:439–58. [5] Ma G, Love N. BTX biodegradation in activated sludge under multiple redox conditions. J Environ Eng 2001;127:509–16. [6] Viliesid F, Lilly MD. Influence of dissolved oxygen tension on the synthesis of catechol 1,2-dioxygenase by Pseudomonas putida. Enzyme Microbial Technol 1992;14:561–5. [7] Boll M, Fuchs G. Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172. Eur J Biochem 1995;244: 860–71. [8] Heider H, Boll M, Breese K, Breinig S, Ebenau-Jehle C, Feil U, et al. Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica. Arch Microbiol 1998;170:120–31. [9] Gottschalk G. Bacterial Metabolism. 2nd ed. New York, USA: Springer; 1986.
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[10] Romanov V, Hausinger RP. Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism 2,4-dichloro- and 2-chlorobenzoate. J Bacteriol 1994;176:3368–74. ¨ , Grady Jr CPL. Aerobic and anoxic biodegradation of [11] C ¸ ınar O benzoate: stability of biodegradative capability under endogenous conditions. Water Res 2001;35:1015–21. [12] Clesceri LS, Greenberg AE, Eaton AD, editors, Standard Methods for the Examination of Water and Wastewater. 20th ed. Washington, DC, USA: APHA, AWWA, and WEF; 1998. [13] Coschigano PW, Haggblom MM, Young LY. Metabolism of both 4chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain. Appl Environ Microbiol 1994;60:989–95. [14] Dorn E, Knackmuss HJ. Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown Pseudomonad. Biochem J 1978;174:73–84. [15] Crawford RL, Hutton SW, Chapman PJ. Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J Bacteriol 1975;121:794–9. [16] Fujisawa H, Hayaishi O. Protocatechuate 3,4-dioxygenase. I. Crystallization and characterization. J Biol Chem 1968;243:2673–81. [17] Ono K, Nozaki M, Hayaishi O. Purification and some properties of protocatechuate 4,5-dioxygenase. Biochim Biophys Acta 1970;220: 224–38. [18] Daniels L, Hanson RS, Philps JA. Methods for General and Molecular Bacteriolgy. Washington, DC, USA: American Society for Microbiology; 1994. [19] Ellis TG, Barbeau DS, Smets BF, Grady Jr CPL. Respirometric technique for determination of extant kinetic parameters describing biodegradation. Water Environ Res 1996;68:917–26. [20] Rudolph JM, Grady Jr CPL. Catabolic enzyme levels in bacteria grown on binary and ternary substrate mixtures in continuous culture. Biotechnol Bioeng 2002;79:188–99. [21] Nakai C, Nakazawa T, Nozaki M. Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1. Arch Biochem Biophys 1988;267:701–13. [22] Nakai C, Horiike K, Kuramitsu S, Kagamiyama H, Nozaki M. 3 Isoenzymes of catechol 1,2-dioxygenase (pyrocatechase), a–a, a–b and b–b, from Pseudomonas arvilla C-1. J Biol Chem 1990;265:660– 5. [23] Schomburg D, Stephan D. Enzyme Handbook. Berlin, Germany: Springer-Verlag; 1994.
Stability of aerobic benzoate-degrading capability of Pseudomonas aeruginosa 142 ¨ zer C¸ınar* O Department of Environmental Engineering, Kahramanmaras¸ Su¨tc¸u¨ I˙mam University, Karacasu, Kahramanmaras¸, Turkey 46601 Received 10 October 2003; accepted 11 June 2004
Abstract Pseudomonas aeruginosa 142 was grown on benzoate as sole carbon source in chemostats under fully aerobic condition and tested for the aerobic ability to degrade benzoate at maximum oxygen uptake rate (OUR) in a batch reactor after exposure to anaerobic conditions in the absence of substrate. Aerobically grown P. aeruginosa 142 lost around 75% initial max OUR in 8 h of anaerobic exposure. Laboratory experiments with aerobic ring-cleaving enzymes showed that a small part of the 75% loss of activity was due to the aerobic ring-cleaving enzymes (i.e. C12DO) while the remainder was due to cytochrome oxidases. # 2004 Elsevier Ltd. All rights reserved. Keywords: Benzoate; Biodegradation; Aromatic biodegradative capability; Enzyme stability
1. Introduction Aromatic compounds are important components in petroleum-based fuels, agricultural chemicals, and consumer products. Most aromatic compounds, particularly those containing single benzene rings, are subject to degradation by bacteria at significant rates. Biological processes therefore play an important role in their destruction in wastewater treatment systems. A critical step in aromatic compound biodegradation is cleavage of the benzene ring, because the nature of the ring-cleavage reactions depends on the oxidation state of the environment in which biodegradation is occurring. If molecular oxygen is present, then many of the peripheral and ring-cleavage reactions are oxidative and involve molecular oxygen as a reactant [1–3]. On the other hand, if molecular oxygen is absent, the ring-cleavage reactions are reductive and require a different set of enzymes [4]. Since many bacteria can degrade aromatic compounds under both oxidizing and reducing conditions, they have the ability to synthesize catabolic enzymes for both conditions. This raises the question of how they regulate their aromatic * Tel.: +90-3442512341; fax: +90-3442512342. E-mail address: [email protected]. 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.035
compound degrading enzyme systems when the environment in which they are living cycles between the two oxidation states, such as in biological nitrogen removal (BNiR) systems. This question is becoming increasingly important as more industrial wastewater treatment systems, which frequently receive significant quantities of aromatic compounds, are required to remove nitrogen. Only limited studies have been conducted on the stability of the biodegradative capability for aromatic compounds under alternating aerobic/anoxic conditions. Ma and Love [5] observed that the activities of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase decreased to very low levels when microorganisms were shifted from aerobic to anoxic environments, but that these aerobic aromatic-degrading enzymes were quickly induced when the environment was shifted from anoxic to aerobic conditions. Viliesid and Lilly [6] reported that both the synthesis and activity of catechol 1,2-dioxygenase are regulated by the dissolved oxygen (DO) concentration, decreasing as the DO concentration is decreased. Ma and Love [5] observed similar, but opposite, results for methyl viologen oxidation potential, which represents the activity of benzoyl-CoA reductase (a key enzyme in the anoxic biodegradation of aromatic compounds), suggesting that high DO concentrations inhibit this
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anoxic aromatic-degrading enzyme in vivo. Boll and Fuchs [7] also found that benzoyl-CoA reductase is extremely sensitive to oxygen because in cell-free extracts it is irreversibly inactivated by oxygen with a half-life of about 20 s under saturated DO conditions. Furthermore, this enzyme is located in the cytoplasm. Since the oxygen concentration in the cytoplasm of bacterial cells is proportional to the DO concentration in the medium in which the cells are growing, one would expect benzoyl-CoA reductase also to be rapidly destroyed in whole cells in aerobic environments. In addition, immunoblot analysis of cell-free extracts from Thauera aromatica grown aerobically on benzoic acid showed trace amounts of inactive benzoyl-CoA reductase, causing Heider et al. [8] to speculate that microorganisms might maintain a backup enzyme level for rapid response when they are exposed to shifts in the redox environment. Because of the lack of information on the stability of the biodegradative capability of microorganisms responsible for aromatic compounds and because of the importance of enzyme stability on the fate of aromatic compounds in BNiR systems, extensive research on this subject has been conducted. This study began with simple screening experiments in which pure cultures were grown on benzoate in a totally aerobic chemostat. The culture was harvested from it, held for various times under anoxic conditions in the absence of substrate and the oxygen uptake rate (OUR) and the activity of aerobic aromatic degrading enzymes measured. Benzoate was chosen as the substrate for these studies because it can be degraded aerobically through any of the three central aerobic intermediates [9], depending on the organisms involved and is degraded anoxically through benzoyl-CoA, the major anoxic central intermediate [4]. Consequently, the response of benzoate degrading enzymes is likely to be representative of the response of many aromatic degrading enzyme systems.
2. Materials and methods 2.1. Reactor setup and operation Experiments were performed with microorganisms from the pure culture grown in an aerobic chemostat. The pure culture was Pseudomonas aeruginosa 142, kindly provided by Dr. Robert P. Hausinger of Michigan State University. This bacterium was selected because it was isolated from contaminated soil and thus is environmentally relevant, utilizes a wide variety of aromatic compounds, has a low rate of spontaneous mutation and degrades benzoate by the chromosomally encoded b-ketoadipate pathway [10]. The chemostat was operated with a hydraulic retention time of 48 h on mineral salts media containing benzoate as the carbon and energy source. All inorganic nutrients were provided in excess, making benzoate the growth limiting substance [11]. The feed benzoate concentration was 2000 mg/L as chemical oxygen demand (COD). Oxygen
was provided in excess to the aerobic chemostat, with a residual effluent concentration approaching saturation. The feed to both chemostats contained 0.026 M phosphate buffer, which was adequate to maintain the pH at 7.3 in the aerobic chemostat. 2.2. Experimental design The design of experiments to test the stability of aerobic benzoate-degrading capability of P. aeruginosa 142 is illustrated in Fig. 1. After the aerobic chemostat had been operated for an extended period of time, aliquots of biomass from the chemostat were removed and placed into anoxic continuously mixed batch reactors with no exogenous substrate. Biomass samples from these batch reactors were then removed after 0, 1, 2, 4, and 8 h exposure to measure the ability of cells to take up oxygen (OUR) and the activity of aerobic catabolic aromatic-degrading enzymes within the bacteria (catechol 1,2-dioxygenase (C12DO), catechol 2,3-dioxygenase (C23DO), protocatechuate 3,4-dioxygenase (P34DO), protocatechuate 4,5-dioxygenase (P45DO), and gentisate 1,2-dioxygenase (G12DO)). 2.3. Analytical methods Routine analyses used in operation of the aerobic chemostat were run according to standard methods [12]. Benzoate was measured using high-pressure liquid chromatography (HPLC) [13]. Total organic carbon (TOC) was measured with a Shimadzu TOC-5000 analyzer, equipped with an ASI-5000 auto sampler. The lower detection limit was 0.02 mg/L as C (1.67 mM). 2.4. Aerobic enzyme assays The activities of all the aerobic catabolic enzymes were quantified by measuring changes in absorbance with time in a Beckman DU-640 (Fullerton, CA) UV recording spectrophotometer. The products of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were monitored when catechol was provided as the substrate [14]. They were cis,cismuconic acid (257 nm) and 2-hydroxy-cis,cis-muconic
Fig. 1. Redox conditions imposed during the various phases of the experimental design.
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semialdehyde (375 nm), respectively. The activity of gentisate 1,2-dioxygenase was quantified by measuring the formation of its product, maleylpyruvate, at 337 nm [15]. The activity of protocatechuate 3,4-dioxygenase was quantified by measuring the consumption of protocatechuate at 290 nm [16], whereas the activity of protocatechuate 4,5-dioxygenase was quantified by measuring the generation of 4-carboxy-2-hydroxy muconic semialdehyde at 410 nm [17]. The total protein concentrations in the cells were determined by the bicinchoninic acid–copper reaction [18] and all enzyme activities were normalized to the total protein concentrations to obtain specific enzyme activities. 2.5. Oxygen uptake rate assay Respirometry was used to assess the ability of P. aeruginosa 142 to respire benzoate when added individually to batch respirometers [19]. Cells were removed from a chemostat and placed in a 40 mL, water-jacketed (25 8C), and stirred respirometer with zero head-space. The respirometer contained a port for a dissolved oxygen (DO) microprobe (Instech Laboratories, Plymouth Meeting, PA) and a port for injection of substrate. The DO microprobe was attached to an YSI model 5300 Biological Oxygen Monitor (Yellow Springs, OH), which was interfaced with a microcomputer for automatic data acquisition. The endogenous oxygen uptake was measured for 2–3 min, after which a test substrate was injected and the resultant oxygen uptake was measured for 15–20 min. Linear least squares analysis was used to establish the endogenous oxygen uptake rate (OUR) before the substrate injection. The remaining data were then adjusted by subtracting an amount equivalent to the oxygen that would have been consumed had endogenous metabolism continued, giving a net oxygen consumption curve due to exogenous substrate. The maximum OUR associated with degradation of the injected substrate was then determined by linear least squares analysis of the linear portion of the curve and the results were normalized with respect to the bacterial concentration as measured by absorbance at 600 nm, giving
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a rate with units of mg O2/(L-min-abs). Three respirometers were run simultaneously, providing three replicate measurements of the maximum OUR on a given substrate.
3. Results and discussions The pure culture, P. aeruginosa 142, was grown in an aerobic chemostat with solid retention time (SRT) of 2 days. All inorganic nutrients and electron acceptors were provided in excess, making benzoate the growth limiting substance. The lack of any detectable benzoate in aerobic chemostat indicates that the bacteria were well adapted (the lower detection limit for benzoate was 0.1 mg COD/L). The chemostat performance was also monitored by measuring TOC. The baseline TOC concentration was approximately 15 mg C/L in the chemostat. Overall, the performance of the chemostat was excellent, demonstrating that bacterial cultures can effectively remove high concentrations of benzoate. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their maximum oxygen uptake rate is presented in Fig. 2. As in Fig. 2, assays performed on three different days are presented to demonstrate the reproducibility of the response. The results show the decline in the cells’ ability to uptake oxygen (as both an electron acceptor and a reactant for oxidases) continued throughout the experimental period, until more than 70% of it had been lost within 8 h. There are some potential reasons for the observed results. The loss was not due to starvation per se because there was no loss of activity when the cells were starved under aerobic conditions [11]. This suggests that the loss was associated with exposure to anaerobic conditions in some way. One possibility is that the loss of ability was associated with loss of aerobic ring-cleavage enzymes (e.g. C12DO), while the other is that the terminal cytochrome oxidase was lost during prolonged exposure to anaerobic conditions. The first possibility was investigated by measuring the change in the activity of aerobic ring-
Fig. 2. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their maximum oxygen uptake rate. Percent maximum oxygen uptake rate is presented as a percentage of the rate prior to anaerobic starvation.
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Fig. 3. Effect of exposure of aerobically grown P. aeruginosa 142 to anaerobic starvation conditions on their activity of C12DO. The activity of C12DO is presented as a percentage of the rate prior to anaerobic starvation.
cleavage enzymes during anaerobic starvation. Since the different types of microorganisms may express different types of aerobic ring-cleavage enzymes to break the stable benzene ring, aerobic ring-cleavage enzymes involved in the metabolism of benzoate in aerobic chemostat was screened and C12DO was the only aerobic ring-cleavage enzyme expressed as Rudolph and Grady [20] observed the activity of C12DO in P. aeruginosa 142 grown on benzoate aerobically. Thus, to be certain that the enzyme assays were working properly and with sufficient sensitivity, tests were performed with pure cultures known to express each of the key enzymes. These tests confirmed that significant levels of the other aerobic ring-cleavage enzymes were indeed absent from the culture. The effects of anaerobic starvation on the level of C12DO present in the culture are presented in Fig. 3. The results from Figs. 2 and 3 show that the rate of decline on the activity of C12DO was much lower than that on the maximum OUR, suggesting that the second possibility; that is, the terminal cytochrome oxidase could be lost during prolonged exposure to anaerobic conditions, was involved in this study. Thus, it is clear that anaerobic starvation caused both a loss in the ability of the cells to catabolize benzoate aerobically via the mild loss of C12DO activity (see Fig. 3) and a significant decrease in the electron transport capacity of the cells. The effect of oxygen availability on C12DO activity was also investigated by Viliesid and Lilly [6] and they found that the activity of catechol 1,2-dioxygenase present in Pseudomonas putida were dependent on the dissolved oxygen (DO) concentration. The activity of catechol 1,2-dioxygenase is influenced by the DO concentration and the halfsaturation coefficient is fairly large, with values of 1.92 and 3.10 mg/L being reported in Pseudomonas putida [21] and Pseudomonas arvilla [22], respectively [23]. Such values suggest that the activity of catechol 1,2-dioxygenase is likely to be low in systems with limited DO concentrations. The response of the aerobic degradative capacity for benzoate via the measurement of max OUR to anaerobic starvation conditions (see Fig. 2) was summarized in three
important ways. First, much more aerobic degradative capacity was lost, with around 75% being lost in 8 h. Second, loss continued over the entire anoxic period. Both of these observations suggest that the hydraulic retention time (HRT) of the anoxic zone in a biological nutrient removal wastewater treatment plant (WWTP) will influence the aerobic degradative capacity remaining in the biomass entering the aerobic zone. Studies are also under way addressing this question. Third, it was clear that some of the loss of aerobic degradative capacity was associated with the loss of catabolic enzymes; that is, C12DO in this study, and some of that was associated with loss of terminal electron transfer capacity. Ma and Love [5] found that both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase (another ring-cleavage enzyme involved in aerobic benzoate biodegradation) were rapidly lost upon initiation of the anoxic phase in a sequencing batch reactor degrading BTEX compounds under alternating anoxic and aerobic conditions, suggesting that some of the loss of activity observed herein were probably due to loss of catabolic enzymes. Regardless of the mechanism, however, it is clear that the ability of the biomass to carry out aerobic biodegradation of benzoate was seriously compromised by exposure to anoxic conditions. Furthermore, the fact that the aerobic degradative capacity was unaffected by aerobic starvation suggests that the loss under anoxic conditions was due to the lack of oxygen and not to starvation. The fact that aerobic degradative capacity for aromatic compounds is lost under anoxic conditions suggests that new aerobic catabolic enzyme synthesis will be required upon return of biomass in a biological nitrogen removal system to the aerobic zone. Furthermore, Viliesid and Lilly [6] found that both the rate of synthesis and the quantity of catechol 1,2-dioxygenase present in Pseudomonas putida are dependent on the DO concentration. This suggests that the DO concentration maintained in the aerobic zone of a biological nitrogen removal system will influence the success of that system in degrading aromatic compounds.
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Acknowledgements This research was supported by an unrestricted grant from the DuPont Educational Aid Program. I would like to thank Emeritus Prof. Dr. C.P. Leslie Grady, Jr. of Clemson University, Clemson, SC, USA, Assoc. Prof. Dr. Metin Digrak of Kahramanmaras, Turkey, and Dr. John M. Rudolph for valuable contributions.
References [1] Altenschmidt U, Oswald B, Steiner E, Herrmann H, Fuchs G. New aerobic benzoate oxidation pathway via benzoyl-coenzyme and 3 hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp. J Bacteriol 1993;175:4851–8. [2] Powlowski J, Shingler V. Genetics and biochemistry of phenol biodegradation by Pseudomonas sp. CF600. Biodegradation 1994;5:219– 36. [3] Harwood CS, Parales RE. The b-ketoadipate pathway and the biology of self-identity. Ann Rev Microbiol 1996;50:553–90. [4] Harwood CS, Burchhardt G, Herrmann H, Fuchs G. Anaerobic metabolism of aromatic compounds via benzoyl-CoA pathway. FEMS Microbiol Rev 1999;22:439–58. [5] Ma G, Love N. BTX biodegradation in activated sludge under multiple redox conditions. J Environ Eng 2001;127:509–16. [6] Viliesid F, Lilly MD. Influence of dissolved oxygen tension on the synthesis of catechol 1,2-dioxygenase by Pseudomonas putida. Enzyme Microbial Technol 1992;14:561–5. [7] Boll M, Fuchs G. Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172. Eur J Biochem 1995;244: 860–71. [8] Heider H, Boll M, Breese K, Breinig S, Ebenau-Jehle C, Feil U, et al. Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica. Arch Microbiol 1998;170:120–31. [9] Gottschalk G. Bacterial Metabolism. 2nd ed. New York, USA: Springer; 1986.
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[10] Romanov V, Hausinger RP. Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism 2,4-dichloro- and 2-chlorobenzoate. J Bacteriol 1994;176:3368–74. ¨ , Grady Jr CPL. Aerobic and anoxic biodegradation of [11] C ¸ ınar O benzoate: stability of biodegradative capability under endogenous conditions. Water Res 2001;35:1015–21. [12] Clesceri LS, Greenberg AE, Eaton AD, editors, Standard Methods for the Examination of Water and Wastewater. 20th ed. Washington, DC, USA: APHA, AWWA, and WEF; 1998. [13] Coschigano PW, Haggblom MM, Young LY. Metabolism of both 4chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain. Appl Environ Microbiol 1994;60:989–95. [14] Dorn E, Knackmuss HJ. Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown Pseudomonad. Biochem J 1978;174:73–84. [15] Crawford RL, Hutton SW, Chapman PJ. Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J Bacteriol 1975;121:794–9. [16] Fujisawa H, Hayaishi O. Protocatechuate 3,4-dioxygenase. I. Crystallization and characterization. J Biol Chem 1968;243:2673–81. [17] Ono K, Nozaki M, Hayaishi O. Purification and some properties of protocatechuate 4,5-dioxygenase. Biochim Biophys Acta 1970;220: 224–38. [18] Daniels L, Hanson RS, Philps JA. Methods for General and Molecular Bacteriolgy. Washington, DC, USA: American Society for Microbiology; 1994. [19] Ellis TG, Barbeau DS, Smets BF, Grady Jr CPL. Respirometric technique for determination of extant kinetic parameters describing biodegradation. Water Environ Res 1996;68:917–26. [20] Rudolph JM, Grady Jr CPL. Catabolic enzyme levels in bacteria grown on binary and ternary substrate mixtures in continuous culture. Biotechnol Bioeng 2002;79:188–99. [21] Nakai C, Nakazawa T, Nozaki M. Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1. Arch Biochem Biophys 1988;267:701–13. [22] Nakai C, Horiike K, Kuramitsu S, Kagamiyama H, Nozaki M. 3 Isoenzymes of catechol 1,2-dioxygenase (pyrocatechase), a–a, a–b and b–b, from Pseudomonas arvilla C-1. J Biol Chem 1990;265:660– 5. [23] Schomburg D, Stephan D. Enzyme Handbook. Berlin, Germany: Springer-Verlag; 1994.