High affinity p-nitrophenol oxidation by Bacillus ... - Wiley Online Library

FEMS Microbiology Letters 166 (1998) 115^120

High a¤nity p-nitrophenol oxidation by Bacillus sphaericus JS905 Venkateswarlu Kadiyala a; *, Barth F. Smets b , Kartik Chandran b , Jim C. Spain b

a

a AFR/MLQR, Tyndall Air Force Base, Tyndall, FL 32403-5323, USA Department of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06269-2037, USA

Received 9 April 1998; revised 8 July 1998; accepted 9 July 1998

Abstract Bacillus sphaericus JS905, isolated from an agricultural soil by selective enrichment, transforms p-nitrophenol (PNP) releasing only nitrite in stoichiometric amounts. The kinetics of PNP oxidation by this strain were successfully measured using an extant respirometric assay. Washed PNP-grown cells displayed very high substrate affinity (Ks ) values of 0.003 and 0.023 mg PNP l31 , and low specific growth rate (Wmax ) values of 0.021 and 0.019 h31 at 30 and 37³C, respectively, at initial substrate concentration of 0.70 and 1.40 mg PNP l31 . Substrate inhibition was only evident at substrate concentration of 2.80 mg PNP l31 ; the measured kinetic parameters were 0.055 and 0.043 h31 . Temperature had a small impact on the measured values for Wmax and Ks . Surprisingly, values for the endogenous decay coefficient (0.015 and 0.020 h31 at 30 and 37³C, respectively) were of the same size as the specific growth rate. This indicates that net growth of B. sphaericus JS905 on PNP when used as a sole substrate is very small. As a result, the advantage of the high affinity PNP removal by this strain must probably depend on its growth on other primary substrates. The O2 /PNP stoichiometry indicates the consumption of 3 mol of molecular oxygen for the oxidation of each mol of PNP. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bacillus sphaericus JS905; Kinetics; p-Nitrophenol oxidation ; Respirometry

1. Introduction p-Nitrophenol (PNP), a priority environmental pollutant [1], occurs in industrial e¥uents [2] posing esthetic and health problems [3]. Pure cultures of aerobic soil bacteria readily metabolize PNP with removal of the nitro group as nitrite [4^8]. Two alternative pathways exist for aerobic PNP biodegradation [9]. Both pathways converge at the level of Lketoadipate. In the ¢rst pathway, hydroquinone is * Corresponding author. Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515003, India. Tel.: +91 (8554) 55367 ext. 223; Fax: +91 (8554) 32432.

formed from PNP via a monooxygenase attack in the para position with concomitant NO3 2 release. The hydroquinone is oxidized subsequently by a ring-cleaving dioxygenase to yield Q-hydroxymuconic semialdehyde which is enzymatically converted to Lketoadipic acid [5]. Consequently, 2 mol of molecular oxygen is required for the conversion of PNP to L-ketoadipic acid. In the second pathway, 4-nitrocatechol or 4-nitroresorcinol is formed from PNP via a monooxygenation reaction at the ortho or meta position followed by a second monooxygenase attack in the para position to yield 1,2,4-benzenetriol (BT) with concomitant release of nitrite. BT is further oxidized by a ring cleavage dioxygenase to yield mal-

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 1 9 - X

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V. Kadiyala et al. / FEMS Microbiology Letters 166 (1998) 115^120

eylacetate which is converted enzymatically to L-ketoadipate [7]. Very recently, a complete pathway for aerobic PNP degradation involving two monooxygenations for conversion of PNP to BT, both reactions catalyzed by a single two-component enzyme system, in Bacillus sphaericus JS905 has been described [10]. Thus, in the second pathway, 3 mol of O2 is required for transformation of each mol of PNP to L-ketoadipate. Among other things, successful application of biological treatment to remove PNP from contaminated soils and waters depends on the biokinetic parameters: the maximum speci¢c removal rate of PNP or the maximum speci¢c growth rate on PNP (qmax [M PNP/M cells/t] or Wmax [1/t]); the half saturation coe¤cient, Ks [M PNP/l3 ]; the self inhibition coe¤cient, Ki [M PNP/l3 ]; and the speci¢c decay coe¤cient, b [1/t]. Very few reports on the kinetics of PNP mineralization by aerobic pure cultures are available [11,12]. Our studies on PNP biodegradation showed that B. sphaericus JS905 has a great potential in transforming PNP releasing only nitrite in stoichiometric amounts [8]. Information on kinetics of PNP biotransformation, measuring activity at low concentrations, is essential to decide whether a biological technology is feasible and to design a biochemical reactor. Respirometry was therefore employed in the present study to quantify the kinetics of whole cell PNP removal/oxidation by the strain B. sphaericus JS905. 2. Materials and methods 2.1. Chemicals PNP was obtained from Aldrich (Milwaukee, WI), and tryptic soy broth (TSB) was from Difco (Detroit, MI). All other chemicals were of the highest purity commercially available. 2.2. Organism and induction with PNP The strain B. sphaericus JS905 was maintained on minimal salts medium (MSB) [13] containing 15 mg of PNP, 200 mg of yeast extract and 18 g of agar per liter. For induction with PNP, cells were grown in 2 l of half-strength TSB overnight at 37³C with shaking

at 250 rpm. The cells were harvested by centrifugation, washed three times with MSB, suspended in MSB containing PNP (150 WM) and yeast extract (0.1%) to an OD600 of 0.87, and incubated at 37³C with shaking (250 rpm). When the yellow color of the PNP disappeared, PNP (150 WM) was added again, the sequence was repeated until the culture received a total of six consecutive additions of PNP within 3 h. PNP disappeared within 15 min after the second addition onwards. Just before every new addition of PNP, samples of the culture medium were withdrawn, centrifuged to remove the cells and analyzed by a colorimetric method for the presence of nitrite [14]. Similar samples were analyzed by HPLC for detection of PNP or its metabolites. HPLC was performed on a Sphersorb C8 column (5 Wm, 250U4.6 mm; Altech, Deer¢eld, IL) with tri£uoroacetic acid (13.5 mM) in water and acetonitrile (60:40) as the mobile phase at a £ow rate of 1.0 ml min31 . UV210 was monitored with an HP 1040A diode array detector (Hewlett-Packard, Palo Alto, CA). 2.3. Respirometry Respirometric experiments were performed essentially as described by Ellis et al. [15]. Washed PNPgrown cell suspensions of B. sphaericus JS905 in MSB at 37³C were diluted 10-fold in prewarmed MSB and kept at 30 or 37³C. Cells were aerated at 30 or 37³C for 30 min prior to use in assays. Measurements were conducted in water-jacketed 2 ml oxygraph ¢tted with Clark-type oxygen electrode and maintained at 30 or 37³C using a recirculating water bath. YSI 5331 dissolved oxygen (DO) probes (YSI, Yellow Springs, OH) were equipped with YSI 5776 high-sensitivity membranes and connected to a YSI 5300 Biological Oxygen Monitor connected via an interface block to an A/D data acquisition board which was operated using the Labtech Notebook software (Laboratory Technologies Corporation, Wilkington, MA) on a Pentium PC. The DO probes were equilibrated and calibrated at 30 or 37³C in deionized water. Data acquisition was performed at 10 Hz, and data were averaged over a 2- or 4-s interval. Subsequent data analysis was performed on data sets that retained one data point every 2 or 4 s. Approximately 2 ml of cell suspension

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was transferred to each oxygraph chamber, which was subsequently sealed with a hollow-core glass stopper. The suspension was mixed vigorously using a micro stir bar and stir plate. Several minutes of background oxygen uptake data were collected before substrate injections were made, and cell suspensions were reaerated whenever needed. Microvolume injections (1, 2, or 4 Wl) were made through the center of the glass stopper from a 10 mM PNP solution in distilled water. Sample dilution due to substrate injection was negligible and could be ignored. Initial PNP concentrations were expressed in chemical oxygen demand (COD) units using the conversion factor of 1.4964 g COD g31 PNP. Thus, initial concentrations of approximately 1.04, 2.08, and 4.16 mg l31 as COD corresponded 0.70, 1.40, and 2.80 mg PNP l31 and injection volumes of 1, 2, and 4 Wl, respectively. The cell concentration was determined in triplicate samples of the cell suspension using 0^150-ppm COD vials (HACH, Loveland, CO). Linear regressions were performed on the portions of the oxygen uptake curves prior to PNP injection. Resulting values for the slopes divided by the cell concentrations provided estimates for the endogenous decay coe¤cient, b [1/t]. Oxygen uptake curves were subsequently transformed by subtraction of this oxygen uptake rate, and the resulting pro¢le was ¢t to a Monod or Andrews kinetic expression by minimizing the residual sum of squares (RSS) between model predicted and experimental data [15].

3. Results and discussion 3.1. PNP oxidation During induction, TSB-grown cells of B. sphaericus JS905 removed PNP completely and released only nitrite in stoichiometric amounts. The accumulation of nitrite after six consecutive additions of PNP, at 150 WM, during induction was the same as the total amount of PNP added to the culture medium within 3 h (Fig. 1). HPLC analysis of the culture £uids prior to each addition of PNP during induction of enzymes for catabolism of PNP revealed the absence of any degradation product. This suggests that PNP was completely oxidized by B.

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Fig. 1. Cumulative accumulation of nitrite from PNP during induction. Cells of B. sphaericus JS905 were grown overnight in half-strength TSB, harvested by centrifugation and suspended in MSB containing 150 WM PNP and 0.1% yeast extract to an OD600 of 2.096. At each sampling time indicated, 150 WM PNP was added immediately after disappearance of the yellow color of PNP.

sphaericus JS905. When the cells were grown on PNP in the presence of 0.1% yeast extract, the OD600 of the culture medium increased from 2.096 initially to 2.124 after induction. 3.2. Kinetics of PNP oxidation The oxygen uptake curves could be described very well using Monod kinetics at injection concentrations of 1.04 and 2.08 mg PNP l31 as COD (Table 1). However, a signi¢cant substrate inhibition was evident at injection concentration of 4.16 mg l31 as COD (see Fig. 2 as well). Those curves were therefore modeled using the Andrews or Haldane kinetic expression. Very high substrate a¤nities were measured at the low substrate injection concentrations: Ks values were 0.003^0.023 mg PNP l31 , corresponding to 1^7U1039 M PNP. Low maximum speci¢c growth rate values of 0.019 to 0.021 h31 were measured. This low speci¢c growth rate is exacerbated by the endogenous decay rate values of 0.020^0.015 h31 . Since the net speci¢c growth rate of any organism is the net e¡ect of growth and decay, the present results suggest that at 37³C, net growth of B. sphaericus

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Fig. 2. Representative oxygen uptake curves in response to PNP injection in B. sphaericus JS905 suspensions. Transformed oxygen uptake pro¢les are shown (subtraction of background oxygen uptake from the raw oxygen uptake curves). Curves from top to bottom are responses to initial concentrations of 2.80, 1.40, and 0.70 mg PNP l31 . Jagged curves are the experimental data, and smooth curves are the best-¢t lines.

JS905 on PNP is not feasible (Wmax 6 b), while at 30³C growth would be feasible (Wmax s b). A good ¢t of the predicted oxygen uptake pro¢le

to the experimental data demanded that the last 10% of the experimental curve be weighted 15-fold in the RSS calculation. Substrate inhibition was very evident from the shape of the oxygen uptake curves (see Fig. 2): the oxygen uptake rate increased as the substrate disappeared. When substrate inhibition occurs (i.e., at initial concentration of 4.16 mg l31 as COD), the a¤nity coe¤cient increased signi¢cantly (by approximately one order of magnitude). This may suggest that a di¡erent type of substrate uptake/removal is used at those concentrations. Also, when substrate inhibition occurs, we can estimate the value of S*=(Ks Ki )1=2 . This is the value of S above which further increases in S cause the speci¢c removal rates to decrease. The calculated value of S* is 0.58 and 0.39 at 37 and 30³C, respectively, and suggests that at concentrations higher than those values substrate inhibition is noticeable. However, with injection concentrations of approximately 1.40 mg PNP l31 , we were not able to measure substrate inhibition because of the very high substrate a¤nity (Ks IKi ). The impact of temperature is small. The biokinetic parameters most signi¢cantly a¡ected by temperature were the a¤nity coe¤cient and the speci¢c decay coe¤cient. Higher a¤nity is measured at 30³C than at 37³C, whereas the speci¢c decay coe¤cient is larger at 37³C. It should be pointed out that the culture was grown and induced at 37³C. Therefore, the experiment at 30³C here does not measure the

Table 1 Best ¢t parameters for PNP removal by B. sphaericus JS905 at 30 and 37³C Wmax (1/h) Temperature, 30³Ca A X 0.021 c 0.004 B X 0.055 c 0.0076 Temperature, 37³Cb A X 0.019 c 0.004 B X 0.043 c 0.0107

Ks (mg PNP l31 )

Y (mg X COD/mg PNP)

Ki (mg PNP l31 )

b (1/h)

O2 /PNP (mol/mol)

n

0.003 0.0006 0.090 0.075

0.771 0.022 0.769 0.080

n/a n/a 1.665 0.511

0.015 0.003 0.017 0.008

3.127 0.111 3.162 0.258

7

0.023 0.017 0.217 0.188

0.877 0.057 0.765 0.079

n/a n/a 1.570 0.750

0.020 0.001 0.020 0.0006

2.694 0.249 3.178 0.344

7

a

4

4

Initial cell concentration, 357 mg l31 COD (S.D. 10 mg l31 COD). Initial cell concentration, 425 mg l31 COD (S.D. 5 mg l31 COD). A refers to initial substrate concentrations of 1.04 and 2.08 mg l31 as COD. B refers to initial substrate concentration of 4.16 mg l31 as COD. X = mean, c = standard deviation. n = number of observations. n/a = not applicable. b

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impact of temperature on growth on PNP, but rather the impact of temperature on the activity of the existing PNP-degrading enzymes. 3.3. O2 /PNP stoichiometry The oxygen uptake measurement allowed calculation of the oxygen/PNP stoichiometry. The average O2 /PNP stoichiometry for all experiments is 3.137 (S.D. 0.152). The demand for molecular oxygen as reactant in the initial oxygenase reactions is 2 mol for the ¢rst (via hydroquinone), and 3 mol for the second (via BT) postulated PNP pathway. The observed ratio of approximately 3 con¢rms that the second pathway, which involves two monooxygenations of PNP and subsequent dioxygenation of THB consuming 3 mol of O2 for each mol of PNP transformed, is operative in B. sphaericus JS905 [10]. In addition, the O2 /PNP stoichiometry at both temperatures is very similar at the lowest and highest substrate injections (see Table 1). This suggests that uncoupling of oxidative phosphorylation was not the cause of the observed inhibition although all nitrophenols act as uncouplers in microorganisms [16]. Uncoupling would result in an increase of the O2 / PNP stoichiometry. Few detailed reports on PNP mineralization by aerobic pure cultures exist. Schmidt et al. [11] studied kinetics of PNP mineralization by a Pseudomonas strain in both growth and no-growth experiments. In the no-growth experiment, Ks value of 0.67 þ 0.08 mg PNP l31 were reported while in the growth experiment Ks value was 1.1 þ 0.2 mg PNP l31 , and Wmax value was 0.31 h31 . Batch studies on PNP mineralization by pure cultures in PNP-spiked sterile industrial wastewater were performed by Zaidi et al. [12]. From the reported values, kinetic parameters could not be retrieved; however, much slower rates of removal were measured when the initial PNP concentration was 26 Wg l31 compared to 20 mg l31 , suggesting severe substrate limitation (SIKs ) at the lower concentration. Thus, the limited literature data base does not allow an extensive comparison of the measured kinetics. It does seem that the examined strain B. sphaericus JS905 has a very high a¤nity for PNP removal (Ks =0.003^0.023 mg l31 at the lower concentrations tested). However, both the very low maximum speci¢c growth rates, and the exist-

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ence of substrate inhibition observed at high concentrations, may render the use of this strain in remediation of PNP contaminated soils ine¡ectual. Remediation of PNP using this strain may depend on the addition of other carbon sources to allow growth, as has been suggested for mineralization of PNP by other strains [11].

Acknowledgments This research was performed while Venkateswarlu Kadiyala held a Visiting Senior Research Associateship of the National Research Council, USA at the USAF/Armstrong Laboratory. This work was also supported, in part, by funds from the Air Force Of¢ce of Scienti¢c Research.

References [1] Keith, L.H. and Telliard, W.A. (1979) Priority pollutants. I ^ a perspective view. Environ. Sci. Technol. 13, 416^423. [2] Shackelford, W.M. and Keith, L.H. (1976) Frequency of organic compounds identi¢ed in water. U.S. Environmental Protection Agency (ERL), Athens, GA (EPA 600/4-76-062). [3] Bruhn, C., Lenke, H. and Knackmuss, H.J. (1987) Nitrosubstituted aromatic compounds as nitrogen source for bacteria. Appl. Environ. Microbiol. 53, 208^210. [4] Raymond, D.G.M. and Alexander, M. (1971) Microbial metabolism and cometabolism of nitrophenols. Pesticide Biochem. Physiol. 1, 123^130. [5] Spain, J.C. and Gibson, D.T. (1991) Pathway for biodegradation of p-nitrophenol in Moraxella sp. Appl. Environ. Microbiol. 57, 812^819. [6] Hanne, L.F., Kirk, L.L., Appel, S.M., Narayan, A.D. and Bains, K.K. (1993) Degradation and induction speci¢city in actinomycetes that degrade p-nitrophenol. Appl. Environ. Microbiol. 59, 3505^3508. [7] Jain, R.K., Dreisbach, J.H. and Spain, J.C. (1994) Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl. Environ. Microbiol. 60, 3030^3032. [8] Venkateswarlu, K. and Spain, J.C. (1997) p-Nitrophenol biodegradation by Bacillus sphaericus JS905 expressing a soluble monooxygenase. Abstr. Q340. 97th General Meeting of the American Society for Microbiology, Miami Beach, FL. [9] Spain, J.C. (1995) Bacterial degradation of nitroaromatic compounds under aerobic conditions. In: Biodegradation of Nitroaromatic Compounds (Spain, J.C., Ed.), pp. 19^35. Plenum Press, New York. [10] Venkateswarlu, K. and Spain, J.C. (1998) A two-component monooxyggenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocate-

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chol in Bacillus sphaericus JS905. Appl. Environ. Microbiol. 64 (in press). [11] Schmidt, S.K., Scow, K.M. and Alexander, M. (1987) Kinetics of p-nitrophenol mineralization by a Pseudomonas sp.: e¡ects of second substrates. Appl. Environ. Microbiol. 53, 2617^ 2623. [12] Zaidi, B.R., Mehta, N.K., Imam, S.H. and Greene, R.V. (1996) Inoculation of indigenous and non-indigenous bacteria to enhance biodegradation of p-nitrophenol in industrial wastewater: e¡ect of glucose as second substrate. Biotechnol. Lett. 18, 565^570. [13] Spain, J.C. and Nishino, S.F. (1987) Degradation of 1,4-dichlorobenzene by a Pseudomonas sp. Appl. Environ. Microbiol. 53, 1010^1019.

[14] Daniels, L., Hanson, R.S. and Phillips, J.A. (1994) Chemical analysis. In: Methods for General and Molecular Bacteriology (Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R., Eds.), pp. 512^554. American Society for Microbiology, Washington, DC. [15] Ellis, T.G., Barbeau, D.S., Smets, B.F. and Grady Jr., C.P.L. (1996) Respirometric technique for determination of extant kinetic parameters describing biodegradation. Water Environ. Res. 68, 917^926. [16] Howard, P.H., Santodonato, J., Saxena, J., Malling, J.E. and Greninger, D. (1976) Investigations of selected potential environmental contaminants: nitroaromatics. U.S. Environmental Protection Agency (O¤ce of Toxic Substances), Washington, DC (EPA 560/2-76-010).

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