Kinetics Of P-nitrophenol Degradation

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Kinetics of p-nitrophenol degradation by Pseudomonas pseudomallei wild and mutant strains Asma Rehman a; Zulfiqar A. Raza a; Muhammad Afzal a; Zafar M. Khalid a a Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Islamic Republic of Pakistan

Online Publication Date: 01 January 2007 To cite this Article: Rehman, Asma, Raza, Zulfiqar A., Afzal, Muhammad and Khalid, Zafar M. (2007) 'Kinetics of p-nitrophenol degradation by Pseudomonas pseudomallei wild and mutant strains', Journal of Environmental Science and Health, Part A, 42:8, 1147 - 1154 To link to this article: DOI: 10.1080/10934520701418656 URL: http://dx.doi.org/10.1080/10934520701418656

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Journal of Environmental Science and Health Part A (2007) 42, 1147–1154 C Taylor & Francis Group, LLC Copyright
ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520701418656

Kinetics of p-nitrophenol degradation by Pseudomonas pseudomallei wild and mutant strains ASMA REHMAN, ZULFIQAR A. RAZA, MUHAMMAD AFZAL and ZAFAR M. KHALID Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Islamic Republic of Pakistan

Pseudomonas pseudomallei EBN-10 strain, previously isolated from a local pharmaceutical industry’s wastewater, was spontaneously adapted to higher p-nitrophenol (PNP) levels, which then was subjected to gamma ray-induced mutagenesis; the efficient isolates hence obtained were designated as EBN-11 and EBN-12, respectively. EBN-12 mutant strain could completely mineralize PNP (100 mg/L) on the minimal media in 24 h while, the parent strain utilized only 6% of it. Addition of glucose as co-substrate further increased the PNP degradation rate; however, phenol inclusion inhibited the degradation process. Ammonium sulphate was experienced as the best of the nitrogen sources used by EBN-12 mutant strain, while degrading PNP. Keywords: Adaptation; biodegradation, kinetics; p-nitrophenol; Pseudomonas pseudomallei.

Introduction Nitroaromatic compounds are produced by incomplete combustion of fossil fuels. They exhibit high toxicity and/or mutagenicity to plants, animals and microbes, and pose health and environmental risks, either directly or through some of their catabolic metabolites.[1] These compounds are important starting materials for the production of aromatic amines, hydrazo- and azo-compounds, isocyanates, benzidin derivatives, haloaromatics, plasticizers, dyes, explosives, synthetic intermediates, pesticides, herbicides and drugs.[2,3] During the manufacture of these industrial products, p-nitrophenol (PNP) enters the environmental bodies e.g., soil, from where it leaches into the surface water and groundwater resources[4] ; then either simply transforms to dead end products, which may at times proved to be more toxic than the original compound; or the microorganisms may actually utilize it as a carbon and/or nitrogen source.[5] PNP is hardly degradable and included in the priority pollutants’ list with a toxicity value (as EC50 ) of 64 mg/L.[6] Traditionally, the contaminated sites are treated by physical and/or chemical methods, which are often difficult to execute, costly and not environment friendly. On the other

Address correspondence to A. Rehman or Z.M. Khalid, Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, P. O. Box-577, Faisalabad, Pakistan; E-mail: [email protected] or [email protected] Received December 5, 2006.

hand, bioremediation is an emerging alternative technology for the restoration of the environment. The bioremediation involves the enrichment of exogenous microorganisms to the contaminated site, which remove or minimize toxins present in the environment. Although several bacteria including Flavobacterium, Pseudomonas, Moraxella, Nocardia, and Arthrobacter exist in the environment which can mineralize nitroaromatic compounds including PNP, through the removal of their nitro- groups as nitrites;[7−10] yet their general recalcitrance and toxic properties pose a threat to the environment.[11] Since the microbes are not able to easily catalyze the degradation of nitroaromatic compounds; this results in progressive accumulation of these contaminants in the environment. One option to biodegrade such persistent compound is to enrich, acclimatize and/or mutate the degrading species. In the present study, a recently isolated and identified bacterial strain Pseudomonas pseudomallei EBN-10,[12] capable of degrading PNP, was acclimated to gradually increasing PNP concentrations, under shake flask conditions, to obtain a spontaneous mutant strain of P. pseudomallei EBN-11 (capable of utilizing higher PNP); which then was under gone gamma ray-induced mutagenesis to get P. pseudomallei EBN-12 mutant strain, a PNP hyperdegrader. The parent and mutant strains were compared on the basis of their capabilities to degrade PNP by using different combinations of carbon and nitrogen sources. The substrate consumption and biomass accumulation data of different strains of P. pseudomallei on the liquid PNP media have been analyzed to optimize the growth parameters.

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1148 Materials and methods Microorganisms The culture of P. pseudomallei EBN-10 parent strain, previously isolated from the wastewater of a local pharmaceutical industry,[12] was used as a source of PNP degraders. The parent strain was capable of growing on the minimal medium of composition given as: (g/L) NH4 H2 PO4 (1.0), K2 HPO4 (1.0), MgSO4 ·7H2 O (0.2), CaCl2 ·2H2 O (0.2) and FeCl3 ·6H2 O (0.05), supplemented with up to 50 mg PNP/L as sole carbon source, set at pH 7.0, and incubated under shake flask conditions (100 rpm and 37◦ C) in an orbital shaker (OSI-503L, OGAWA SEIKI Co. Ltd., Japan). Adaptation of parent strain to higher PNP levels The EBN-10 parent strain (tolerating PNP up to 50 mg/L) was gradually adapted to higher PNP concentrations, before it might face a growth inhibiting substrate concentration. For that purpose, 1% (v/v) cells suspension of EBN-10 culture (prepared as mentioned under “Preparation of inocula”) was inoculated to the minimal media, containing serially increasing concentrations of PNP as 50, 60, 70, . . . , up to 150 mg/L in 250 mL Erlenmeyer flasks and incubated in the orbital shaker at 100 rpm and 37◦ C. The disappearance of yellowish green color of the media indicated the utilization of PNP, which was confirmed by a spectrophotometric method (as described under “Analytical methods”) and the culture was then transferred to the next higher PNP concentration. The best spontaneously adapted mutant isolate, able to grow on 130 mg PNP/L, was designated as P. pseudomallei EBN-11. Gamma ray-induced mutagenesis of PNP adapted strain The cell suspension (OD 0.7 at 600 nm) of PNP-adapted EBN-11 strain was taken in a McCartney vial (30 mL capacity) and to exposed to the best 60 Co gamma radiation (Gamma Cell 220, Atomic Energy Canada Ltd.) dose of 300 Gy (found suitable for 3 log kill) at an exposure rate of 40 Gy/h. The mutated culture was plated on PNP agar plates and incubated at 37◦ C for 24 h. The fermentation process was visually examined for a heavy microbial growth and disappearance of yellowish green color of PNP. Ten morphologically different mutant isolates observed under an optical microscope, were transferred to separate minimal media supplemented with 100 mg PNP/L in 250 mL Erlenmeyer flasks, and incubated under shake flask conditions (100 rpm, 37◦ C). Among 10 isolates, one most efficiently removed PNP from the culture medium making it completely decolorized within 24 h, as compared to the parent, spontaneous mutant and other tested gamma ray mutated strains. This particular culture was serially diluted, plated on the agar plates of minimal media supplemented with 100 mg PNP/L,

Rehman et al. incubated at 37◦ C and examined from time to time for 72 h. Several bacterial colonies with similar morphology appeared, surrounded by off white halos against yellowishgreen background of PNP-agar plates. The mutant strain from the largest off-white halo was picked up, streaked on nutrient agar plate and designated as P. pseudomallei EBN12 mutant strain. The purity of the strain was checked several times by streaking it on fresh plates of nutrient agar and incubated overnight. All the chemicals used were of reagent grade. Preparation of inocula The parent and mutant strains were transferred, separately, to nutrient broth (0.8%, w/v; Oxoid) media in 250 mL Erlenmeyer flasks and incubated in the orbital shaker (100 rpm, 37◦ C) for 24 h. The cells were harvested by centrifugation (7,740 g, 4◦ C) for 10 min. The cell pellet was resuspended in sterile normal saline to set an absorbance of 0.7 at 600 nm. The aliquots (1%, v/v) of these cell suspensions were separately used as inocula. Culture media setups The shake flask studies were conducted under four experimental approaches as described below: 1. For the mineralization of PNP, the minimal media (50 mL) (of the composition as mentioned earlier), supplemented with filter-sterilized 20, 50 or 100 mg PNP/L as sole carbon source in 250 mL Erlenmeyer flasks, were inoculated, separately, with the cell suspensions of the EBN-10 parent, EBN-11 spontaneous mutant and EBN-12 gamma ray mutant strains. 2. Glucose (100 mg/L) was added as co-substrate to the minimal media containing 100 mg PNP/L and inoculated with EBN-12 mutant strain. 3. Phenol (100 mg/L) was added as co-substrate to the minimal media containing 100 mg PNP/L and inoculated with EBN-12 mutant strain. 4. Same molar concentrations of different nitrogen sources (viz. ammonium dihydrogen phosphate, ammonium sulphate, ammonium nitrate and ammonium chloride) were added to the minimal media containing 100 mg/L of each of PNP and glucose, and inoculated with EBN-12 mutant strain. In each case, the control flasks (without inocula) were also incubated in parallel to determine abiotic PNP degradation effects. The fermentation was carried out under shake flask conditions (100 rpm and 37◦ C). Analytical methods Aliquots (5 mL) of the culture broth samples were taken at specified time intervals and centrifuged (at 15,300 g, 4◦ C for 15 min). The cell-free culture broth (CFCB) was saved

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p-Nitrophenol degradation by Pseudomonas for the determination of the substrate utilization. The cell pellet was washed and resuspended in sterile normal saline, centrifuged (at 15,300 g, 4◦ C for 15 min) again, and desiccated in an electric oven at 60◦ C until constant dry cell biomass (DCBM) was obtained. The PNP contents of the culture media were determined following the method of Heitkamp et al.[13] Briefly, pH value of the CFCB was adjusted at 8.0 with 1 N NaOH, vortexed in a screw-capped test tube and then its absorbance was measured at 414 nm using a spectrophotometer (Spectra 22; LaboMed, Inc.); while a simple control media, missing PNP, was used as blank. Changes in the glucose contents of the CFCB were determined according to the standard phenol-sulphuric acid method,[14] and phenol and nitrate levels following the standard methods.[15] Kinetic data analyses The kinetics of the fermentative degradation was studied by determining the process parameters such as the specific growth rate (µ, h−1 ), volumetric substrate uptake rate (QS , g/L/h) and doubling time (td , h), following the standard methods.[16] Experimentation and data All the experiments were conducted in three independent replicates and the data reported are the mean of three concordant readings.

Results and discussion Adaptation of PNP degrader to more severe conditions Nitroaromatic compounds, including PNP, being toxic, are not readily taken up by the microbes. One option, however, is to first acclimatize the biomass to such toxic compounds and then to use in bioremediation. The parent strain was checked whether it could grow on serially increasing concentrations of PNP like 30, 40, 50, 60 and 70 mg/L. Only that bacterial culture was transferred to the next higher concentration of PNP, which showed good growth on the preceding one; and it was found as able to grow only up to 50 mg PNP/L in the minimal media. After spontaneous adaptation, its mutant strain EBN-11 could use PNP up to 130 mg/L (i.e. 160% enhanced ability to degrade PNP). Arcangeli and Arvin[17] also used the culture adaptation technique to get some bacteria capable of degrading a mixture of aromatic pollutants. Tomei et al.[18] achieved the complete removal of PNP by using an acclimatized bacterial biomass. As a result of PNP degradation, the culture medium became turbid and its color vanished. Zaidi and Narinder[19] also experienced the disappearance of yellowish green color of PNP as an indication of its degradation.

The visual observations of PNP utilization were further checked spectrophotometrically, following the method of Heitkamp et al.[13] From 100 to 130 mg PNP/L, a dynamic equilibrium growth mode of EBN-11 mutant strain was observed, i.e., the growth enhanced due to the substrate availability was cancelled because of its growth inhibition nature, while the residual substrate was available to maintain the catabolic activities in the limited growth cells. Above 130 mg PNP/L, EBN-11 mutant strain could not grow due to severe substrate inhibition. However, EBN-11 mutant strain eliminated the lag phase of about 12 h, which was exhibited by EBN-10 parent strain on 50 or 100 mg PNP/L. The results indicated that at 20 mg/L, PNP was degraded without a significant difference in lag phase periods irrespective of adaptation of the bacterial cells to higher PNP concentrations. The PNP degrading strains (EBN-10, EBN-11 and EBN12) were separately streaked on the agar plates of minimal media containing 20, 30, 40, and so on up to 130 mg PNP/L. The observations, i.e., the appearance of bacterial growth and discoloration of solid media, indicated that all the strains had ability to degrade PNP but to different extents, while the color of PNP-agar plate for abiotic control remained unchanged. Also, the stab cultures of EBN-11 and EBN-12 mutant strains on the solid agar media containing 100 mg PNP/L were decolorized as growth appeared, whereas the color of control and that of EBN-10 parent strain inoculated media remained yellowish green. PNP mineralization study The comparison of time-course studies of PNP utilization by EBN-10, EBN-11 and EBN-12 strains grown on the minimal media containing 20, 50 and 100 mg PNP/L as sole carbon sources is given in Figure 1a. The EBN-11 mutant strain, although, possessed greater ability to degrade PNP (100 mg/L) as compared to its parent strain yet EBN-12 mutant strain degraded same amounts of PNP in the least time period. Parallel to substrate consumption, the time-course study of DCBM formation was also conducted on the respective PNP concentrations. The growth mode of different strains on different PNP concentrations is shown in Figure 1b. On entering the exponential growth phase, PNP concentration’s profile exhibited substrate inhibition kinetics with an initial low PNP removal rate, when its concentration was high; and subsequent faster kinetics, when it dropped to lower values. The increase in the DCBM and decrease in PNP concentration of the media with time, particularly in the exponential phase, indicated that the degradation process was growth-associated (Fig. 1a, 1b); thereafter, in the stationary phase, the uptake of PNP continued but of lower order. The substrate consumption data indicated that once the microorganism had adapted to PNP, its PNP degrading ability rapidly increased. Firstly, very slow bacterial growth

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Fig. 1. PNP utilization (a) and growth profiles (b) of EBN-10 (—), EBN-11 (– – –) and EBN-12 ( – ···– ) strains grown on the minimal media containing 20 mg PNP/L (•), 50 mg PNP/L (◦) and 100 mg PNP/L (
) under shake flask conditions (37◦ C and 100 rpm). All the values are averages of three readings from three independent experiments.

rates were observed but after 12–16 h of lag phase, the cell biomass entered the exponential growth phase (16–24 h) and from 24 to 72 h, it gradually became constant, accompanied by parallel substrate consumption (Fig. 1a, 1b). The DCBM yield of EBN-12 mutant strain was higher than that of other strains. The comparison of three strains revealed that EBN-12 mutant strain was faster PNP degrader and had ability to withstand the PNP at higher concentrations. In contrast, the growth of EBN-10 parent strain remained suppressed on all the tested concentrations of PNP (Fig. 1b) because of non-pre-exposure of its cells to higher PNP concentrations. A lag phase of 24 h with EBN-10 parent strain on 50 mg PNP/L was not unexpected since the microbial degradation of PNP has been reported to occur after a lag phase of 7 d in the soil,[20] 6–12 d in sewage,[21] 40 h to 2 weeks in sediment water ecocores[22,23] and 6 d in ponds.[24] Kinetics of PNP degradation The comparison of DCBM productivity of all the strains on different PNP concentrations under different experimen-

Rehman et al. tal setups is given in Table 1. Being toxic to cells, PNP severely inhibited its utilization and hence the bacterial growth; which is also evident from low QS values (Table 1). Generally, PNP degradation was found to be concentration dependent. EBN-10 parent strain, while growing on 20 mg PNP/L, utilized 99.8% of substrate in 72 h followed by 90 and 6% on 50 and 100 mg PNP/L, respectively. So, the optimal rate of PNP degradation by EBN-10 parent strain occurred at an initial concentration of 20 mg/L above which, the rate and extent of PNP removal gradually reduced up to 50 mg PNP/L. At an initial concentration of 50 mg/L, 90% of PNP were degraded in 72 h of incubation by EBN-10 parent strain as compared to EBN-11 and EBN-12 mutant strains which preferably removed PNP as much as 99 and 100%, respectively, under the same conditions. EBN-10 parent strain degraded ≥90% PNP (of 50 mg/L) at >72 h; whereas, EBN-11 and EBN-12 mutant strains degraded the same concentrations in 24 h of incubation. Heitkamp et al.[13] observed that 45–70% PNP contents of the culture medium were mineralized at 4 d of incubation. Herman and Costerton[25] reported almost 60% PNP mineralization by a bacterial species capable of utilizing PNP as sole carbon and energy source. The kinetic results, given in Table 1, show that the maximum µ (0.29 h−1 ) was observed with EBN-12 mutant strain on 100 mg PNP/L as sole carbon source, followed by 0.28 h−1 on 50 mg PNP/L with the same strain. However, in the case of EBN-10 parent strain, as the initial PNP concentration of the media increased, its growth inhibition effects were more pronounced as evidenced from the decrease in its µ from 0.22 to 0.20 h−1 on the minimal media containing 50 and 100 PNP mg/L, respectively. This was also supported by the td (3.1 h) data of EBN-10 mutant strain (Table 1), which increased (to 3.4 h) on shifting the strain from 20 to 100 mg PNP/L. The greatest QS (10.8 mg/L/h) and DCBM yield (34.0 mg/L) were observed with 100 mg PNP/L as sole carbon source used by EBN-12 mutant strain (Table 1). These results indicated that EBN-12 mutant strain was excellent in tolerating and degrading PNP at higher concentrations than its parent isolate and/or spontaneously mutated strain. So, EBN-12 mutant strain was continued for further study. Effect of co-substrates The effect of addition of glucose (100 mg/L) or phenol (100 mg/L) as co-substrate on the kinetics of PNP degradation was studied. In the presence of glucose, EBN-12 mutant strain completely degraded 100 mg PNP/L in 20 h (Fig. 2) rather than in 24 h in its absence. The glucose addition also improved the kinetics of fermentative degradation of PNP. Different growth parameters with EBN-12 mutant strains, calculated in the presence and absence of glucose, are reported in Table 1. The µ (0.29 h−1 without glucose addition) was increased to 0.32 h−1 (in the presence of 100 mg glucose/L). Karim and Gupta[26] studied the effect of

1151

20

50

EBN-10 100

20

50

EBN-11 20

50

100

EBN-12 100∗

100∗∗

18.0 ± 0.5 11.0 ± 0.4 25.0 ± 0.8 34.0 ± 0.9 38.5 ± 0.9 1.0 ± 0.1 0.27 ± 0.09 0.26 ± 0.08 0.28 ± 0.09 0.29 ± 0.09 0.32 ± 0.10 0.01 ± 0.00 2.6 ± 0.2 2.6 ± 0.2 2.5 ± 0.2 2.4 ± 0.2 2.1 ± 0.1 9.3 ± 0.5 10.7 ± 0.5 1.9 ± 0.1 4.8 ± 0.2 10.8 ± 0.5 10.2 ± 0.5 0.1 ± 0.01 >72 ± 2 20 ± 1 24 ± 1 24 ± 1 20 ± 1 na

100

Glucose (100 mg/L) was added as co-substrate. ∗∗ Phenol (100 mg/L) was added as co-substrate. DCBM = dry cell biomass; µ = specific growth rate; td = doubling time; Qs = volumetric substrate uptake rate; na = not achieved. The results are averages of three replicates from three independent experiments.

∗

DCBM (mg/L) 3.9 ± 0.2 8.0 ± 0.3 2.0 ± 0.1 9.0 ± 0.3 14.0 ± 0.4 0.24 ± 0.08 0.22 ± 0.07 0.20 ± 0.06 0.25 ± 0.08 0.26 ± 0.08 µ (h−1 ) td (h) 3.1 ± 0.2 3.3 ± 0.2 3.4 ± 0.02 2.8 ± 0.2 2.6 ± 0.2 Qs (mg/L/h) 2.3 ± 0.1 5.4 ± 0.3 0.8 ± 0.1 2.2 ± 0.1 5.3 ± 0.3 Time of ≥90% 36 ± 1 >72 ± 2 na 24 ± 1 24 ± 1 PNP reduction (h)

Growth factors init. PNP (mg/L)

Table 1. Comparison of PNP mineralization by various P. pseudomallei strains grown on various concentrations in the minimal media under shake flask conditions (37◦ C and 100 rpm)

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Rehman et al.

Fig. 2. Utilization of PNP (100 mg/L) (•), glucose (Gl, 100 mg/L) (
) and phenol (Ph, 100 mg/L) (
), and DCBM (◦) formation of EBN-12 mutant strain grown on the minimal media containing only PNP (–···–), PNP+glucose (——) and PNP+phenol (– – –) under shake flask conditions (37◦ C and 100 rpm). All the values are averages of three readings from three independent experiments.

Fig. 3. Effect of different nitrogen sources [(NH4 )2 SO4 (
), NH4 NO3 (
), NH4 H2 PO4 (◦) and NH4 Cl (•)] on PNP degradation and DCBM formation of EBN-12 mutant strain grown on the minimal media containing PNP (100 mg/L) and glucose (100 mg/L) under shake flask conditions (37◦ C and 100 rpm). All the values are averages of three readings from three independent experiments.

alternative carbon sources on the biological transformation of nitrophenols. Schmidt et al.[27] observed an enhanced PNP degradation by a Pseudomonas sp. in the presence of glucose as co-substrate. Glucose addition to minimal media also showed positive effect on the biodegradation of other nirtoaromatics. The phenol addition, however, inhibited the PNP degradation process (Fig. 2). Schmidt et al.[27] and Cho et al.[28] also observed the inhibitory effect of phenol on PNP utilization by other strains.

termediate from PNP, which gradually were taken up by the cells while synthesizing the biomass, hence the nitrate levels of the minimal media overall decreased. Tomei et al.[18] also observed the nitrate production during PNP degradation. At the end of incubation period (72 h), the culture media though contained nitrogen in the form of nitrates but the growth had already been ceased due to depletion of carbon sources, required for the microbial population. Two enzymatic routes exist for the removal of nitro substitutes from nitroaromatic compounds. First involves an initial reduction of the nitro-substitute by a nitro-reductase, followed by a release of ammonium.[29,30] In the second route, the nitro substitutes are directly removed in the form of nitrite from the aromatic core. Such direct removal was

Effect of nitrogen sources The glucose was proved to be the best co-substrate so the biodegradation of PNP by EBN-12 mutant strain was also studied by changing the nitrogen sources of the mineral medium containing glucose (Fig. 3). When ammonium sulphate, ammonium nitrate, ammonium dihydrogen phosphate and ammonium chloride were separately supplemented to the minimal media containing 100 mg PNP and glucose/L, EBN-12 mutant strain completely degraded 100 mg/L of PNP within 16, 20, 24 and 36 h of incubation, respectively. The DCBM concentrations of the media also increased, accordingly (Fig. 3). Ammonium sulphate was observed as the best nitrogen source as in its presence EBN-12 mutant strain degraded PNP (100 mg/L) with the µ of 0.39 h−1 followed by 0.35 h−1 with ammonium nitrate as nitrogen source. Other kinetic parameters with the culture broths of EBN-12 mutant strain and different nitrogen sources are given in Table 2. The changes in the nitrate levels of the culture media of EBN-12 mutant strain grown on the minimal media containing PNP (100 mg/L) and glucose (100 mg/L), and supplemented with separate nitrogen sources are shown in Fig. 4. In all the cases, the nitrate contents of the media initially increased when released as an in-

Table 2. Effect of different nitrogen sources on PNP degradation and DCBM formation by P. pseudomallei EBN-12 mutant strain grown on the minimal media containing PNP (100 mg/L) and glucose (100 mg/L) under shake flask conditions (37◦ C and 100 rpm) Growth factors

(NH4 )2 SO4

NH4 NO3

NH4 H2 PO4

NH4 Cl

DCBM 49.0 ± 1.3 45.0 ± 1.1 38.5 ± 0.9 36.0 ± 0.8 (mg/L) 0.39 ± 0.13 0.35 ± 0.11 0.32 ± 0.10 0.27 ± 0.09 µ (h−1 ) 1.7 ± 0.1 2.0 ± 0.1 2.1 ± 0.1 3.2 ± 0.2 td (h) 11.2 ± 0.5 10.7 ± 0.5 10.2 ± 0.5 9.7 ± 0.5 QS (mg/L/h) DCBM = dry cell biomass; µ = specific growth rate; td = doubling time; QS = volumetric substrate uptake rate. The results are averages of three replicates from three independent experiments.

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p-Nitrophenol degradation by Pseudomonas References

Fig. 4. Changes in the nitrate contents of the culture media of EBN-12 mutant strain initially supplied with PNP (100 mg/L) and glucose (100 mg/L) and different nitrogen sources [(NH4 )2 SO4 (
), NH4 NO3 (
), NH4 H2 PO4 (◦) and NH4 Cl (•)] under shake flask conditions (37◦ C and 100 rpm). All the values are averages of three readings from three independent experiments.

demonstrated by Spain and Gibson,[10] who isolated an enzyme that converts PNP to hydroquinone and nitrite. The reaction required oxygen and NADPH, and the enzymatic activity was stimulated by FAD. The nitro group, from a nitro aromatic compound like PNP is removed by using a monoxygenase reaction. PNP monoxygenase from B. sphaericus JS905 also sequentially hydroxylates nitroaromatics.[31] This enzyme hydroxylates the ring adjacent to the hydroxyl group first and then displaces the nitro group from the aromatic ring. The conversion of phenol to catechol by Pseudomonas strain CF600 is catalyzed by a multicomponent phenol hydroxylase.[32]

Conclusions The adaptation followed by gamma ray-induced mutation of P. pseudomallei wild strain improved the PNP degradation kinetics. The EBN-12 mutant stain could withstand and mineralize PNP at high concentration in lesser time period, which constitutes the basic requirement in the waste water treatment systems. The results also showed that through the addition of glucose as co-substrate and a suitable nitrogen source like ammonium sulphate, the degradation kinetics of PNP could be optimized.

Acknowledgments The authors acknowledge Dr. M.I. Rajoka, S.I., Deputy Chief Scientist, NIBGE, for some valuable suggestions. Ms. Asma Rehman is highly indebted to Ms. Javaria Qazi, Research Scientist, NIBGE for her constant encouragement throughout her research.

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