Vol. 16, No. 1, 49–52, 2001
Isolation and Characterization of Pseudomonas Strain for Degradation of 4-nitrophenol ASIFA QURESHI1, S. K. PRABU1 and HEMANT J. PUROHIT*1 1
National Environmental Engineering Research Institute, Nagpur-20 India
(Received May 2, 2000—Accepted October 22, 2000) A Pseudomonas strain capable of growing on 4-nitrophenol (4NP) as a sole source of carbon was isolated from a pharmaceutical waste dumpsite after selective enrichment. Experiments revealed that the strain, SF1, utilizes 4-nitrophenol after 6h of adaptation and could degrade up to 2.1 mM of 4NP. Oxygen consumption studies demonstrated that strain SF1 utilized 1,4-benzoquinone and hydroquinone, as well as 4NP. On partial 16S-rDNA sequence analysis, strain SF1 showed highest homology with the Pseudomonas putida strain based on the FASTA program. Key words: biodegradation, 4-nitrophenol, Pseudomonas strain
Nitroaromatic compounds exist in nature due to their extensive use in the production of dyes, plastics, and explosives; and also as intermediate products from partially transformed insecticides, organophosphorous pesticides, and herbicides5). These chemicals are toxic and mutagenic, and their accumulation results in soil and groundwater pollution. There are various reports on the isolation of microorganisms for degradation of 4-nitrophenol (4NP)1,10). In many instances, an extended lag phase has been reported between the addition of substrate and the onset of growth in the case of degradation of 4NP6). The reports showed degradation of up to 1 mM 4NP (final concentration) as a carbon source in the medium2,8). We report here an isolate, which has a reduced lag period and degrades high concentrations of 4NP as a sole source of carbon. The strain was isolated from contaminated alkaline soil collected at a pharmaceutical waste dumpsite. The ethylacetate extract of the soil on GC-MS analysis gave peaks for chloronitrobenzene, nitrophenols, catechols and a few unidentified peaks, perhaps generated by partial transformations under the environmental conditions. The soil was suspended as slurry in Na-K phosphate buffer pH 7 and used as * Corresponding author; E-mail: [email protected]
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an inoculum for the enrichment of microorganisms. Initially, the enrichment was performed in two-liter capacity flasks containing 500 ml of DLB (diluted Luria broth: up to 10 fold) with 0.35 mM 4NP and 5 g (wet weight) of contaminated soil sample as slurry. The pH of DLB was adjusted to 7±0.2. The enrichment flask was incubated at 30°C with agitation (150 rpm). 4NP was decolorized after 5 days of incubation with an increase in turbidity up to an OD620 nm of 0.6, suggesting that the cells were acclimatized on 4NP as substrate4,9). The total enrichment culture of 500 ml was harvested and subsequently used as a seed for a reactor having 35 l of medium containing 0.5 g l-1 KH2PO4 and 0.35 mM 4NP as carbon and nitrogen sources with continuous aeration at a flow rate of 5 l h-1. The enrichment was continued for two months with regular spiking with 0.35 mM 4NP and 0.5 g l-1 KH2PO4 and a change the color of the medium from yellow to colorless; indicating that 4NP was being degraded by the biomass. Initially, for the first 30 days, complete decolorization was observed after three days of incubation. Subsequently, for 30 days, regular spiking every 24 hours was performed. With time, the 4NP decolorization occurred faster, within 6 hours, suggesting a reduction in the acclimation period11,12). To isolate the microorganisms, the enriched culture was serially diluted with Na-K phosphate buffer (pH 7) and plated out on a min-
QURESHI et al.
imal medium with 4NP (0.35 mM) as a sole carbon source and on DLB plates (pH 7±0.2) with 4NP (0.35 mM). A mineral medium with the following composition (g l-1) was used: Na2HPO4 (5.24), KH2PO4 (2.7), NH4Cl (0.25), CaCl2.2H2O (0.1), MgSO4.7H2O (0.2), and FeSO4.7H2O (0.001): pH 7±0.2. Each dilution of the enrichment culture (10-2 to 10-6) was plated by the spread plate method. The inoculated plates were incubated at 30°C for 20 hours. Colorless zones appeared around the colonies degrading 4NP. Colonies were selected and checked for decolorization in liquid mineral medium containing 4NP (0.35 mM). The isolate SF1 was selected from one of the mineral medium plates containing 4NP. It was maintained on LB plate with 0.07 mM 4NP. SF1 was identified as Pseudomonas by a biochemical method as described earlier3). The isolate is a gram negative rod shaped bacterium, which is highly motile and aerobic, shows positive activity on catalase and oxidase, and is indole test-negative and citrate-positive. The isolate SF1 has also been identified by 16S rDNA sequencing3). In brief, using conserved eubacterial primers, a 1.5 kb fragment was amplified with SF1 total DNA. The amplified fragment was sub-cloned and sequenced in both directions. The sequence data was analyzed using the FASTA program, showed 97.3% homology with Pseudomonas putida (GenBank Accession No D85993). The partial sequence data for the 16S rDNA gene of strain SF1 has been deposited in GenBank under the accession no. AF135269. The degradation experiments were carried out as follows. The cultures were prepared in flasks containing 20 ml of LB medium with 0.07 mM 4NP and were incubated at 30°C for 18 h on a rotary shaker at 150 rpm. The cells were pelleted, washed once with mineral medium, suspended in 20 ml of mineral medium with 0.07 mM 4NP and incubated for 6 h. This step allowed the cells to utilize higher concentrations of 4NP. At this step, also, there was complete decolorization of 4NP. These pre-cultured cells were pelleted and used as an inoculum for all the degradation experiments requiring more than 0.07 mM of 4NP as a sole source of carbon. The experiments were performed in nitrogen-free mineral media with 0.35 mM 4NP and an initial cell density of 0.05 (OD620 nm) to study the growth pattern. The isolate SF1 is capable of utilizing 4NP not only as a carbon and energy source but also as a nitrogen source since the absence of ammonium chloride in the growth medium did not affect its growth. Growth was monitored by cell density at OD620 nm. The disappearance of 4NP and formation of nitrite were measured in the culture as shown in Figure 1. The experiments were performed using specific controls for comparison: one containing SF1 cells without 4NP and an-
Degradation of 4NP by Pseudomonas strain SF1. Symbols: , 4NP; , nitrite; , biomass. Cells were grown in medium with 0.35 mM 4NP as a sole source of carbon and nitrogen. Data used in the figure were derived from three independent experiments and plotted as mean values with standard deviations.
other with 4NP as substrate but without SF1 cells in the media. 4NP levels in the medium were measured by spectrophotometric analysis (Perkin Elmer double beam spectrophotometer, Model Lambda 900) at 420 nm and High Performance Liquid Chromatography (Perkin Elmer series 200 LC system) at 420 nm. Separations were performed on a C18 column (Merck, Lichrosphere-100, RP-18, and 5 mm with acetonitrile: water (70 : 30 v/v) as the mobile phase at a flow rate of 0.5 ml min-1. Nitrite concentrations in the supernatant were assayed by using the nitrite ion detection kit (Aquaquant, E. Merck, Darmstadt, Germany) according to the instructions of the supplier. A standard curve was made using KNO2 as stock solution in deionized (resistance, 18 MW) water. Biodegradation experiments were carried out with different concentrations of 4NP in the medium. Figure 2 shows the concentration, 0.7 mM, 1.4 mM or 2.1 mM, of 4NP used as a substrate in the medium. For the experiments with higher concentrations of 4NP (0.7– 2.1 mM), the initial cell density was 0.5 (OD620 nm). The results demonstrated that although SF1 could degrade high concentrations of 4NP, i.e. 2.1 mM, the rate of degradation was slow with 95% or more removal in 20–25 h. For the oxygen uptake assays, the cells were grown on 4NP (1 mM) and citrate (1 mM) as a substrate. The cells were pelleted by centrifugation at 15°C and 5000 rpm, and suspended in 20 mM Na-K phosphate buffer pH 7. The oxygen consumption was measured with a Digital Oxygen sys-
Isolation and characterization of Pseudomonas strain
51 Table 1. Rate of oxygen consumption by whole cells of Pseudomonas strain SF1 towards different concentrations of possible intermediate compounds of the 4NP pathway.
Oxygen uptake (nmol O2 min-1 mg-1 dry wt cells) Cells grown on 4NP
Cells grown on Citrate
Hydroquinone Fig. 2. Degradation of 4NP by Pseudomonas strains SF1. Cells were grown in medium with different concentrations viz., , 0.7 mM; , 1.4 mM; , 2.1 mM of 4NP as a sole source of carbon. Data used in the figure were derived from three independent experiments and plotted as mean values with standard deviations.
tem (Model 10, Rank Brothers, Bottisham, UK) at 30°C. The reaction mixture contained cells at an OD620 nm of 1.5 in a total volume of 3 ml of Na-K phosphate buffer per reaction. The endogenous oxygen uptake rate was recorded for the reaction. Once the rate had stabilized, the reaction was initiated with addition of substrates. For each set of reactions, fresh cells were used. Each reaction was continued for 3 minutes after the oxygen uptake rate had stabilized and assay substrate was added. The uptake rate was expressed as nmol of oxygen consumed per min per mg dry weight of the cells7) . Dry weight was determined by drying cell pellets at 70°C until a constant weight was obtained. The rates were corrected for endogenous oxygen consumption (0.89 nmol O2 min-1mg-1 dry wt cells). The results on oxygen uptake are shown in Table 1. The oxygen uptake rates of cells grown on 4NP as well as citrate were similar when 4NP was used as the assay substrate. The maximum oxygen uptake rate was observed for hydroquinone (0.5 mM) with 4NP-exposed cells i.e., 76.6 nmol min-1mg-1 dry weight of cells. However, with 4nitrocatechol as a substrate, cells showed the lowest oxygen uptake rates indicating that 4-nitrocatechol does not serve as a preferred substrate for strain SF1. The oxygen consumption rates towards 4NP (0.05 mM) in cells grown on 4NP and citrate seem to be relatively similar, indicating constitutive expression of the 4NP pathway in the isolate SF1. The
The results are the mean±SD of triplicate determinants. Cells were grown on 4NP or citrate as a sole source of carbon at 1 mM in mineral medium.
oxygen consumption assays were carried out using cells grown as seed culture, hence, no lag period in the analysis was observed. The results suggested that strain SF1 uses the hydroquinone pathway for degradation, which was confirmed by GC-MS analysis, as reported earlier3). The isolate SF1 showed varied degradative potential for 4NP as a sole source of carbon and energy as compared to several other strains, such as Moraxella sp. isolated from activated sludge10), Arthrobacter aurescens isolated from soil1) and Pseudomonas cepacia isolated from pesticidecontaminated soil8). This suggested that in the case of soil from a pharmaceutical dumpsite, where there was a direct load of 4NP on the system, the environment selected for a strain capable of degrading a higher concentration of 4NP. The isolation of strain SF1, which could degrade up to 2.1 mM of 4NP, can be attributed to the enrichment strategy. The protocol involved repeatedly providing the substrate and then starving the cells to maximize the degradative capacity of the system. Since the culture was spiked regularly with 4NP, the population that was still expressing the enzymes grew immediately. This yielded a strain with a
strong degradative phenotype.
References 1) Hanne, L.F., L.L. Kirk, S.M. Appel, A.D. Narayan and K.K. Bains. 1993. Degradation and induction specificity in actinomycetes that degrade p-nitrophenol. Appl. Environ. Microbiol. 59: 3505–3508. 2) Jain, R.K., J.H. Dreisbach and J.C. Spain. 1994. Biodegradation of p-nitrophenol via 1,2,4-Benzenetriol by an Arthrobacter sp. Appl. Environ. Microbiol. 60: 3030–3032. 3) Kutty, R., H.J. Purohit and P. Khanna. 2000. Isolation and Characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol. Can. J. Microbiol. 46: 211–217. 4) Linkfield, T. G., J.M. Suflita and J.M. Tiedje. 1989. Characterisation of the acclimation period before anaerobic dehalogenation of halobenzoates. Appl. Environ. Microbiol. 55: 2773–2778. 5) Nelson, L.M. 1982. Biologically induced hydrolysis of parathion in soil: isolation of hydrolysing bacteria. Soil. Bio. Biochem. 14: 219–222. 6) Nishino, S.F. and J.C. Spain. 1993. Cell density dependent adaptation of Pseudomonas putida to biodegradation of p-nitrophenol.
QURESHI et al. Environ. Sci. Technol. 27: 489–494. 7) Nicholas, C.M. and W.A. Venables. 1986. Adaptation of Pseudomonas putida mt-2 to growth on Aromatic amines. J. Gen. Microbiol. 132: 2209–2218. 8) Prakash, D., A. Chauhan and R.K. Jain. 1996. Plasmid encoded degradation of p-nitrophenol by Pseudomonas cepacia. Biochem. Biophy. Res. Comm. 224: 375–338. 9) Spain, J.C. and P.A. Van Veld. 1983. Adaptation of natural microbial communities to degradation of xenobiotic compounds: Effects of Concentration, Exposure, Time, Inoculum and Chemical Structure. Appl. Environ. Microbiol. 45: 428–435. 10) Spain, J.C. and D.T. Gibson. 1991. Pathway for biodegradation of p-nitrophenol in a Moraxella sp. Appl. Environ. Microbiol. 57: 812–817. 11) Wiggins, B.A. and M. Alexander. 1988. Role of chemical concentration and second carbon sources in acclimation of microbial communities for biodegradation. Appl. Environ. Microbiol. 54: 2803–2807. 12) Wiggins, B.A., S.H. Jones and M. Alexander. 1987. Explanations for the acclimation period preceding the mineralization of organic chemicals in aquatic environments. Appl. Environ. Microbiol. 53: 791–796.