Environ. Sci. Technol. 2005, 39, 3330-3337
Pot and Field Studies on Bioremediation of p-Nitrophenol Contaminated Soil Using Arthrobacter protophormiae RKJ100 SUMEET LABANA, GUNJAN PANDEY, DEBARATI PAUL, NARINDER K. SHARMA, APARAJITA BASU, AND RAKESH K. JAIN* Institute of Microbial Technology, Sector 39-A, Chandigarh-160036, India
Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. Bioremediation of PNP-containing soil was first carried out in pots using immobilized and free cells of Arthrobacter protophormiae RKJ100 in order to ascertain the role of a suitable carrier material. Results showed that stability of the introduced strain was enhanced upon immobilization and that the rate of PNP depletion decreased with increasing depth of soil. Small-scale field studies (in one square meter plots) were then conducted in which PNP-contaminated soil from an agricultural field was bioaugmented with strain RKJ100 under natural environmental conditions. PNP was totally depleted in 5 days by immobilized cells, whereas free cells were able to deplete 75% of PNP in the same time period. The fate of the released strain as monitored by plate counts, hybridization studies, and realtime polymerase chain reaction revealed fairly stable population of the cells upon immobilization on corncob powder throughout the period of study.
Introduction Nitroaromatic compounds (NACs) are widely used in a range of industries and are generally recalcitrant to biological treatment. p-Nitrophenol (PNP), an important member of the NAC family, is used for the manufacture of pesticides, dyestuffs, pharmaceuticals, and as a fungicide to protect leather from fungal mould. PNP is released into the soil as a result of hydrolysis of the organophosphate pesticides parathion, methyl parathion (1), and herbicides dinoseb and dinitrocresol (2, 3), particularly in developing countries where there is widespread use of these pesticides. From the soil, PNP can leach into the groundwater resources thus contaminating them. Even in a developed country such as the U.S.A., illegal commercial application of methyl parathion in hundreds of residences and businesses across several states a few years ago resulted in exposure of residents to high levels of this pesticide (4-7). Urinary PNP levels in these individuals were found to be well in excess of the general population prompting the authorities to relocate the affected residents and decontaminate their homes (4-7). Because of its high toxicity, it is listed as a priority pollutant by the United States Environmental Protection Agency (USEPA) (8). * Corresponding author phone: +91-172-2690713/2695215; fax: +91-172-2690585/2690632; e-mail:
[email protected]. 3330
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Traditionally, contaminated sites are treated by physical or chemical methods which are often difficult to execute, costly, and not environmentally friendly. Bioremediation, an emerging alternative technology for restoration of contaminated environments, includes bioaugmentation which involves the addition of exogenous micro-organisms to a contaminated site. However, for successful bioaugmentation, it is important to determine the environmental conditions that influence survival and biodegradative activity of introduced organisms. Although several PNP degrading strains have been isolated (9-11), very few studies have been carried out on PNP degradation in soils (12, 13). Since biodegradation and fate of organic pollutants can best be predicted by laboratory scale test systems, bioaugmentation was carried out in pots containing 3.5 kg of soil in which immobilized cells of strain RKJ100 were added and then in small (one square meter) field plots. The optimization of the process during scale-up provided valuable information for field implementation on a larger scale. Quantification of microbial populations is important to study their survival in soil, particularly using molecular methods (14-16). In this study colony hybridization, slot-blot, and real-time polymerase chain reaction (PCR) analyses were performed for rapid and specific detection of this strain in soil samples collected from all plots at different time intervals. Simultaneously, depletion of PNP was quantified in these samples to determine the effectiveness of the bioremediation strategy.
Experimental Section Organism and Culture Conditions. Arthrobacter protophormiae RKJ100 used for this study and culture conditions has been described previously (17). Immobilization of Bacterial Cells. Corncob powder was used as a carrier material to immobilize bacteria owing to its properties of being highly absorbent, granular, high volume low weight, biodegradable, and inexpensive. The major components of corncob are cellulose, hemicellulose, and lignin with small amounts of protein, starch, and lipids. Locally available corncobs were ground in a milling machine, air-dried, and sieved through a 2-mm mesh to obtain a particle size of approximately 0.5-2 mm. Cells of strain RKJ100 grown in 1 L of minimal medium (MM) (17) containing 2% sodium succinate and 0.5 mM PNP up to an OD of 1 were harvested, washed with phosphate buffer, resuspended in 200 mL MM to get approximately 6 × 1011 cells, and mixed with 300 g of corncob powder. Additional water was added to get cell a suspension:corncob ratio of 6:1 (v/w) which allowed homogeneous mixing of bacterial cells with the corncob powder. Stability of Bacterial Cells Immobilized on Corncob Powder. To check the stability of immobilized cells they were mixed with corncob powder (autoclaved three times) as explained above, divided into aliquots, and kept at three different temperatures, viz., room temperature (RT), 4 °C, and -20 °C. The viability of cells was checked at 1 week, 1 month, 2 months, 3 months, and 6 months by colony-forming unit (CFU) enumeration. For this, 50 mg of corncob powder containing immobilized cells was suspended in 500 µL of sterile normal saline (0.9% NaCl), vortexed vigorously for 2 min, and allowed (the corncob powder) to settle down. The supernatant was diluted serially and plated on nutrient agar plates. Pot Studies. Nine pots, each pot measuring 20 cm in length and having top and bottom diameters of 20 and 12.5 cm, respectively, were filled with 3.5 kg of soil to which 70 ppm PNP was added exogenously. For this, air-dried soil samples 10.1021/es0489801 CCC: $30.25
2005 American Chemical Society Published on Web 03/19/2005
FIGURE 1. The experimental site for bioremediation of PNPcontaminated soil. This site was located at 76.54 E longitude and 30.42 N latitude. Dimensions of the experimental plots were 1 m × 1 m × 30 cm, separated from each other by a distance of 2 m, and containing approximately 300 kg of contaminated soil. were sieved (2 mm) and aqueous PNP solution was added to the soil to form a thick slurry (to give final PNP concentration of 70 ppm). The slurry was air-dried at 40-45 °C for 3-4 days, and the dried soil was pulverized prior to being used in the pots. The pots were then divided into three sets of three pots containing soil as described below: Set A: Soil Mixed with Bacteria. Twenty milliliters MM containing cells of strain RKJ100 was mixed with 3.5 kg of soil such that there were approximately 2 × 106 bacterial cells per gram of soil. Set B: Soil Mixed with Bacteria Immobilized on Carrier Material. Twenty milliliters of cell suspension premixed with corncob powder (please see earlier) was further mixed with 3.5 kg of soil to give approximately 2 × 106 cells/g soil. Set C: Soil without Corncob Powder or Bacteria. PNPamended soil was added to the pots without cells of strain RKJ100 or carrier material. This served as the control. The moisture level of soil in all the pots was maintained at 40-50% of the water holding capacity of soil throughout the period of the experiment by sprinkling tap water periodically whenever necessary. The pots were incubated at 30 °C for 30 days. Five grams of soil samples were collected from each pot in triplicate from three different depths of the soil, i.e., 5, 10-15, and 15-20 cm using a hollow pipe of ∼1 in. diameter. Samples were collected every 24 h until 7 days and thereafter at 10, 15, 20, 25, and 30 days. Field Studies. An agricultural site sprayed with parathion/ methyl parathion for several years and therefore contaminated with PNP (initial PNP level of ∼9 ppm) was identified, and soil from this site was used for carrying out small-scale field studies. The soil contained clay 42%, silt 20%, sand 38%, organic carbon 0.84%, phosphorus/ha 34.4 kg, and potash/ ha 347 kg with a pH of 7.2 (18). Four plots measuring 1 m × 1 m and 30 cm deep were prepared in an isolated area. Each plot was separated from the other by a distance of 2 m and each, was lined on all the sides by a plastic sheet (Figure 1) and the whole experimental area was fenced. Agricultural soil contaminated with PNP was collected, transported to the site of study, and added to the plots as described below. Plot A: Soil mixed with inoculum immobilized on corncob powder. Cells of strain RKJ100 (∼6 × 1011) suspended in 2 L of MM were mixed with 300 g of corncob powder and subsequently with ∼5 kg of soil. This inoculated soil was then thoroughly mixed with the rest of the soil (∼300 kg in each plot) with the help of a cement mixer.
Plot B: Soil mixed with the free cells of strain RKJ100 only suspended in 2 L of MM as indicated above. Plot C: Soil mixed with 300 g of corncob powder only suspended in MM as indicated above. Plot D: Soil mixed with MM only as indicated above. The moisture level of the soil was adjusted to 40-50% of its water holding capacity of the soil and maintained throughout the period of study by irrigating with water the plots when required. The ambient temperature was monitored daily throughout the period of study in order to determine the changes in the weather. Collection of Samples. Soil samples were collected from a depth of ∼10 cm using a hollow pipe of ∼1 in. diameter from three different random positions, pooled, mixed thoroughly, and used for further analysis. Three such samples were collected from each plot. Sampling was done at the following time points: 0, 1, 2, 3, 5, 7, 10, 20, and 30 days. Analysis of PNP Concentration. Soil samples were extracted for PNP in both pot and field studies and analyzed for residual PNP by high-performance liquid chromatography (HPLC). For this, 1 g of soil sample was suspended in 10 mL of 5% NaOH, vortexed for 10 min, and centrifuged at 1500 rpm for 10 min. The supernatant was acidified to pH 2.0 with HCl and extracted with double the volume of ethyl acetate, and the extract was evaporated to dryness under vacuum. This exercise was repeated in triplicate, and the residues were then dissolved in 1 mL of methanol and quantified by HPLC using a Waters 600 model equipped with a Waters 996 photodiode array detector operating at 315 nm. Separation was carried out with a Waters Spherisorb 5 µm C8 column, and the mobile phase was acetonitrile:water (20:80 v/v) containing 0.1% trifluoroacetic acid with a flow rate of 1.0 mL/min. Monitoring of Bacteria in the Field. (i) Selective Plate Counting Method. The unique properties of strain RKJ100 to utilize PNP, o-nitrobenzoate (ONB), p-hydroxybenzoate (PHB), and nitrocatechol (NC) as the sole source of carbon and energy, as well as the ability to tolerate cobalt (19), was exploited to monitor the fate of this strain after it was added to soil. Soil samples (1 g) were suspended in normal saline, serially diluted, and plated on nutrient agar plates. Suspected colonies of RKJ100 were picked and patched on selective plates of ONB (15 mM), PHB (15 mM), NC (0.2 mM), PNP (0.4 mM), and ONB (15 mM) + Co (250 µg/mL). Colonies that grew on all the selective plates were calculated since no other organism from contaminated soil was able to grow on all these compounds. (ii) Slot-Blot and Colony Hybridization. Bacterial genomic DNA from strain RKJ100 was extracted from 1 mL of culture using the DNeasy Tissue Kit (Qiagen, Valencia, CA) following the instructions of the manufacturer. DNA was eluted with 100 µL of elution buffer from the same kit. Total soil DNA was extracted from samples collected at different time intervals from the four plots using a FastPrep DNA isolation kit according to the manufacturer’s instructions (BIO101). The DNA was further purified by gel extraction using a Qiaquick gel extraction kit (Qiagen GmbH, Hilden, Germany). The concentration of extracted DNA was quantified spectrophotometrically, and approximately 500 ng of DNA was obtained from 1 g of soil. To quantify strain RKJ100 in soil using hybridization techniques, a specific probe was designed that hybridized specifically to genomic DNA of strain RKJ100. This probe, a 300-bp segment of the “benzenetriol dioxygenase gene” involved in the degradation of some nitroaromatic compounds (unpublished data), was amplified using a set of primers by PCR. The reaction mixture consisted of 50 ng of genomic DNA of RKJ100, 1 U of Taq DNA polymerase (New England Biolabs, MA), 1X buffer (10 mM Tris-HCl [pH 9.0]), VOL. 39, NO. 9, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Gel photograph showing the 300-bp fragment of benzenetriol dioxygenase amplified using gene-specific primers. Lane M depicts the 100-bp marker; Lanes 1 and 2 shows no amplification from related strains of RKJ100; lane 3 shows the amplication of the 300-bp fragment of benzenetriol dioxygenase gene from strain RKJ100. 1.5 mM MgCl2, 10 mM dNTPs, and 100 ng of each primer (BtD-F: 5′ acc gtc ctc gga ccg ttc 3′; BtD-R: 5′ cca tca gaa gga atc gga ta 3′). Initial DNA denaturation and enzyme activation steps were performed at 95 °C for 30 s, annealing at 50 °C for 1 min, extension at 72 °C for 1 min, and a final extension for 5 min at 72 °C in a Personal Cycler (Eppendorf, Germany). The PCR product was visualized by gel electrophoresis using 2% agarose (Figure 2). Total genomic DNA isolated from the plot soil was blotted onto a Hybond N+ membrane using PR648 slot-blot filtration manifolds (Amersham Pharmacia Biotech., Inc., USA) and probed with the 300-bp fragment of benzenetriol gene. The membrane was then washed two times at RT in 2X SSC (diluted from 20X stock [3 M sodium chloride, 0.3 M sodium citrate pH 7.0]) followed by two washes at 50 °C in 0.1X SSC (20).
The specificity of the probe was determined by hybridization with some of the other strains of Arthrobacter (data not shown) and also with DNA from control plots. To quantify DNA from any source a standard curve based on known concentrations of DNA is generally used (21). A standard curve was constructed by plotting counts/unit area generated by known amounts of DNA using molecular imager FX system (BioRad, Hercules, CA) and the Quantity One Software (BioRad, Hercules, CA). The DNA concentration of strain RKJ100 from soil samples was determined from the standard curve. For colony hybridization, 200 mg of soil was taken from each soil sample and suspended in 1 mL of MM by vortexing. After serial dilutions, 50 µL was plated on nutrient agar plates and incubated at 30 °C overnight. The colonies were transferred onto a Hybond N+ membrane sized according to the Petri plate to be probed. Three spots were marked on both the membrane and plate for orientation. The nylon membrane was then treated sequentially with Sol A (10% SDS), Sol B (0.5 N NaOH, 1.5 M NaCl), Sol C (0.5 M Tris-Cl, 1.5 M NaCl, pH 7.4), and Sol D (2X SSC) for 15 min each. The membrane was then UV-cross-linked, hybridized with the 300-bp fragment of benzenetriol dioxygenase gene, and washed as mentioned above. (iii) Quantitative Real-Time PCR/TaqMan PCR. This technique requires an internal fluorescence-labeled probe that hybridizes to the target sequence and measures fluorescence emitted continuously during the amplification. Fluor intensity was correlated with the number of accumulated PCR amplicons. The universal primers 357F and 518R were used to amplify the variable region (V3) of bacterial 16S rRNA gene. Probe P1 was designed by aligning 16S rRNA gene sequences of several Arthrobacter spp. (obtained from GenBank) using ClustalX, followed by the construction of a dendrogram (Figure 3) showing the phylogenetic relatedness among them. The 16S rRNA gene sequences of strain RKJ100 and three closely related strains were re-aligned in ClustalX, and based on the mismatches among their nucleotide sequences in the region 357-518 a strain-specific probe (P1) was constructed. Strain RKJ100 was quantified by real-time PCR using a probe labeled with FAM at the 5′ end and TAMRA
FIGURE 3. Dendrogram showing the phylogenetic relatedness of strain RKJ100 with other strains belonging to Arthrobacter spp. The nucleotide sequences were aligned in ClustalX, and the dendrogram was constructed using NJ Plot program. Bootstrap values have been indicated at the nodes. 3332
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FIGURE 4. Stability of strain RKJ100 cells as a function of time during different storage conditions: (a) cells immobilized on corncob powder as a carrier material; (b) free cells. The viability was checked at three different temperatures, i.e., room temperature, 4 °C, and -20 °C. at the 3′ end (P1: 5′ aag aag ccc ttc ggg gtg acg g 3′) and primers to amplify the V3 region (161 bp) of 16S rRNA gene (357F: 5′ ctc cta cgg gag gca gca g 3′; 518R: 5′ gta tta ccg cgg ctg ctg g 3′). The primers and the probe used in this study were synthesized by Biobasics, Canada. The specificity of the probe was further tested using other strains of Arthrobacter spp. and on soil samples of control plots by real-time PCR. Real-time PCR was performed in a 25-µL reaction mixture that consisted of template DNA (6-78 ng from standard and 100 ng from soil DNA), 12.5 µL of Platinum Quantitative PCR SuperMix UDG (a 2X-concentrated mixture of 40 mM Tris HCl, 100 mM KCl, 6 mM MgCl2, 400 µM dATP, 400 µM dCTP, 400 µM dGTP, 800 µM dUTP, 60 U/mL PE Taq DNA Polymerase, 40 U/mL UDG), primers (250 nM each), and probe P1 (300 nM). The PCR protocol for amplification of the 150 bp fragment of 16S rRNA gene was as follows: 5 min at 95 °C, 30 cycles consisting of 15 s at 95 °C, 1 min at 50 °C, and 2 min at 72 °C. Reactions were performed in an Icycler (Biorad, CA). The parameter Ct (threshold cycle) is the cycle number at which the fluorescence emission crosses a threshold within the logarithmic increase phase. Data were analyzed using Icycler iQReal-time PCR Detection System software (Version 3.0A). The Ct values were obtained for the samples containing 100 ng of soil DNA isolated at different time points from the four plots as template DNA. Consequently, genomic DNA of strain RKJ100 was calculated using a standard curve drawn by plotting the Ct values vs known concentrations of genomic DNA of strain RKJ100. Thereafter, the amount of genomic DNA of strain RKJ100 was calculated per reaction and per gram of soil (approximately 500 ng of genomic DNA was isolated from 1 g soil). Nucleotide Sequences and Accession Numbers. 16S rRNA gene sequences of Arthrobacter spp. obtained from GenBank are AY773328, AY773327, AY833102, AJ315071, AJ315070, AJ315069, AJ316305, and the GenBank accession number of 16S rRNA gene sequence of strain RKJ100 is AY577525. Statistical Analysis. Data were analyzed using the Student’s t test to determine the significance of the difference between the means. P values of
