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Molecular and biochemical profiling of pentachlorophenol utilizing bacteria from pulp and paper mill effluent irrigated soil in Northern India.

Discharge of pulp and paper mill include many environmental pollutants among which Chlorophenols (CPs) are major ones (Chandra et al. 2009), which are generated as the by-products when chlorine is used for bleaching of pulp and as water disinfectant. Pentachlorophenol (PCP) and its sodium salt have been widely used as wood and leather preservative owing to their toxic effect on bacteria, mould, algae and fungi (Kaoa et al. 2004). Its frequent and widespread use has led to contamination of aquatic and terrestrial ecosystems (Jensen 1996).

Pulp paper industries are the sixth largest effluent generating industries of the world (Ugurlu et al. 2007). Since early fifties the number of paper pulp mills in India has increased from 17 to more than 406 in 2008, with simultaneous increase in paper production from 0.13 to 1.9 million tons per annum. Paper mill generates as low as 1.5 [m.sup.3] of effluent per ton to as high as 60 [m.sup.3] per tonne of paper produced (Asghar et al. 2007). The safe permissible limit of PCP in water is 0.30 ig [l.sup.-1]. However, in our country, the large units of pulp and paper mills discharge their effluent, having residual PCP in high concentrations (> 80 mg [l.sup.-1] effluent), in local water ways.

Pulp and paper mill effluent irrigation to crops is a cheap and attractive option compared to discharge of this effluent into natural waterways (Muthukumar and Vediyappan 2010). Local farmers irrigate their agricultural fields on regularly basis from these water channels and thereby contaminating them with PCP. Being highly chlorinated, PCP is expected to be recalcitrant to aerobic biodegradation as in general, aromatic compounds with higher amounts of chlorine are more resistant to biodegradation (Anandarajah et al. 2000). Due to persistence of PCP in soil and water environments, both the European and US Environment Protection Agencies have classified PCP as a 'priority pollutant' and have recommended restricted use to minimize its further accumulation and to circumvent toxicity of the ecosystem.

Biological treatment of PCP attracts more attention than physical and chemical methods, because a variety of microorganisms are known to utilize it as their sole carbon source and the reaction products are [Cl.sup.-] ions, C[O.sub.2] and biomass. Several microorganisms possessing the ability to metabolize various industrial pollutants have been isolated from the environment (Tripathi et al. 2011). Aerobic PCP degradation by mixed microbial cultures is important since most PCP-contaminated sites are surface soil or sediments which may support growth and activity of aerobic microbial consortia. Bioremediation protocols for soil contaminated with high concentration of PCP can be achieved only by using efficient indigenous PCP degrading microorganisms.

We analysed PCP utilizing bacteria in agricultural soils irrigated with pulp and paper mill effluent discharged from Century Pulp and Paper mill, LalKuan, Uttrakhand, India. Chlorophenol-degrading bacterial isolates were biochemically characterized and identified by partial 16S rRNA gene sequencing.

MATERIALS AND METHODS

Field site and sample collection

The effluent from the Century Pulp and Paper mill (CPM), LalKuan, Uttrakhand, India (79[degrees]100E longitude and 29[degrees]30N latitude), which is discharged in local waterways is being used as source of irrigation to the sugarcane fields since last 25 years. A field was selected from this site for sampling. A total of 5 composite soil samples were collected at 0-15 cm depth during the month of March using a soil auger. Each composite sample was made of five sub samples, collected from along the zigzag paths (Zigzag sampling) to account for the randomness. The collected soil samples were properly labelled and stored in polythene bags and transported to the laboratory in the insulated container at 4[degrees]C.

Soil physico-chemical analysis

The soil samples were analyzed for various physical and chemical characteristics such as texture, pH, electrical conductivity (EC), organic carbon (OC), available N, Olsen P and exchangeable K as per methods described by Page et al. (1982). Residual PCP in soil samples was estimated using HPLC as described.

Enrichment of soil samples with PCP

The enrichment of PCP degrading bacteria was carried out as per method described by Karn et al. (2010). From each of the soil samples, 10 gm of soil was added in 90 ml of mineral salt (MS) medium having PCP @ 50 [micro]g [ml.sup.-1] (Dams et al., 2007). The composition of MSM (in gm[L.sup.-1]) was K[H.sub.2]P[O.sub.4], 0.68; [K.sub.2]HP[O.sub.4], 1.73; MgS[O.sub.4].7[H.sub.2]O, 0.1; Ca[Cl.sub.2], 2[H.sub.2]O, 0.02; N[H.sub.4]S[O.sub.4], 0.017; and 1 ml of trace metal solution which includes (in mg[L.sup.-1]) FeS[O.sub.4], 7[H.sub.2]O, 200; ZnS[O.sub.4], 7[H.sub.2]O, 10.0; MnS[O.sub.4]. 4[H.sub.2]O, 3.0; NiCl.6[H.sub.2]O, 2.0; [H.sub.3]B[O.sub.3], 30.0; Co[Cl.sub.2].6[H.sub.2]O, 1.0; Zn[Cl.sub.2], 10.0; and EDTA, 2.5. PCP was added to the medium after autoclaving. The pH was adjusted to 7.3 [+ or -] 0.2 prior to autoclaving.

The flasks were incubated for 7 days at 30[degrees]C on rotary shaker at 200 rpm. After 7 days of incubation, 10 ml soil suspension was taken from the flask and transferred aseptically to the flask containing fresh MS medium having PCP @ 50 pg [ml.sup.-1] and again incubated for 7 days. This step was repeated for 4 more weeks.

Isolation of PCP degrading bacteria

After 6 weeks of enrichment, the potential PCP degrading bacterial strains were isolated by serial dilution technique on MS medium containing 50 [micro]g PCP [ml.sup.-1]. From each of the sample, single colonies were purified by repeated streaking. All the purified isolates were morphologically characterized based on their colony shape, size, colour, pigmentation, margin and elevation after 48 h of incubation (Seeley and VanDenmark, 1972). Representative morphotypes were purified, sub-cultured and maintained on MS medium agar slants having PCP @ 50 [micro]g [ml.sup.-1].

Biochemical characterization of the selected isolates

The biochemical characterization of the 22 selected isolates was done in accordance with Bergey's Manual of Systematic Bacteriology. Each pure culture was tested for Gram reaction. The catalase activity was determined based on formation of bubbles in the presence of 3% [H.sub.2][O.sub.2] solution. Oxidase was performed on paper discs using tetramethyl-p-phenylenediamine. Nitrate reductase was detected on nitrate agar plates with methyl green as an indicator. Urease test was carried out by method described by Christensen (1946). Urea broth containing phenol red indicator was inoculated with test cultures and incubated for 5-6 days at 37[degrees]C. Presence of yellow color indicated the presence of urease. Starch hydrolysis was demonstrated from clearing zones formed around the colonies grown on starch containing agar.

Growth of selected bacterial isolates at different concentrations of PCP

The biochemically characterized isolates were further screened for their ability to grow at 100, 300, 500 and 700 ppm of PCP in MS broth. Desired concentration of filter sterilized PCP from its stock solution was added to sterile MS broth in order to get final concentration of 100, 300, 500 and 700 ppm. Three replications were maintained for each isolate. The flasks were inoculated with different bacterial isolates individually and incubated at 30[degrees]C at 200 rpm for 48 h. After incubation, pH of the broth was measured. Growth of the cells was measured in terms of total protein. The total protein of the bacterial isolates was estimated by Bradford's method (Bradford, 1976), which involves the binding of Coomassie Brilliant Blue G-250 dye to proteins. When the dye binds to protein, it is converted to a stable unprotonated blue form and is detected at 595 nm using a spectrophotometer.

16S rRNA gene sequencing and phylogenetic analysis

Genomic DNA extraction from the isolates showing growth above 100 ppm PCP was carried out by modified method of Charles and Nester, 1993. Briefly, 1.5 ml overnight grown cultures in TY broth were centrifuged at 12000 rpm for 10 min and washed with 1.5 mL of 0.85% NaCl. Washed pellet was suspended in 0.4 mL Tris-EDTA buffer ([T.sub.10][E.sub.25]). Cell Lysis was done by adding 20 mL of 25% SDS, 50 mL of 1% lysozyme and 50 mL of 5M NaCl followed by incubation at 68[degrees]C for 30 min in a circulatory water bath. Proteins were precipitated by 260 mL of 7.5 M ammonium acetate solution followed by centrifugation at 12000 rpm for 10 min. Supernatant was pipetted out in fresh sterile microfuge tube in which 1mL RNase (4 mg [mL.sup.-1]) was added followed by Incubation at 37[degrees]C for 20 min. Equal volume of chloroform was added and RNA was precipitated by centrifugation for 1 min at 12000 rpm. The top layer containing total cell DNA was pipetted out and precipitated by adding 0.8 vol. of isopropanol followed by incubation on ice for 30 min and pelleted by centrifuging at 10000 rpm for 15 mins. DNA was further washed with 0.5 mL of 70% ethanol and spun down at 10000 rpm for 1 min. Traces of ethanol were removed by air drying the tubes in inverted position. Pure DNA sample was then suspended in 20 mL Tris-EDTA buffer ([T.sub.10][E.sub.1]) and stored at 4[degrees]C for further use.

The gene encoding 16S rRNA was amplified by PCR using the pair of universal primers pA (5'-AGA GTT TGA TCC TGG CTC AG-3') and pH (5'-AAG GAG GTG ATC CAG CCG CA-3') and conditions described in Edwards et al. (1989). 16S rRNA gene was used as a template in cycle sequencing reactions with fluorescent dye-labelled terminators (Big Dye, Applied Biosystems). Both primers pA and pH were used for sequencing and run in 3130xl ABI prism automated DNA sequencer. All the sequences were compared with 16S rRNA gene sequences available in the GenBank databases by BLASTn search. Identification to the species level was determined based on 16S rRNA gene sequence similarity (>97%) with that of a prototype strain sequence. Multiple sequence alignment of approx 1500-bp sequences was performed using CLUSTAL W, version 1.8. A phylogenetic tree was constructed using the neighbor-joining method. Tree topologies were evaluated through bootstrap analysis of 1,000 data sets by MEGA 4.0 package.

Data Analysis

Statistical analyses of the data was performed using STATISTICA 10. Analysis of variance and separation of means by least significant differences were performed by using the general linear models (GLM). Unless indicated otherwise, differences were considered only when significant at P = 0.05.

RESULTS AND DISCUSSION

Physico-chemical characteristics of soil samples

The soil was sandy loam in texture with alkaline pH and electrical conductivity of 0.73 dS[m.sup.-1] (Table 1). Soil was having moderate organic carbon content (0.95%) and good in available N (64.85 kg [ha.sup.-1]) and extractable K (130.62 kg [ha.sup.-1]) content but poor in P content (16.86 kg [ha.sup.-1]). Significant amount of residual PCP was present in soil (113.34 mg [Kg.sup.-1]) reflecting the toxic levels of PCP in soil. There is no prescribed set limits for PCP in soil, however, The United States Environmental Protection Agency (EPA) has registered PCP in the list of priority of pollutants and the safe permissible limits of PCP in water is 0.30 [micro]g [L.sup.-1] (US EPA, 1999). The major source of PCP in agricultural soil at the farmer's field at Lal Kuan, Uttrakhand is due to irrigation with water containing effluent discharged from Century pulp and paper mill, Lal Kuan. The PCP is generated as by-product due to bleaching of pulp with chlorine (Vallecillo et al, 1999) and is released with effluent in environment.

Isolation of bacterial isolates and their morphological characterization

By enrichment of all the five soil samples with mineral salt medium containing 50 ppm of PCP as sole carbon source, 188 isolates were selected for morphological characterization. These isolates were purified to single colony for morphological characterization. Based on morphological characterization, 22 isolates were selected for further studies (Table 2). All the isolates were found to be rod shaped except LK4 which was cocci. Most of the colonies were whitish in colour, however, variation in white colour was observed from dull, creamy white to off-white. Two colonies were shiny with blackish shade. Margins of colonies varied from circular, irregular to punctiform with entire, curled and undulated margin. Some of colonies were pigmented. The margins of colonies were either flat or raised.

Biochemical Characterization of selected isolates

All the 22 isolates were biochemically characterized and results are shown in table 3. All the isolates were found to be Gram negative except LK 156 which showed variable Gram reaction. Similarly, all the isolates were oxidase positive except LK 156 which showed variable oxidase test results and LK 188 which was oxidase negative. All the isolate were catalase positive and only 12 isolates viz. LK 39, 41, 43, 47, 51, 54, 59, 60, 72, 128, 142 and 147 were urease positive. Isolates LK 1, 4, 5, 23, 32, 59, 81, 124, 141, 147 and 188 were able to carry out starch hydrolysis. Nitrate reduction ability was found in isolates LK 1, 39, 41, 43, 47, 51, 54, 60, 72, 124, 142, 147, 150, 188. Based on morphological and biochemical characterization most of the isolates showed resemblance with Pseudomonas sp. Based on morphological and biochemical characterization PCP utilizing bacteria was also identified by Shukla et al., (2001),Sharma and Thakur, (2008) and Tewari et al. (2011). Utilization of PCP in form of sole source of carbon by Pseudomonas sp and Arthrobacter sp was reported by Shukla et al. (2001) and Sharma and Thakur (2002). Sharma and Thakur (2008) characterized the Pseudomonas sp from paper mill and studied the potency of the isolated strains for PCP reduction in sequential bioreactor.

Growth of bacterial isolates at different concentrations of PCP

All the 22 isolates were further screened for their ability to grow at 100, 300, 500 and 700 ppm of PCP in MS broth. Growth was measured in terms of protein per ml of broth. All the 22 isolates could grow from 100 to 700 ppm of PCP in the MS broth, however, low growth was observed at 700 ppm (Table 4).

Significant variation was observed in growth as total protein among isolates at all the concentrations of PCP after 48 h of incubation. Out of 22 isolates, 8 isolates viz. LK 1, LK 4, LK 39, LK 81, LK 124, LK 141, LK 147 and LK 156 showed significantly higher growth at all the concentrations of PCP (Table 4). Isolate LK 156 showed maximum growth in terms of protein (548.67 [micro]g m[L.sup.-1] at 100 ppm PCP; 782.00 [micro]g m[L.sup.-1] at 300 ppm PCP; 665.33 [micro]g m[L.sup.-1] at 500 ppm PCP and 345.33 [micro]g m[L.sup.-1] at 700 ppm PCP). Isolate LK 5 showed significantly low growth as compared to other isolates. It was observed that all the isolates could grow well at 300 ppm of PCP in medium as compared to 100 ppm of PCP as evident by significantly higher protein at 300 ppm of PCP. Similar observation was observed for few isolates which grew well at 500 ppm of PCP as compared to 100 ppm of PCP (Table 4). The cultures were initially isolated by enrichment method and continuously maintained at 50 ppm of PCP in MA agar slants. When these isolates were grown at higher PCP concentration under shaking condition they exhibited higher growth in terms of protein. It could be due to higher availability of PCP as sole C source in the medium.

The results indicated that PCP concentrations less than 500 ppm in medium were utilized by acclimated culture after 48 h of incubation. However, when the PCP concentration was above 500 ppm the utilization of PCP by the culture was low as indicated by low growth. The aerobic pathway of PCP degradation is: C6[Cl.sub.5]OH + 4.5[O.sub.2] + 2[H.sub.2]O [right arrow] 6[O.sub.2] + 5HCl (Crawford and Crawford, 1996). The equation showed that PCP degradation leads to a decrease in pH. The pH of broth after 72 h of incubation did not decrease significantly in the medium containing PCP at 100 to 500 ppm of PCP and ranged in between 6.8 to 7.0. In contrast, at 700 ppm of PCP, the pH in medium decreased significantly and ranged in between 5.9 to 6.1. It could be the reason for low growth of isolates at 700 ppm of PCP in the medium. Similar observation was observed by Yang et al (2006) where the lag phase of bacterial isolate increased at higher concentration of PCP (>200 ppm) as compared to lower concentrations of <200 ppm and resulted in low growth at >200 ppm of PCP even after 45 h of incubation.

16S rRNA gene sequencing and phylogenetic analysis

From the genomic DNA of all the 22 bacterial isolates 16S rRNA gene was amplified using both forward and reverse primer (pA and pH, respectively). The reverse and forward purified 16S rRNA gene from all the isolates was sequenced. After obtaining the forward and reverse sequences of 16S rRNA gene (approx. 600-700 bp), contigs were made using online software CAP3. All the sequences were compared with 16S rRNA gene sequences available in the GenBank databases by BLASTn search and the identity of isolates is given in table 5. The partial sequences of 16S rRNA gene sequences after analysis were submitted to NCBI GenBank database under accession numbers KF261572 to KF261593 (Table 5). Multiple sequence alignment of approx 1500-bp sequences was performed using CLU STAL W, version 1.8 and phylogenetic was constructed (Fig 1). The phylogenetic relationships of the isolates as inferred from comparison of partial sequences (approx 1500bp) of the 16S rRNA genes showed that these isolates fell into three major lineages of domain Bacteria; the [alpha],[gamma]-Proteobacteria and Firmicutes.

The 19 isolates of [gamma]-Proteobacteria, matched with sequences of Pseudomonas aeruginosa (LK 1), Pseudomonas citronellolis (LK 5, LK 81 and LK 141), Pseudomonas putida (LK 41, LK 43, LK 47, LK 51, LK 54, LK60, LK 72, LK 142 and LK 150), Pseudomonas plecoglossicida (LK 124 and LK 147) and Enterobacter sp (LK 188). Pseudomonas is a well-known PCP degrading genera reported to degrade high concentration of PCP (Karn et al 2010, Kaoa et al. 2005). Karn et al (2011) also reported degradation of PCP by Enterobacter sp isolated from distillery dumpsite using enrichment method. Karan (2011) observed Enterobacter sp was able to degrade 70% of PCP at 100 mg [L.sup.-1] in growth medium. In our study, Enterobacter sp although could grow at 700 ppm of PC but the growth was low as compared to other isolates (Table 4).

Only 2 isolates viz. Ochrobactrum sp (LK 59) and Ensifer adhaerens (LK 4) belonged to [alpha]-Proteobacteria. Ochrobactrum anthropi was reported to degrade chlorophenol (Muller et al. 1998), whereas no such report is available regarding Ensifer adhaerens. Therefore this could also be a new PCP degrading bacterial genera. The single isolate of Firmicutes phyla was identified as Lysinibacillus fusiformis (LK 156) which could show maximum growth at 300 and 500 ppm of PCP in MS medium (Table 4). No reports are available in literature about Lysinibacillus sp degrading PCP, however, Chandra et al (2006) reported degradation of high concentration of PCP up to 300 mg[L.sup.-1] by Bacillus sp. Hence, along with Ensifer adhaerens, Lysinibacillus fusiformis is also reported for the first time in present study.

CONCLUSION

Our results show that the ability to degrade pentachlorophenol is widely distributed among phylogenetically very different bacteria in agricultural soils irrigated with water contaminated with effluent discharged from pulp and paper mill. Bacterial isolates utilizing PCP up to 500 ppm obtained in this study can be used for developing consortium for degrading PCP in contaminated soils.

REFERENCES

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Dharmendra Kumar, Rajeev Kaushik and Surender Singh

Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012, India.

(Received: 06 September 2015; accepted: 11 November 2015)

* To whom all correspondence should be addressed.

E-mail: dharmendra851@gmail.com

Caption: Plate 1. Purified colonies of few selected PCP utilizing bacteria isolated from pulp and paper mill effluent irrigated soil

Caption: Plate 2. Biochemical characterization of bacterial isolates

Caption: Plate 3. Visualization of genomic DNA isolated from selected bacterial isolates obtained from pulp and paper mill effluent irrigated soil

Caption: Plate 4. Visualization of PCR amplified 16S rDNA product of selected bacterial isolates obtained from pulp and paper mill effluent irrigated soils

Caption: Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of PCP degrading isolates and their closest phylogenetic relatives. The numbers on the tree indicate the percentage of bootstrap sampling derived from 1000 replicates
Table 1. Physicochemical properties of soil

Parameters                            Average Values (1)

pH                                    8.16 ([+ or -]0.1)
EC (dS [m.sup.-1])                   0.73 ([+ or -]0.04)
OC (%)                               0.95 ([+ or -]0.16)
Available N (kg [ha.sup.-1])         64.85 ([+ or -]10.9)
Olsen P (kg [ha.sup.-1])             16.81 ([+ or -]0.21)
Exchangeable K (kg [ha.sup.-1])     164.5 ([+ or -]18.12)
Residual PCP (mg [Kg.sup.-1] soil)  113.34 ([+ or -]11.36)

* Mean of 5 replications and figure in parenthesis are
Standard deviation from mean

Table 2. Morphological characterization of selected bacterial isolates
obtained from pulp and paper mill effluent irrigated soil

Strain   Colony Colour    Size and Pigmentation      Margin
Number

LK-1     Shiny blackish   Punctiform pigmented       Entire
LK-4     Creamy white     Circular non pigmented     Undulate
Lk-5     Off white        Punctiform non pigmented   Lobate
LK-23    Off white        Punctiform non pigmented   Entire
LK-32    Shiny whitish    Circular pigmented         Lobate
LK-39    Creamy white     Irregular pigmented        Curled
LK-41    Creamy white     Circular pigmented         Undulate
LK-43    Dull             Circular pigmented         Lobate
LK-47    White            Punctiform non pigmented   Undulate
LK-51    Creamy white     Punctiform non pigmented   Undulate
LK-54    White off        Irregular non pigmented    Entire
LK-59    Dull mucoid      Irregular non pigmented    Curled
LK-60    Creamy white     Circular non pigmented     Entire
LK-72    Dull             Irregular pigmented        Undulate
LK-81    Shiny            Punctiform pigmented       Curled
LK-124   Creamy white     Irregular non pigmented    Entire
LK-141   Shiny            Irregular non pigmented    Lobate
LK-142   Creamy white     Circular non pigmented     Entire
LK-147   Creamy white     Irregular non pigmented    Undulate
LK-150   White off        Circular non pigmented     Curled
LK-156   Creamy white     Circular pigmented         Entire
LK-188   Creamy white     Circular non pigmented     Curled

Strain   Elevation    Shape
Number

LK-1     Flat         Rods
LK-4     Convex       Cocci
Lk-5     Raised       Rods
LK-23    Flat         Rods
LK-32    Flat         Rods
LK-39    Raised       Rods
LK-41    Flat         Rods
LK-43    Umbonate     Rods
LK-47    Umbonate     Rods
LK-51    Raised       Rods
LK-54    Pollinated   Rods
LK-59    Raised       Rods
LK-60    Raised       Rods
LK-72    Lobat        Rods
LK-81    Raised       Rods
LK-124   Pollinated   Rods
LK-141   Raised       Rods
LK-142   Convex       Rods
LK-147   Flat         Rods
LK-150   Raised       Rods
LK-156   Raised       Rods
LK-188   Flat         Rods

Table 3. Biochemical Characterization of selected bacterial
isolates obtained from pulp and paper mill effluent irrigated soil

Strain     Gram     Oxidase   Catalase   Urease     Starch
Number   reaction                                 hydrolysis

LK-1       -ve         +         +         -          +
LK-4       -ve         +         +         -          +
Lk-5       -ve         +         +         -          +
LK-23      -ve         +         +         -          +
LK-32      -ve         +         +                    +
LK-39      -ve         +         +         +          -
LK-41      -ve         +         +         +          -
LK-43      -ve         +         +         +          -
LK-47      -ve         +         +         +          -
LK-51      -ve         +         +         +          -
LK-54      -ve         +         +         +          -
LK-59      -ve         -         +         +          +
LK-60      -ve         +         +         +          -
LK-72      -ve         +         +         +          -
LK-81      -ve         +         +         -          +
LK-124     -ve         +         +         +          +
LK-141     -ve         +         +         -          +
LK-142     -ve         +         +         +          -
LK-147     -ve         +         +         +          +
LK-150     -ve         +         +         +          -
LK-156     -ve        +/-        +        +/-         -
LK-188     -ve         -         +         -          +

Strain   N[O.sub.3]
Number   reduction
            test

LK-1         +
LK-4         -
Lk-5         -
LK-23        -
LK-32        -
LK-39        +
LK-41        +
LK-43        +
LK-47        +
LK-51        +
LK-54        +
LK-59        -
LK-60        +
LK-72        +
LK-81        -
LK-124       +
LK-141       -
LK-142       +
LK-147       +
LK-150       +
LK-156       -
LK-188       +

Table 4. Growth of selected bacterial isolates at
different concentration of PCP

Isolate              Protein ([micro]g [mL.sup.-1]) at different
                               concentration of PCP

No.                  100 ppm   300 ppm   500 ppm   700 ppm

LK 1                 357.00    625.33    335.50    145.33
LK 4                 273.67    500.33    367.00    122.00
LK 5                  28.67    145.33     95.33     13.67
LK 7                 152.00    185.33     68.67     45.33
LK-32                 92.00    175.33    158.67     42.00
LK-39                310.33    543.67    527.00    120.33
LK-41                158.67    208.67    142.00     77.00
LK-43                 27.00    477.00    143.67     55.33
LK-47                145.33    162.00     58.67     37.00
LK-51                 83.67    283.67    183.67     85.33
LK-54                113.67    413.67     80.33     57.00
LK-59                232.00    182.00    115.33     75.33
LK-60                143.67    193.67    160.33     48.67
LK-72                115.33    265.33    267.00     38.67
LK-81                315.33    148.67    448.67    175.33
LK-124               275.33    595.33    448.67    232.00
LK-141               280.33    582.00    415.33    152.00
LK-142               162.00    420.33    270.33     55.33
LK-147               372.00    552.00    318.67    152.00
LK-150               108.67    375.33     42.00     95.33
LK-156               548.67    782.00    665.33    345.33
LK-188               103.23     58.77     43.48     37.11
[LSD.sub.(p=0.05)]   43.13     34.61     38.53     21.79

Table 5. Nucleotide identity (%) of isolates to the closest phylogenetic
neighbour and classification of the isolates

Strain   Accession   Identity                    % Similarity
Number   Number

LK 1     KF261572    Pseudomonas aeruginosa      96%
LK 4     KF261573    Ensifer adhaerens           98%
LK 5     KF261574    Pseudomonas citronellolis   97%
LK 23    KF261575    P. citronellolis            99%
LK-32    KF261576    P. citronellolis            97%
LK-39    KF261577    Pseudomonas putida          99%
LK-41    KF261578    P. putida                   99%
LK-43    KF261579    P. putida                   99%
LK-47    KF261580    P. putida                   99%
LK-51    KF261581    P. putida                   99%
LK-54    KF261582    P. putida                   99%
LK-59    KF261583    Ochrobactrum sp             99%
LK-60    KF261584    P. putida                   99%
LK-72    KF261585    P. putida                   99%
LK-81    KF261586    Pseudomonas citronellolis   99%
LK-124   KF261587    P. plecoglossicida          99%
LK-141   KF261588    P. citronellolis            99%
LK-142   KF261589    P. putida                   99%
LK-147   KF261590    P. plecoglossicida          99%
LK-150   KF261591    P. putida                   97%
LK-156   KF261592    Lysinibacillus fusiformis   99%
LK-188   KF261593    Enterobacter sp             99%

Strain   Classification
Number

LK 1     [gamma]-Proteobacteria
LK 4     [alpha]-Proteobacteria
LK 5     [gamma]-Proteobacteria
LK 23    [gamma]-Proteobacteria
LK-32    [gamma]-Proteobacteria
LK-39    [gamma]-Proteobacteria
LK-41    [gamma]-Proteobacteria
LK-43    [gamma]-Proteobacteria
LK-47    [gamma]-Proteobacteria
LK-51    [gamma]-Proteobacteria
LK-54    [gamma]-Proteobacteria
LK-59    [alpha]-Proteobacteria
LK-60    [gamma]-Proteobacteria
LK-72    [gamma]-Proteobacteria
LK-81    [gamma]-Proteobacteria
LK-124   [gamma]-Proteobacteria
LK-141   [gamma]-Proteobacteria
LK-142   [gamma]-Proteobacteria
LK-147   [gamma]-Proteobacteria
LK-150   [gamma]-Proteobacteria
LK-156   Firmicutes: Bacilli
LK-188   [gamma]-Proteobacteria
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Author:Kumar, Dharmendra; Kaushik, Rajeev; Singh, Surender
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2016
Words:5205
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