IJAB ( Volume No : 23 Issue 01 )

Evaluation of Microsatellite Markers for Genotyping of Markhor (Capra falconeri) Populations PP: 1-6

Keywords:Markhor; Microsatellites; Genetic diversity; Population structuring; Caprinae


Textile industry is a continuous source of colored wastewater. This wastewater frequently used for irrigation purpose in many underdeveloped countries including Pakistan. In this study, we isolated the bacterial strains capable of decolorizing dyes and promote the plant growth. Hence to decolorize the reactive red 120 (RR120), the strain WS-D/183 was optimized following response surface methodology (RSM) based modeling approach. Moreover, strain WS-D/183 was also assessed for plant growth promoting characteristics. Results revealed that the strain WS-D/183 showed a good potential for decolorization of structurally diverse types of azo dyes on reaction with a mixture of heavy metal ions (Cr6+, Cd2+, Zn2+, Pb2+). This strain concurrently removed reactive dyes (100 mg L-1) and reduced Cr(VI). Results showed that each dye was decolorized up to 90% except reactive yellow-2 which was decolorized up to 57.4%. Furthermore, the bacterium reduced Cr(VI) by 41 to 95% along with concurrent decolor


In textile industry, a major proportion of dyes used for dyeing fabrics are azo-dyes. These dyes are preferred over other classes of synthetic dyes due to their low price, ease of application and availability in a variety of colors (Shah et al. 2014). However, a high fraction of the applied quantity ranging between 15–50% is wiped out in dyeing and washing processes and forms the colored wastewaters (Pratum et al. 2011). Release of colored wastewaters also causes entry of azo dyes in water and soil resources (Tony et al. 2009). Such dyes disrupt light penetration into water, which effect photosynthetic rate of hydrophytes (Shah et al. 2014). It is reported that azo dyes and their metabolites can cause cancer and add poisonous materials in the environment (Carneiro et al. 2010). In addition, entry of such wastewaters in soil resources results into a disturbance in soil microbial communities, soil processes and crop productivity (Arif et al. 2016; Rehman et al. 2018). Azo dyes and meta

Materials and Methods

Growth media for bacterial growth

A mineral salt medium (MSM) spiked with four heavy metals was used to isolate metal-tolerant, dye degrading bacterial isolate. The ingredients of MSM used in this study are given in Maqbool et al. (2016). The mineral salt medium was spiked with four different heavy metal salts as previously reported in Maqbool et al. (2016). However, pH of MSM was maintained at 7.2 using basic and acidic solutions.

Isolation, Screening and molecular identification of isolate WS-D/183

For bacterial isolation, effluent samples were taken from outlets of various textile industries. These industries were located near Sargodha Road, Faisalabad, Pakistan. Serial dilution of collected wastewater samples was performed and about 100 μL of 10-3–10-6 dilutions was poured on MSM agar plates. These plates were spiked with selected heavy metals and placed in static incubator at 30°C. The bacterial isolates having variable size, color and shape were purified through streaking thrice using MSM agar plates. To prepare inoculum of each isolate, the cultures of all bacterial isolates were inoculated in Erlenmeyer flasks and incubated at 30°C for 72 h. By using sterile MSM, the OD600 of all bacterial culture was adjusted as 0.5. Then, 18 mL of MSM containing mixture of heavy metals and RR120 were added separately along 2 mL of isolates in test tubes and incubated at 30°C. After two days of incubation, removal of initially added dye was determined (Maqbool et al. 2016). The isola

Metal tolerance of isolate WS-D/183

The metal tolerance of the bacterium was determined by estimating MIC of selected heavy metals. For this purpose, nutrient agar medium was spiked with cobalt, chromium, zinc, lead, nickel, cadmium salts to develop 0–5000 mg L-1 of given heavy metals. The strain WS-D/183 was streaked on each plate and preserved for five days to check the growth of WS-D/183. The minimum concentration of a particular metal that inhibited growth of the strain WS-D/183 was considered as MIC for the strain WS-D/183.

Optimization of RR120 decolorization by isolate WS-D/183

Bio-decolorization of structurally diverse azo dyes: The ability of WS-D/183 for decolorizing different reactive dyes and direct dyes [orange direct (OD), Congo red (CRD) and blue direct (BD)] was assessed in MSM spiked with mixture of metal ions. The general characteristics of these dyes are given in Table 1. The experiment was conducted in glass vials. The cells of the strain WS-D/183 were added to MSM and OD600 was adjusted at 0.05. After filter sterilization, each dye was added to maintain 100 mg/L conc. Triplicates of inoculated vials along with un-inoculated control were kept at 30°C. After 24, 48, 72 and 144 h, decolorization was determined as described above. Optimization of RR120 decolorization by the strain WS-D/183 using RSM: Effect of pH, yeast extract, sodium chloride and metal mixture on removal of RR120 via WS-D/183 was assessed following RSM. Each selected variable was studied at five levels as previously explained in Maqbool et al. (2016). Small composite design (SCD)

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Where represents expected response given vector x of predictor variables, β˳ is a regression constant, βі is linear regression coefficient, βіi is quadratic regression coefficient and βіj is bilinear regression coefficients.

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Screenshot (128).png

The model adequacy was assessed by lack of fit technique. Lack-of-fit of second-order model indicated that quadratic model lack-of-fit is highly insignificant with low F-value and high p-value. Thus, quadratic polynomial model was more appropriate to describe the relation of response (i.e., decolorization) to the input factors (i.e., pH, NaCl, yeast extract and heavy metal mixture content). The evaluation of variable significance of whole model was checked by R2 and F-test. Moreover, confidence levels were determined to check significance of R2. Variance inflation factor was also calculated to measure extent of multicollinearity among two or more input variables of polynomial regression model.

Concurrent removal of RR120 and hexavalent chromium using WS-D/183

The strain WS-D/183 was also checked for concurrent elimination of RR120 and Cr(VI) in MSM in the presence of Zn2+, Pb2+ and Cd2+ mixture. In first step, the bacterium was checked for Cr(VI) removal in MSM taken in glass vials. The supernatants taken over time were analyzed for Cr(VI) content with diphenylcarbazide (DPC) method (Anwar et al. 2014). After confirmation of Cr(VI) reduction, it was evaluated for concurrent elimination of RR120 and hexavalent chromium. For this purpose, filter sterilized RR120 was added to MSM spiked with Cr+6, Zn2+, Pb2+ and Cd2+. After inoculation, vials along with

un-inoculated controls were placed in an incubator. The supernatant was collected after 0, 30, 60, 90, 120, 150, 180 and 210 h. It was used to measure decolorization and removal of Cr(VI) by using DPC method, whereas, bacterial pellet was used to measure growth of the bacterium. The bacterial pallets were rinsed and suspended in distilled water. OD600 was determined using a CO800 Cell Density Meter (Biochrom, England).

Assessment of PGPR traits of WS-D/183

We studied the indole acetic acid (IAA) and phosphorus solubilization as indices of PGPR traits. Gordon and Weber (1951) method was employed to determine IAA production by the bacterial strain. Briefly, LB broth containing L-tryptophan, IAA precursor, was inoculated with the strain WS-D/183. After 48, 120 and 240 h of incubation, IAA content and pH of the supernatant were determined. For IAA measurement, two drops of H3PO3 and 1 mL Salkowski reagent were added to supernatants and kept at room temperature for few minutes. The strength of pink color developed was assessed at 530 nm. A calibration plot was developed using IAA standards and used to determine IAA. For measuring phosphate solubilization, NBRIP broth medium having tri-calcium phosphate as insoluble phosphorus source was prepared. The broth was inoculated with the culture of the strain WS-D/183 and incubated under static conditions. After 48, 120 and 240 h of incubation, phosphorus content and pH of the filtrate were determine

Phytotoxicity Evaluation of the wastewater treated with WS-D/183

Three MSM flasks containing 500 mg RR120 L-1, 25 mg Cr(VI) L-1 and RR120+Cr(VI) were inoculated with the strain WS-D/183 for 72 h in order to obtain treated water for phyto-toxicity evaluation of the strain. The untreated RR120, Cr(VI) and RR120+Cr(VI) along with their respective treated counterparts were used to determine their toxicity on seed germination of mung bean [Vigna radiata (L.) Wilczek]. Ten seeds were sown in sand taken in petri plates. The sand plates containing seeds according to the below given eight treatments were watered. Experiment was conducted in triplicate in growth chamber where plants received the light for 16/8h light/dark periods at temperature of 25/30°C and day/night humidity of 70/90%. After 10 days, germination (%), radical length and plumule length was measured. Treatments: T0 = No dye, T1 = RR120 @ 500 mg L-1, T3 = Cr(VI) @ 25 mg L-1, T4 = RR120 @ 500 mg L-1 + Cr(VI) @ 25 mg L-1, T5 = No dye + WS-D/183, T6 = RR120 @ 500 mg L-1 + WS-D/183, T7 = Cr(VI) @

Statistical analysis


Molecular identification of the isolate WS-D/183

More than 200 bacteria were isolated and checked for decolorization of RR120 in MSM spiked with a mixture of Cr6+, Pb2+, Cd2+ and Zn2+. The decolorization ranged from 1.3±1.1 to 95.6±3.2% for various bacterial isolates. The maximum decolorization of the RR120 was shown by the isolate WS-D/183. Bioinformatic analysis of 16S rRNA gene of the isolate WS-D/183 through BLASTn revealed that it had the highest similarity with several species of genus Pseudomonas. Moreover, during phylogenetic analysis the isolate WS-D/183 (GeneBank Accession No. MG547881) was clustered with bacterial spp. belonging to genus Pseudomonas (Fig. 1). Moreover, phylogenetic tree revealed that WS-D/183 belongs to genus Pseudomonas and designated as Pseudomonas sp. WS-D/183 (Fig. 1).

MIC of metal ions for Pseudomonas sp. WS-D/183

The MIC of six metals for growth of Pseudomonas sp. WS-D/183 is presented in Table 2. The strain WS-D/183 showed variable resistance towards metal ions. The MIC of Co2+, Cr6+, Zn2+, Pb2+, Ni2+ and Cd2+ for the bacterium were 13.58, 9.62, 30.59, 4.83, 3.41 and 44.48 mM, respectively.

Potential of WS-D/183 to decolorize structurally diverse textile dyes

Pseudomonas sp. WS-D/183 showed excellent removal of selected reactive and direct dyes (Table 3). However, it was observed that it showed higher decolorization of reactive than direct dyes. After 24 h treatment, the strain WS-D/183 showed maximum decolorization of RB5 (53.5±1.1%) followed by RR120 (18.3±1.3%) and RY2 (17.7±3.2%) as compared to 14.7% (±1.7), 7.5% (±2.7) and 3.4% (±1.1) decolorization of BD, CRD, OD, respectively. However, after 144 h, more than 96% of the added RR120

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was decolorized followed by RB5 (89.2±1.1%), RY2 (64.2±2.1%) and RO16 (63.7±2.4%). The other dyes CRD, BD and OD dyes were decolorized up to 72.5 (±1.5%), 48.2 (±2.6%) and 34.6 (±3.2%) respectively, over the same treatment period.

Optimization of RR120 dye decolorization by Pseudomonas sp. WS-D/183 using RSM

Validation and importance of RSM model: Response surface methodology was used to optimize four different variables for biodecolorization of RR120. The quadratic model showed lowest sequential p-value (0.0001). Data for sequential model selection suggested preferring quadratic over two-factor interaction model because it had very low p-value (Table 4). Moreover, cubic model terms were found different from each other. The model validation showed a high value of R2=0.8531 with adjusted-R2=0.6736. This indicated a big share of variation in response that can be addressed by quadratic polynomial model. Furthermore, “Adeq precision” ratio was found as 9.846. Predicted residual sum of square (PRESS) determines how well the hypothesized model is being estimated by the design. Lower PRESS value showed good performance of the model. Quadratic model showed lower PRESS as compared to other models. The estimated regression model is presented below which shows the contribution of different second