Effective Bioremediation of Heavy Metals for Environmental Sustainability Approach using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 isolated from Hindon River, India

Effective Bioremediation of Heavy Metals for Environmental Sustainability Approach using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 isolated from Hindon River, India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effective Bioremediation of Heavy Metals for Environmental Sustainability Approach using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 isolated from Hindon River, India Deepak Kumar, Sangeeta Kumari This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4652732/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The goal of this work was to isolate, identify, and characterize heavy metals degrading bacterial strains from the contaminated Hindon river (Yamuna's tributary) in India. Hindon river water quality was also assessed using physico-chemical and heavy metals analyses, revealing the river's degraded state. The bacterial isolates were isolated from Hindon river. Two bacterial isolates coded as HIB2 and HIB7 were screened and selected for further study based on the resistance to heavy metals (Cd, Ni, and Pb). Finally, these were identified based on morphological, biochemical tests and 16S rRNA sequencing. Biodegradation potential for heavy metals was assessed using inductively coupled plasma mass spectrometry (ICP-MS). The bacterial isolates HIB2 and HIB11 from Hindon river were identified as Bacillus subtilis and Pseudomonas aeruginosa respectively. At 10 mg/L initial concentration, results of biodegradation experiments showed the removal competences of 53.9% for Cd 2+ and 68.6% for Ni 2+ and 71.7% for Pb 2+ using Bacillus subtilis HIB2, where 57.6% for Cd 2+ and 66.4% for Ni 2+ and 70.7% for Pb 2+ using Pseudomonas aeruginosa HIB11 after 14 days of incubation at pH 7.0 and 37°C. Both isolates revealed the similarity in removal efficiency for these three heavy metals. The ability of Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 degrading heavy metals was found to be through curing experiments. The study showed that the Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 could be involved in effective degradation of heavy metals (Cd, Ni, and Pb). Biodegradation Hindon river Heavy Metals Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Rising river water pollution and environmental degradation are attributable to fast-growing populations, land expansion along river basins, and urban and industrial growth. Several sectors, such as electroplating and battery, leather, and paint producing units, are now using diverse heavy metals, which could result in many heavy metals being released into the environment (Satyavathi et al ., 2014). Heavy inorganic metals bioaccumulation and consequent biomagnification such as cadmium (Cd), lead (Pb), chromium (Cr) and nickel (Ni) which can reach the level that are toxic to living organisms in the river, emphasis on requirement for removal of heavy metals (Khatri et al ., 2020 : Kalaimurugan et al., 2019 ). River Hindon, originating from the upper Shivalik’s in Uttar Pradesh in Saharanpur, Uttar Pradesh, India, streams 265 km through six districts including Muzaffarnagar, Meerut, Baghpat, Ghaziabad and Gautam Budh Nagar, till merge with the Yamuna, is a main resource of water to the vastly colonized and largely populated rural area, where, its water is applied for in-house, farming and industrialized related activities, henceforth the current fast development in urbanization and industrialization along the course of Hindon has caused numerous problems of contaminations in Hindon river ( Kumar et al ., 2016 and Suthar et al., 2009 ). Several physical, chemical, and biological approaches have been projected for the removal of heavy metals from polluted soil and water of which microorganisms-based bioremediation is well-thought-out as a hopeful and worthwhile technique. Verma et al.,2006 and Oziegbe et al., 2021 discussed that microorganisms-based bioremediation is economical and eco-friendly to mitigate the heavy metal contamination. Bioremediation can be described as the application of microorganisms for removal of contaminants in water and soil. The bioaugmentation approach, adding microorganisms with efficacious degradation ability in the polluted areas, has projected and measured to increase bioremediation of pollutants (Govarthanan et al ., 2016; Janbandhu and Fulekar, 2011 ; Cerquiera et al ., 2011 ). The engagement of a bacterial consortium has confirmed to be extra valuable comparted with single isolate due to synergistic relations amongst members of the relatives, which may result in the comprehensive degradation of the pollutants ( Nokman et al., 2019 ). The application of microorganisms with verified degradation competency and resistance in the polluted environment is crucial for a successful bioaugmentation. The heavy metal degradation process applying microorganisms includes biosorption, bioleaching, biomineralization, immobilization and redox reactions ( Fig. 1 ) . Biosorption which is one of the bioremediation processes, is most effective in heavy metal detoxification (Hargreaves et al ., 2015 ; Alabssawy and Hashem, 2024 ). Many studies have found that bacteria isolated from different sources had the capability of flourish in the harsh metal pollution (Bestawy et al ., 2012: Bhakta et al ., 2014 and Guo et al ., 2010). Hamid et al., 2023 isolated heavy-metal tolerant bacterial strains from industrial sites and scrap yards in Kashmir, India. To increase their effectiveness in the degradation of heavy metals as a sustainable environment technology, the current investigation targeted at the isolation, screening, identification, and characterisation of heavy metals degrading bacteria isolated from the Hindon River. . Materials and Methods Sample Collection Water samples were collected from five different sites of Hindon river (Fig 2) i.e. Atali village (S1)-29°12'43.4"N 77°31'18.4"E, Baparsi village (S2)-29°11'07.2"N 77°29'42.6"E, Shekpura village (S3)-29°05'34.4"N 77°25'21.1"E, Kalina village (S4)-29°04'27.1"N 77°27'43.6"E and Kinauni village (S5)-29°03'05.2"N 77°27'15.9"E, in sterilized bottles in month of Jan -Jun 2017 according to standard method (APHA,2012 and CPCB,2009). These samples were kept into thermo boxes immediately after collection, followed by transportation to laboratory. Further, the samples were stored at 4◦C after taking to the laboratory for subsequent analysis. Physical and chemical characterizations of the Hindon river water Hindon river water samples were analyzed for physico-chemical parameters i.e. pH, electrical conductivity (EC), chemical oxygen demand (COD), biological oxygen demand (BOD), total dissolved solids (TDS), alkalinity, hardness, sulfate (SO²⁻₄), nitrate (NO⁻₃) and chloride (Cl⁻) were estimated according to American Public Health Association methods ( APHA,2012 ). For heavy metals concentrations (Cd, Ni, and Pb), the samples were digested with concentrated nitric acid followed by filtration through 0.45 µm filter paper and were analyzed by using ICP-MS. Isolation and screening of heavy metal resistant bacteria The collected Hindon river water samples were serially diluted up to 10 −6 dilution with distilled water and inoculated on the nutrient agar medium containing 200 mg/kg of heavy metal (Cd, Ni and Pb) by spread plate method. Plates were kept in the incubator for incubation at 37 o C for 24 hours. Pure culture of morphologically different colonies was isolated and purified by repeated streaking and then maintained at 4 o C on nutrient agar slants for further experiment. More, screening of bacterial isolates was done based on resistance against heavy metals. To check the heavy metal resistance level of all selected isolates were assessed by inoculating isolates in nutrient agar plates containing different concentrations of concerned heavy metals ranging from 100 to 500 mg/L. The plates were incubated 24h at 37°C and observed for bacterial growth on the culture medium and then highly resistive bacterial strains were selected. Identification of Selected Bacterial Isolate These isolates were primarily characterized through morphological characteristics such as gram’s staining, shape, motility test. Additionally, these were identified by biochemical characteristics based on Bergey’s Manual of Systematic Bacteriology. The biochemical tests performed to assess the capability of the isolates were Indole production, methyl red (MR) test, voges proskauer (VP) test, citrate utilization test and H 2 S production. Additionally, carbohydrate fermentation tests were conducted to evaluate the capacity of the isolates to ferment sugars such as glucose and sucrose, where a color transformation from pink to light yellow showed the fermentation of sugar. Moreover, the enzymatic hydrolysis tests such as starch hydrolysis, catalase test, oxidase test, urease test, and nitrate reduction test were also studied for the identification of isolates. The isolates identified by morphological and biochemical characterization were grown on nutrient broth. Conclusively, the bacterial isolates were further examined for their molecular characteristics by 16S rRNA gene sequencing. Genomic DNA was extracted from bacterial isolates and processed for 16S rRNA sequencing, where Realtime-PCR (polymerased chain reaction) was used to amplify extracted DNA using the universal primer in a thermal cycler. The forward primer sequence was 16SF (2f) 5'- AGAGTTTGATCMTGGCTCAG-3' and the reverse primer sequence was 16SR (1492r) 5'-TACGGYTACCTTGTTACGACTT-3'. The standalone PCR-primers and dNTPs were removed from PCR-products by using a cleaning kit (Montage-PCR made by Millipore). The PCR-product was processed for sequencing using the primers (27F/1492R). Sequencing reactions were carried out using an ABI-PRISM® BigDyeTM Terminator Cycle-sequencing kits with AmpliTaq®DNA polymerase (FS enzyme) (Applied Biosystems). Single-pass sequencing was conducted on individual template using above universal primers of 16S rRNA. The fluorescent labelled fragments were separated from the standalone terminators with ethanol. The samples were re-suspended in distilled-water and further processed for electrophoresis in an ABI 3730xl-sequencer (Applied-Biosystems). The 16s rRNA sequences were blasted using BLASTA tool at NCBI site (http://www.ncbi.nih.gov/BLAST/). The guide tree into a text file in Newick format was downloaded which was recognized by phylogenetic software named Mega V software (version-5.0) and then a phylogenetic tree was created using the neighbor-joining method. The gene sequences of two bacterial isolates were deposited to the National Centre for Biotechnology Information (NCBI) Gene bank and then the accession numbers were obtained for further communication. Biodegradability assay of bacterial isolates for Heavy Metals Chemical reagents In this study, certified reference material (CRM) of individual heavy metal (Cd, Ni and Pb) and supra-pure nitric acid (HNO 3 ) of analytical grade were purchased from Merck. Working solution standard was made by dissolving stock solution (1000 mL) in deionized distilled water. Experimental study Individual isolate was inoculated into the flask containing nutrient broth which was incubated for 24 to 48 hours in the shaking incubator at 150 rpm, while pH and temperature were adjusted 7.0 and 37 ̊C, respectively. Once optical density (O.D.) attained 0.5 (k = 600 nm: for identical enzymatic activity), then 5, 10, 50 & 100 mg/L of sterilized heavy metals (Cd, Ni and Pb) was supplemented individually in each inoculated flask of 250 mL and incubated another time at the same condition for 14 days. After defined time interval of incubation, 10 ml of culture broth from shaking flask was collected and centrifuged (Remi, R-8M/CDLC-3111, India) at 5000 rpm for 15 min to acquire a cell-free medium, then 1 mL of supernatants was separated and digested with 5 mL of concentrated HNO 3 in microwave digester (Anton paar, ECO/81331413, ‎Graz, Austria) to accomplish acid digestion. The solution was filtered using a filter paper (Whatman No. 42) to eliminate any insoluble material and transferred into a centrifuge tube and then made-up by 50 ml. To measure reduction, this solution of total heavy metal was analyzed by ICP-MS (Thermo Scientific, ICAP Q (2271R)) and the results were calculated for heavy metal degradation capacity (%) as per below: HM= Heavy Metal Statistical analysis All experiments were made in triplicate and the data was expressed as the means ± SD. ANOVA were used to study the statistically significant differences (p<0.05) for heavy-metal removal after treatment. The results of each experiment were assumed to be independent with different variance. Results & Discussion Characteristic of Hindon river water The results obtained at the characterization of various parameters in the water samples collected from five different sites of Hindon River are summarized in Table 1. The mean values of pH in water samples from five sites, ranged from 7.25 to 7.69, were found within the permissible limits. The pH values of water samples were marginally alkaline at all five sites, showing that there is neither acid nor alkaline pollution in the water samples from Hindon river (Gupta et al. , 2013). Electrical conductivity (EC) of water samples at all sites, where mean values varied from 1667.0 to 1916.75 μS/cm, were beyond the acceptable limit, where, what sample from Atali village site exhibited highest EC compared to that of other four sites. High EC was attributed to the existence of high number of dissolved ions ( Verma et al., 2006 ). River water pollution by release of domestic and industrial waste rises the EC. High number of dissolved ions in water causes unpleasant taste and moreover distresses animals and plants (Kumar et al .,2018) . Total dissolved solid (TDS) ranged from 773.25 to 1238.0 mg/L as mean value, was also beyond the permissible limit at all five sites of river. High level of TDS in river water is primarily due to the occurrence of carbonates (CO 3 2− ), bicarbonates (HCO 3 − ), chlorides (Cl − ), phosphates (PO 4 3− ) and nitrate compound such as Ca (NO 3 ) 2 , Mg (NO 3 ) 2 , KNO 3 and ‎Mn (NO 3 ) 2 , organic salts, and other solid elements. Contagion of Hindon river by industrial effluent could be the responsible for increase in TDS as the dissolved ions are significantly existing in Industrial waste. The existence of high level of TDS in river water also results in unfit for drinking. The mean values of alkalinity varied from 261.75 to 368.75 mg/L was also significantly higher than the permissible limits at the all sites of Hindon river. High concentration of dissolved ions rises the alkalinity of river water, which possibly can also be one of the factors for enlarged alkalinity of water samples at all five sites. High alkalinity of river water results in unfit for drinking as well as for irrigation 1 . Hardness of water samples (mean value from 319.25 to 374.50 mg/L) exceeded the permissible limit at the all five sites. Well, hardness indicates the soap forming ability of a water sample where the two cations primarily Ca + and Mg + are accountable. Existence of other ions such as sulphate (SO²⁻₄), nitrate (NO⁻₃), and chloride (Cl⁻) in river water was found within the permissible limit. Sulphate is mostly natural in nature attributed to primarily by mineral sources like gypsum, etc. Verma et al .,2006 studied on high concentration sulphate in drinking water may cause several intestinal diseases. Chloride is extensively dispersed in nature in the form of salts of Na (NaCl), K (KCl) and Ca (CaCl 2 ). COD and BOD values for Hindon river water were above the acceptable values at all five sites of river. A high BOD and COD values showed that the water has highly oxygen demanding waste attributed to the presence of organic pollutants which causes the depletion of dissolved oxygen (DO) which is a fundamental requirement for aquatic life. The high value of COD gives valuable information about the pollution potential of the Hindon river water (Gupta et al ., 2017). The heavy metals Cd, Ni and Pb were found above permissible limits for drinking water as per BIS. The physicochemical characterization of Hindon river water at five places revealed that the river had high levels of organic and inorganic pollution, which was most likely attributable to trash produced by companies located along the river's banks. Table 1 Physicochemical analysis of the water samples collected along four sites of the Hindon river (March-June 2017). Parameters Sites BIS/ CPCB* Unit S1 Mean±SD S2 Mean±SD S3 Mean±SD S4 Mean±SD S5 Mean±SD pH 7.25±0.49 7.25±0.47 7.33±0.33 7.48±0.57 7.69±0.44 6.5-8.5 EC 1916.75±327.90 1901.25±85.45 1667±178.41 1767.75±332.28 1843.75±423.86 - μS/cm TDS 1178.50±223.92 1238±34.12 1044±93.40 1117.25±136.27 773.25±84.70 500 mg/L Alkalinity 344.75±21.23 368.75±33.24 327.75±56.14 348±103.19 261.75±44.97 200 mg/L Hardness 319.25±35.94 362.50±35.42 374.50±71.92 359.25±55.4 339±121.57 200 mg/L COD 356.50±66.52 365.25±92.88 307.25±42.51 327.25±34.95 339.5±67.13 - mg/L BOD 109.50±17.05 91.5±21.43 84.5±16.15 92.75±10.03 92.5±25.47 ≤ 2* mg/L NO⁻₃ 40.75±10.85 48.25±4.82 44±18.23 31±13.69 25±9.08 45 mg/L SO²⁻₄ 48.25±9.36 56.00±7.68 46.75±0.83 51±11 48±6.75 200 mg/L Cl⁻ 178.25±21.11 199.25±20.04 168±36.80 163.75±47.37 108±25.84 250 mg/L Cd 0.022±0.02 0.037±0.02 0.027±0.02 0.04±0.06 0.002±0.00 0.003 mg/L Ni 0.048±0.01 0.021±0.01 0.144±0.22 0.068±0.05 0.012±0.00 0.02 mg/L Pb 0.052±0.04 0.046±0.04 0.051±0.04 0.054±0.04 0.016±0.01 0.01 mg/L Selection of isolates In the present investigation total number of 32 bacteria were isolated initially from the water samples following enrichment in nutrient media containing 200 mg/L heavy metals (Cd, Ni and Pb). When these strains were further studied for resistance and degradation of higher concentrations of heavy metals, two strains, viz., strain HIB2 and HIB11 were found to utilize and grow in nutrient containing up to 500 mg/L heavy metals and were selected for further study. The morphological and biochemical characteristics attentively suggested that these isolates were Bacillus sp . and Pseudomonas sp. (Tables 2). Biochemical and Molecular characterization (16S rRNA) As shown in Table 2, the bacterial isolate B2 was found to be gram-positive, small rod-shaped and motile. This isolate was able to ferment sucrose and glucose. It was MR negative and VP positive, and showed starch hydrolysis, but did not show H 2 S production. It showed positive results for the catalase test, nitrate reduction, oxidase test and citrate utilization tests. However, it showed negative results for urease test and indole test. The isolate B11 was found rod-shaped, gram negative and motile. It did not produce acid with sucrose and glucose. It showed negative results of MR test, indole test, urease test, and VP test, while positive results for catalase test, oxidase test, nitrate test, and citrate test. Further did not show starch hydrolysis and H 2 S production. Biochemical test results are also shown in Fig. 2. Molecular identification was performed for the confirmation of selected isolates by amplification and sequencing of the 16S rRNA gene. A pure culture of selected isolates was grown on nutrient agar for 24h and the identification of isolates was further confirmed using 16S rRNA gene sequencing using the universal primers, the forward primer sequence 16SF (2f) 5'- AGAGTTTGATCMTGGCTCAG-3' and the reverse primer sequence was 16SR (1492r) 5'-TACGGYTACCTTGTTACGACTT-3'. The resulting sequences were compared with their closest relatives available in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the NCBI-BLAST tool. The nucleotide BLAST similarity search analysis showed that the two isolates matched with their closest relatives as follows: the closest sequence identity of Isolate B2 was found to be with Bacillus subtilis strain based on nucleotide homology and phylogenetic analysis ( Fig. 3a). Similarly, isolate B11 had the closest sequence identity with Pseudomonas aeruginosa strain ( Fig. 3b). Additionally, these bacterial sequences were deposited in the NCBI Gene bank and got assigned the accession numbers for further communication. Isolate B2 was named as Bacillus subtilis strain HIB2 with Accession Number: MK936323.1 and isolate B11 was named as Pseudomonas aeruginosa strain HIB11 with Accession Number: MK937645.1 (Table 3) . The phylogenetic tree of each strain was constructed using the neighbor-joining algorithm from MEGA-V (Version 5.0) software and their phylogenetic relationship were inferred ( Fig. 4). The approximate phylogenetic position of the strains B2 and B11 are depicted in ( Fig 4) . Table 2 Morphological & Biochemical characteristic of the selected bacterial isolates. Test Bacillus Subtilis HIB2 Pseudomonas aeruginosa HIB11 Gram staining + - Shape Rods Rods Motility + + Catalase + + Oxidase + + Indole production - - Methyl red - - VP (Voges Proskauer) + - Citrate utilization + + Urease test - - Nitrate + + H 2 S - - Starch hydrolysis + - Sucrose + - Glucose + - Table 3 Identification of effective bacterial isolates by 16S rRNA gene sequence analyses Isolate Organism Accession no. Identity (%) HIB2 Bacillus subtilis MK936323.1 99% HIB11 Pseudomonas aeroginosa MK937645.1 99% Bioremediation of Heavy metals In this study, metal removal was carried out using bacterial strains isolated from Hindon river water and after screening based on resistance against metal ions. Isolated bacteria in this study have unique metal accumulation characteristics and easy to cultivate. Tolerance and removal of Cd, Ni and Pb were determined in the aqueous medium. The amount of heavy metals removal was evaluated by comparing initial metal concentration and final concentration after treatment by bacterial isolates and their consortium (Table 4 to 9). The bacterial isolates in the medium were studied for: (i). The reduction rate of heavy metals at different concentrations from 5 to 100 mg/L at 14 days incubation period (Fig. 5 B – 7B). (ii). The reduction rate of heavy metal (10 mg/L concentration) at different time intervals of the incubation period for 14 days (Fig.5 A- 7A). Heavy metal concentration was analyzed using the ICP-MS instrument. (Thermo fisher). Removal of Lead (Pb 2+ ) The results displayed in Table 4 & 5 shows that both isolates Bacillus subtilis HIB2 . and Pseudomonas aeruginosa HIB11 were well competent for removal of lead (Pb 2+ ) from polluted water. There was significant influence on the removal efficacy of Pb 2+ using Pseudomonas aeruginosa HIB11 when increased the initial concentration from 5 to 100 mg/L. The remaining values of Pb 2+ in the media were ranged from 1.21±0.16 to 61.49±5.65 mg/L at rising the initial concentration from 5 to 100 mg/L respectively. In case of Bacillus subtilis HIB2, the removal efficacy of Pb 2+ was also influenced at rising the initial concentration from 5 to 100 mg/L. The removal efficacy significantly varied from 78.8% to 41.9% at rising the initial concentration from 5 to 100 mg/L respectively. However, this showed that Bacillus subtilis HIB2 had slightly high removal efficiency of Pb 2+ in comparison of Pseudomonas aeruginosa HIB11 (Fig 5). Similar findings were reported by Azzam et al., (2015) who found that Bacillus sp. and Pseudomonas sp. had capability of eliminating Pb 2+ 99.7% and 99.6%. The key process for removal of Pb 2+ by bacterial isolates includes primarily neutralization and adsorption. Dabir et al., (2019) reported Microbacterium oxydans CM3 and Rhodococcus sp. AM1 to reduce 39% and 58% of lead (Pb) at 400 mg/L after 72 h, respectively. Vimalnath et al., (2018) reported Pseudomonas aeruginosa cells to uptake 71.7% of Pb concentration ranging from 10 to 250 mg/L. Murthy et al., (2012) studied the biosorption of lead ions using Bacillus cereus at different concentrations of lead from 100 to 500 mg/L and found decrease in removal with increase in Pb concentration . Guo et al., (2010) studied the Bacillus sp. L14 which showed the 80.48% removal of Pb under the initial concentration of 10 mg/L concentration within 24 hrs. incubation period. Table 4. The efficiency of Bioremediation of Lead (Pb 2+ ) at 10 mg/L at different time interval. Residual values (mg/L) Time (days) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 1 7.19 a ±0.29 28.1 7.15 a ±0.72 28.5 3 5.56 a ±0.67 44.4 6.47 b ±0.55 35.3 5 4.60 a ±0.55 54.0 5.01 b ±0.14 49.9 7 3.39 a ±0.59 66.1 4.14 b ±0.51 58.6 14 2.83 a ±0.53 71.7 2.93 a ±0.08 70.7 Table 5. The efficiency of Bioremediation of Lead (Pb 2+ ) at different time interval at different concentration after 14 incubation period. Residual values (mg/L) Conc. (mg/L) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 5 1.06 b ±0.04 78.8 1.21 a ±0.16 75.8 10 2.83 a ±0.53 71.7 2.93 a ±0.08 70.7 50 25.64 a ±4.35 48.7 27.17 b ±7.86 45.6 100 58.04 a ±7.88 41.9 61.49 a ±5.65 38.5 Note: 1 Mean ± SE; n=3, %R: Percent Removal 2 Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test). Removal of Cadmium (Cd 2+ ) The results for Cadmium (Cd 2+ ) exposed that Pseudomonas aeruginosa HIB11 attained better removal efficacy in comparison to Bacillus subtilis HIB2 at the same initial concentrations (Table 6 & 7). Furthermore, the removal efficacy of Cd 2+ using Pseudomonas aeruginosa HIB11 was also somewhat influenced (72.4–37.9%) at mostly varied the initial concentration from 5 to 100 mg/L (Fig 6). Similarly, the removal efficacy of Cd 2+ using Bacillus subtilis HIB2 was also affected at rising the initial concentration from 5 to 100 mg/L. This revealed that both Bacillus subtilis HIB2, and Pseudomonas aeruginosa HIB11 were significant competent for removal of Cd 2+ from polluted water. However, the remaining concentration of Cd 2+ after treatment were quite low using Pseudomonas aeruginosa HIB11 as compared to Bacillus subtilis HIB2 (Table 6 & 7). This metal biosorption efficiency of both isolates HIB2 and HIB11 could possibly be involvement of bio-flocculant formed by these bacteria. The bacterial constituent like cell wall and extracellular polysaccharide attributed to Ion uptake, has significant roles in curbing heavy metal contamination in the treatment procedures 4 . Similar studies were carried. Dabir et al., (2019) informed lead reduction of 200 mg/L concentration up to 20% by Bacillus sp. CM4. Li et al., (2019) reported average 54.7%, 43.2% and 7.34% removal of Cd at concentrations of 0.05mg/L, 0.5mg/L and 5mg/L by B. subtilis r espectively after 24 days incubation period. Guo et al., (2010) studied the Bacillus sp. L14 which showed 75.78%, removal of 10 mg/L concentration of Cd within 24 hrs. incubation period. Khan et al., (2015) reported E. coli P4 to reduce 18.8%, 37%, and 56% Cd 2+ after 48h, 96h, and 144h, respectively . Zeng et al., (2009) studied the removal efficiency of Pseudomonas aeruginosa for Cd and found 43.3% removal at initial concentration of 110.2 mg/L with 24 hours incubation period. Ziagova et al., (2007) reported Pseudomonas sp. Removed the 75% Cd at 200 mg/L initial concentration after 5 hours of incubation . Table 6. Bioremediation efficiency of Cadmium (Cd 2+ ) at 10 mg/L at different time interval. Residual values (mg/L) Time (days) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 1 8.75 a ±0.48 12.5 8.57 a ±0.73 14.3 3 8.02 a ±0.58 19.8 8.26 a ±0.75 17.4 5 6.36 a ±0.49 36.4 6.42 a ±0.43 35.8 7 5.56 a ±0.81 44.4 5.80 a ±0.22 42.0 14 4.61 a ±0.85 53.9 4.24 a ±1.26 57.6 Table 7. The efficiency of Bioremediation of Cadmium (Cd 2+ ) at different time interval at different concentration after 14 incubation period. Residual values (mg/L) Conc. (mg/L) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 5 1.40 a ±0.46 72.0 1.38 a ±0.45 72.4 10 4.61 a ±0.85 53. 4.24 a ±1.26 57.6 50 26.81 a ±1.64 46.3 25.04 a ±2.72 49.9 100 58.86 a ±13.67 41.1 62.02 a ±7.80 37.9 Note: 1 Mean ± SE; n=3, %R: Percent Removal 2 Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test). Removal of Nickel (Ni 2+ ) Similar patterns were observed for the reduction of Nickel (Ni 2+ ) ions using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 as presented in Table 8 and 9. The removal in Ni 2+ value was considerably higher using Bacillus subtilis HIB2 in comparison to Pseudomonas aeruginosa HIB11 Nonetheless, the removal efficacy of Ni 2+ was highly declined at rising the initial concentration from 5 to 100 mg/L. Bacillus subtilis HIB2 had the highest nickel removal proportion (69.4%) than Pseudomonas aeruginosa HIB11 (68.8%) at initial concentration of 5 mg/L. The removal efficacy of Ni 2+ was significantly declined from 68.8% to 33.8% at rising the initial concentration from 5 to 100 mg/L using Pseudomonas aeruginosa HIB11. Similarly, in case of Bacillus subtilis HIB2, the removal efficacy of Ni 2+ was significantly declined from 69.40% to 37.36% (Fig.7). This showed that the efficacy of both isolates is initial concentration dependent. Similar studies were reported. Das et al., (2014) observed the removal of Ni by Bacillus thuringiensis where observed a substantial percentage (82%) removal of Ni from the medium during in vitro culture. Similarly, there are other studies on the removal of Ni such as 95% Ni removal by Microbacterium sp. (Sathyavathi et al., 2014), biosorption of Ni & Cd, by E. coli sp. (Ansari & Malik, 2007) and removal of Ni by P roteus vulgaris strain, Stenotrophomonas sp. and Bacillus thuringiensis respectively (Kumar et al., 2016) . Table 8. Bioremediation efficiency of Nickel (Ni 2+ ) at 10 mg/L at different time interval. Residual values (mg/L) Time (days) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 1 8.95 a ±0.75 10.5 8.10 b ±0.24 19.0 3 7.77 a ±0.66 22.3 5.56 b ±0.10 44.4 5 7.01 a ±0.14 29.9 4.96 c ±0.10 50.4 7 6.39 a ±0.11 36.1 4.47 c ±0.12 55.3 14 3.14 a ±0.08 68.6 3.36 a ±0.34 66.4 Table 9. The efficiency of Bioremediation of Nickel (Ni 2+ ) at different time interval at different concentration after 14 incubation period. Residual values (mg/L) Conc. (mg/L) Bacillus subtilis HIB2 %R Pseudomonas aeruginosa HIB11 %R 5 1.53 a ±0.39 69.4 1.56 a ±0.36 68.8 10 3.14 a ±0.08 68.6 3.36 a ±0.34 66.4 50 23.89 a ±7.43 52.2 30.38 c ±6.49 39.2 100 62.64 b ±10.14 37.3 66.14 a ±7.10 33.8 Note: 1 Mean ± SE; n=3, %R: Percent Removal 2 Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test). Conclusions A physicochemical examination indicates the river's dire state and recommends taking immediate and decisive measures to restore its water quality. Hindon river has heavy metal resisting bacteria pertaining to genus Bacillus sp., Pseudomonas sp., which have the competence to be utilized for bioremediation of metals from river water. The purified bacterial isolates were proficient of surviving in high concentration of Cd, Ni and Pb. Bacillus sp. and Pseudomonas sp. were previous reported to be highly competent in removal of metals Cd, Ni and Pb, which supports their utilization for cost-saving and ecofriendly removal of these metals into less toxic form from river water. Results of biodegradation experiments shown the removal competences of 53.9% for Cd 2+ and 68.6% for Ni 2+ and 71.7% for Pb 2+ using Bacillus subtilis HIB2, where 57.6% for Cd 2+ and 66.4% for Ni 2+ and 70.7% for Pb 2+ using Pseudomonas aeruginosa HIB11 after 14 days of incubation at pH 7.0 and 37°C. However, the removal effectiveness of these three heavy metals was similar in both isolates. Ex-situ application for the removal of metals from polluted locations requires more research. Declarations Acknowledgments We are thankful to the faculty of Amity School of Biotechnology, AUH, Gurgaon, India, for providing the support and grateful to ITC Lab, Gurgaon for providing necessary facilities for this research. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. The authors declare that they have no competing interests. Intellectual Property We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. References Alabssawy, A.N., Hashem, A.H. (2024) Bioremediation of hazardous heavy metals by marine microorganisms: a recent review. Arch Microbiol 206 , 103 https://doi.org/10.1007/s00203-023-03793-5 Ansari, M. 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Rasayan Journal of Chemistry , 11 (4), 1477–1484. https://doi.org/10.31788/rjc.2018.1143075 Kumar, M., Kumar, V., Varma, A., Prasad, R., Sharma, A. K., Pal, A., and Singh, J. (2016). An efficient approach towards the bioremediation of copper, cobalt, and nickel contaminated field samples. Journal of soils and sediments , 16 (8), 2118-2127. Murthy, S., Bali, G., & Sk, S. (2012). Lead Biosorption by A Bacterium Isolated from Industrial Effluents. International Journal of Microbiology Research , 4 (3), 196–200. https://doi.org/10.9735/0975-5276.4.3.196-200 Narmadha, D. and Kavitha, M. S. (2012). Treatment of domestic wastewater using natural flocculants. International Journal of Life Sciences Biotechnology and Pharma Research , 1 (3), 206-213. Nokman, W., Benluvankar, V., Packiam, S. M. and Vincent, S. (2019). Screening and molecular identification of heavy metal resistant Pseudomonas putida S4 in tannery effluent wastewater. Biocatalysis and agricultural biotechnology , 18, 1010-52. https://doi.org/10.1016/j.bcab.2019.101052 Oziegbe, A.O. Oluduro, E.J. Oziegbe, E.F. Ahuekwe, S.J. Olorunsola (2021) Assessment of heavy metal bioremediation potential of bacterial isolates from landfill soils. Saudi Journal of Biological Sciences, Vol 28 :7,3948-3956. https://doi.org/10.1016/j.sjbs.2021.03.072 Sathyavathi, S., Manjula, A., Rajendhran, J. and Gunasekaran, P. (2014). Extracellular synthesis and characterization of nickel oxide nanoparticles from Microbacterium sp. MRS-1 towards bioremediation of nickel electroplating industrial effluent. Bioresource technology , 165, 270-273. 10.12691/ijebb-9-1-2 Singh KP, Mohan D, Sinha S, Dalwani R (2004) Impact assessment of treated/untreated wastewater toxicants discharged by sewage treatment plants on health, agricultural, and environmental quality in the wastewater disposal area. Chemosphere 55:227–255. https://doi.org/10.1016/j.chemosphere.2003.10.050 Suthar, S., Nema, A. K., Chabukdhara, M., & Gupta, S. (2009). Assessment of metals in water and sediments of Hindon River, India: Impact of industrial and urban discharges. Journal of Hazardous Materials , 171 (1–3), 1088–1095. https://doi.org/10.1016/j.jhazmat.2009.06.109 Verma, S., Bhargava, R., & Pruthi, V. (2006). Oily sludge degradation by bacteria from Ankleshwar, India. International Biodeterioration & Biodegradation , 57 (4), 207–213. https://doi.org/10.1016/j.ibiod.2006.02.004 Vimalnath, S., Ravishankar, H., Schwandt, C., Kumar, R. V., & Subramanian, S. (2018). Mechanistic studies on the biosorption of Pb(II) by Pseudomonas aeruginosa. Water Science and Technology , 78 (2), 290–300. https://doi.org/10.2166/wst.2018.296 Zeng, X. X., Tang, J. X., Liu, X. D., & Jiang, P. (2009). Isolation, identification, and characterization of cadmium-resistant Pseudomonas aeruginosa strain E1. Journal of Central South University of Technology , 16 (3), 416–421. https://doi.org/10.1007/s11771-009-0070-y Ziagova, M., Dimitriadis, G., Aslanidou, D., Papaioannou, X., Litopoulou Tzannetaki, E., & Liakopoulou-Kyriakides, M. (2007). Comparative study of Cd (II) and Cr(VI) biosorption on Staphylococcus xylosus and Pseudomonas sp . in single and binary mixtures. Bioresource Technology , 98 (15), 2859–2865. https://doi.org/10.1016/j.biortech.2006.09.043 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4652732","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329149698,"identity":"1ffe447e-b496-475f-9c09-d41d415d3940","order_by":0,"name":"Deepak Kumar","email":"","orcid":"","institution":"Amity University Haryana","correspondingAuthor":false,"prefix":"","firstName":"Deepak","middleName":"","lastName":"Kumar","suffix":""},{"id":329149699,"identity":"53e5e84e-2419-47cd-88ce-7e3a1c4992e1","order_by":1,"name":"Sangeeta Kumari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYJACZgYGCQZ+ECuhgBQtkg0gLQbEa2FgMDgAJolQLt9+9vDnwh0W8sbnVyd+eGDAIM8vdgC/FoMzeWnSM89IGG678XazBNBhhjNnJxDQwpBjxszbJpFgduPsBpCWBIPbBLTI978x/gzSYjzj7OYfRGlhuJFjIA3SYsDfu404WwxuvDEDaTGccYN3m0WCgQRhv8j354AcVifP3392880fFTby/NKEHAYHEmCVEsQqBwH+A6SoHgWjYBSMgpEEANwRPzuAvfwiAAAAAElFTkSuQmCC","orcid":"","institution":"Amity University Haryana","correspondingAuthor":true,"prefix":"","firstName":"Sangeeta","middleName":"","lastName":"Kumari","suffix":""}],"badges":[],"createdAt":"2024-06-28 07:09:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4652732/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4652732/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60843161,"identity":"72eb3c9e-b3c0-44ea-a23a-d4b38c86c960","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17855,"visible":true,"origin":"","legend":"\u003cp\u003eBioconversion mechanism for hazardous and recalcitrant chemicals by microbiota in simpler Less toxic derivatives.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/2ce550e00a5d9dc9489397de.jpg"},{"id":60843160,"identity":"3a64286c-93c0-40b1-80ad-adaa357d82f7","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42983,"visible":true,"origin":"","legend":"\u003cp\u003eMap of sampling sites at the Hindon River\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/ebc49b16341e1292446ed89a.jpg"},{"id":60843495,"identity":"c76215e9-de69-41b4-b33d-c9a15a0ea739","added_by":"auto","created_at":"2024-07-22 18:05:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 3 a. \u003c/strong\u003ePure cultures of two (B2 and B11) bacterial isolates\u003c/p\u003e","description":"","filename":"3a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/a5ba011cafe006f8f09c1000.jpg"},{"id":60843162,"identity":"40f358e7-340d-4e0a-8dc0-f29f9c8b6e85","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 3b.\u003c/strong\u003e Biochemical characterization of \u003cem\u003eBacillus subtilis \u003c/em\u003eHIB2 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e","description":"","filename":"3b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/d57f40612d146997e301c3ae.jpg"},{"id":60843167,"identity":"729b87d8-a129-4195-ae59-1ccd63672807","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 4.\u003c/strong\u003e Phylogenetic tree showing the genetic relationship among the cultivated bacteria and reference 16S rRNA sequences from the GenBank based on partial 16S ribosomal RNA gene sequences. Scale bar 0.001 = 0.1% difference among nucleotide sequences.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/aa6cc251cad30297508fda08.jpg"},{"id":60843164,"identity":"e3fbc1f9-4ffc-414e-b04f-0533425f6c9b","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 5.\u003c/strong\u003eBatch mode study of Pb degradation \u003cstrong\u003eA. \u003c/strong\u003eat different\u003cstrong\u003e \u003c/strong\u003eIncubation time\u003cstrong\u003e, B. \u003c/strong\u003eat different\u003cstrong\u003e c\u003c/strong\u003eoncentration\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/16c8f51fcbea8e30b4d95a2d.jpg"},{"id":60843165,"identity":"fbfeed30-3b16-447d-91f7-8e6cb746dc89","added_by":"auto","created_at":"2024-07-22 17:57:24","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":26774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 6.\u003c/strong\u003eBatch mode study of Cd degradation \u003cstrong\u003eA. \u003c/strong\u003eat different\u003cstrong\u003e \u003c/strong\u003eIncubation time\u003cstrong\u003e, B. \u003c/strong\u003eat different\u003cstrong\u003e c\u003c/strong\u003eoncentration\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/65bfab0c02277d19655ffd82.jpg"},{"id":60843496,"identity":"53da56f0-e9be-45dd-b811-d733061d83af","added_by":"auto","created_at":"2024-07-22 18:05:24","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":25906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 7.\u003c/strong\u003e Batch mode study of Ni degradation \u003cstrong\u003ea. \u003c/strong\u003eIncubation time\u003cstrong\u003e, b. \u003c/strong\u003eConcentration\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/036d1b97340090d2311cf670.jpg"},{"id":88332962,"identity":"ecaaf2d8-b0a2-4943-9e95-0aa670829ad2","added_by":"auto","created_at":"2025-08-05 11:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1804013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4652732/v1/b7343bb0-907a-4fd6-a5ee-96c222c225f9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effective Bioremediation of Heavy Metals for Environmental Sustainability Approach using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 isolated from Hindon River, India","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRising river water pollution and environmental degradation are attributable to fast-growing populations, land expansion along river basins, and urban and industrial growth. Several sectors, such as electroplating and battery, leather, and paint producing units, are now using diverse heavy metals, which could result in many heavy metals being released into the environment \u003cb\u003e(Satyavathi\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2014).\u003c/b\u003e Heavy inorganic metals bioaccumulation and consequent biomagnification such as cadmium (Cd), lead (Pb), chromium (Cr) and nickel (Ni) which can reach the level that are toxic to living organisms in the river, emphasis on requirement for removal of heavy metals \u003cb\u003e(Khatri\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2020\u003c/b\u003e: Kalaimurugan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). River Hindon, originating from the upper Shivalik\u0026rsquo;s in Uttar Pradesh in Saharanpur, Uttar Pradesh, India, streams 265 km through six districts including Muzaffarnagar, Meerut, Baghpat, Ghaziabad and Gautam Budh Nagar, till merge with the Yamuna, is a main resource of water to the vastly colonized and largely populated rural area, where, its water is applied for in-house, farming and industrialized related activities, henceforth the current fast development in urbanization and industrialization along the course of Hindon has caused numerous problems of contaminations in Hindon river \u003cb\u003e( Kumar\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2016 and\u003c/b\u003e Suthar et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Several physical, chemical, and biological approaches have been projected for the removal of heavy metals from polluted soil and water of which microorganisms-based bioremediation is well-thought-out as a hopeful and worthwhile technique. \u003cb\u003eVerma et al.,2006\u003c/b\u003e and Oziegbe et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e discussed that microorganisms-based bioremediation is economical and eco-friendly to mitigate the heavy metal contamination.\u003c/p\u003e \u003cp\u003eBioremediation can be described as the application of microorganisms for removal of contaminants in water and soil. The bioaugmentation approach, adding microorganisms with efficacious degradation ability in the polluted areas, has projected and measured to increase bioremediation of \u003cb\u003epollutants (Govarthanan\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2016;\u003c/b\u003e Janbandhu and Fulekar, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; \u003cb\u003eCerquiera\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2011\u003c/b\u003e). The engagement of a bacterial consortium has confirmed to be extra valuable comparted with single isolate due to synergistic relations amongst members of the relatives, which may result in the comprehensive degradation of the pollutants \u003cb\u003e(\u003c/b\u003eNokman et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The application of microorganisms with verified degradation competency and resistance in the polluted environment is crucial for a successful bioaugmentation. The heavy metal degradation process applying microorganisms includes biosorption, bioleaching, biomineralization, immobilization and redox reactions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Biosorption which is one of the bioremediation processes, is most effective in heavy metal detoxification \u003cb\u003e(Hargreaves\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2015 ;\u003c/b\u003e Alabssawy and Hashem, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMany studies have found that bacteria isolated from different sources had the capability of flourish in the harsh metal pollution \u003cb\u003e(Bestawy\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2012: Bhakta\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2014 and Guo\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e.,\u003cb\u003e2010).\u003c/b\u003e Hamid et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e isolated heavy-metal tolerant bacterial strains from industrial sites and scrap yards in Kashmir, India.\u003c/p\u003e \u003cp\u003eTo increase their effectiveness in the degradation of heavy metals as a sustainable environment technology, the current investigation targeted at the isolation, screening, identification, and characterisation of heavy metals degrading bacteria isolated from the Hindon River.\u003c/p\u003e \u003cp\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater samples were\u0026nbsp;collected from five different sites of Hindon river \u003cstrong\u003e(Fig 2)\u003c/strong\u003e i.e. Atali village (S1)-29\u0026deg;12\u0026apos;43.4\u0026quot;N 77\u0026deg;31\u0026apos;18.4\u0026quot;E, Baparsi village (S2)-29\u0026deg;11\u0026apos;07.2\u0026quot;N 77\u0026deg;29\u0026apos;42.6\u0026quot;E, Shekpura village (S3)-29\u0026deg;05\u0026apos;34.4\u0026quot;N 77\u0026deg;25\u0026apos;21.1\u0026quot;E, Kalina village (S4)-29\u0026deg;04\u0026apos;27.1\u0026quot;N 77\u0026deg;27\u0026apos;43.6\u0026quot;E\u0026nbsp;and Kinauni village (S5)-29\u0026deg;03\u0026apos;05.2\u0026quot;N 77\u0026deg;27\u0026apos;15.9\u0026quot;E, in sterilized bottles in month of Jan -Jun 2017 according to standard method \u003cstrong\u003e(APHA,2012 and CPCB,2009).\u003c/strong\u003e These samples were kept into thermo boxes immediately after collection, followed by transportation to laboratory. Further, the samples were stored at 4◦C after taking to the laboratory for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical and chemical characterizations of the Hindon river water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHindon river water samples were analyzed for physico-chemical parameters i.e. pH, electrical conductivity (EC), chemical oxygen demand (COD), biological oxygen demand (BOD), total dissolved solids (TDS), alkalinity, hardness, sulfate (SO\u0026sup2;⁻₄), nitrate (NO⁻₃)\u0026nbsp;and chloride (Cl⁻)\u0026nbsp;were estimated according to American Public Health Association\u0026nbsp;methods (\u003cstrong\u003eAPHA,2012\u003c/strong\u003e).\u0026nbsp;For heavy metals concentrations (Cd, Ni, and Pb), the samples were digested with concentrated nitric acid followed by filtration through 0.45 \u0026micro;m filter paper and were analyzed by using ICP-MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and screening of heavy metal resistant bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected Hindon river water samples were serially diluted up to 10\u003csup\u003e\u0026minus;6\u003c/sup\u003e dilution with distilled water and inoculated on the nutrient agar medium containing 200 mg/kg of heavy metal (Cd, Ni and Pb) by spread plate method. Plates were kept in the incubator for incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours. Pure culture of morphologically different colonies was isolated and purified by repeated streaking and then maintained at 4\u003csup\u003eo\u003c/sup\u003eC on nutrient agar slants for further experiment. More, screening of bacterial isolates was done based on resistance against heavy metals. To check the heavy metal resistance level of all selected isolates were assessed by inoculating isolates in nutrient agar plates containing different concentrations of concerned heavy metals ranging from 100 to 500 mg/L. The plates were incubated 24h at 37\u0026deg;C and observed for bacterial growth on the culture medium and then highly resistive bacterial strains were selected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of Selected Bacterial Isolate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese isolates were primarily characterized through morphological characteristics such as gram\u0026rsquo;s staining, shape, motility test. Additionally, these were identified by biochemical characteristics based on Bergey\u0026rsquo;s Manual of Systematic Bacteriology. The biochemical tests performed to assess the capability of the isolates were Indole production, methyl red (MR) test, voges proskauer (VP) test, citrate utilization test and H\u003csub\u003e2\u003c/sub\u003eS production. Additionally, carbohydrate fermentation tests were conducted to evaluate the capacity of the isolates to ferment sugars such as glucose and sucrose, where a color transformation from pink to light yellow showed the fermentation of sugar. Moreover, the enzymatic hydrolysis tests such as starch hydrolysis, catalase test, oxidase test, urease test, and nitrate reduction test were also studied for the identification of isolates.\u003c/p\u003e\n\u003cp\u003eThe isolates identified by morphological and biochemical characterization were grown on nutrient broth. Conclusively, the bacterial isolates were further examined for their molecular characteristics by\u0026nbsp;16S rRNA gene sequencing. Genomic DNA was extracted from bacterial isolates and processed for 16S rRNA sequencing, where Realtime-PCR (polymerased chain reaction) was used to amplify extracted DNA using the universal primer in a thermal cycler. The forward primer sequence was 16SF (2f) 5\u0026apos;- AGAGTTTGATCMTGGCTCAG-3\u0026apos; and the reverse primer sequence was 16SR (1492r) 5\u0026apos;-TACGGYTACCTTGTTACGACTT-3\u0026apos;. The standalone PCR-primers and dNTPs were removed from PCR-products by using a cleaning kit (Montage-PCR made by Millipore). The PCR-product was processed for sequencing using the primers (27F/1492R). Sequencing reactions were carried out using an ABI-PRISM\u0026reg; BigDyeTM Terminator Cycle-sequencing kits with AmpliTaq\u0026reg;DNA polymerase (FS enzyme) (Applied Biosystems). Single-pass sequencing was conducted on individual template using above universal primers of 16S rRNA. The fluorescent labelled fragments were separated from the standalone terminators with ethanol. The samples were re-suspended in distilled-water and further processed for electrophoresis in an ABI 3730xl-sequencer (Applied-Biosystems). The 16s rRNA sequences were blasted using BLASTA tool at NCBI site (http://www.ncbi.nih.gov/BLAST/). The guide tree into a text file in Newick format was downloaded which was recognized by phylogenetic software named Mega V software (version-5.0) and then a phylogenetic tree was created using the neighbor-joining method. The gene sequences of two bacterial isolates were deposited to the National Centre for Biotechnology Information (NCBI) Gene bank and then the accession numbers were obtained for further communication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiodegradability assay of bacterial isolates for Heavy Metals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, certified reference material (CRM) of individual heavy metal (Cd, Ni and Pb) and supra-pure nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e) of analytical grade were purchased from Merck. Working solution standard was made by dissolving stock solution (1000 mL) in deionized distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndividual isolate was inoculated into the flask containing nutrient broth which was incubated for 24 to 48 hours in the shaking incubator at 150 rpm, while pH and temperature were adjusted 7.0 and 37 ̊C, respectively. Once optical density (O.D.) attained 0.5 (k = 600 nm: for identical enzymatic activity), then 5, 10, 50 \u0026amp; 100 mg/L of sterilized heavy metals (Cd, Ni and Pb) was supplemented individually in each inoculated flask of 250 mL and incubated another time at the same condition for 14 days. After defined time interval of incubation, 10 ml of culture broth from shaking flask was collected and centrifuged (Remi, R-8M/CDLC-3111, India) at 5000 rpm for 15 min to acquire a cell-free medium, then 1 mL of supernatants was separated and digested with 5 mL of concentrated HNO\u003csub\u003e3\u003c/sub\u003e in microwave digester (Anton paar, ECO/81331413, \u0026lrm;Graz, Austria) to accomplish acid digestion. The solution was filtered using a filter paper (Whatman No. 42) to eliminate any insoluble material and transferred into a centrifuge tube and then made-up by 50 ml. To measure reduction, this solution of total heavy metal was analyzed by ICP-MS (Thermo Scientific, ICAP Q (2271R)) and the results were calculated for heavy metal degradation capacity (%) as per below: HM= Heavy Metal\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"400\" height=\"108\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were made in triplicate and the data was expressed as the means \u0026plusmn; SD. ANOVA were used to study the statistically significant differences (p\u0026lt;0.05) for heavy-metal removal after treatment. The results of each experiment were assumed to be independent with different variance.\u003c/p\u003e"},{"header":"Results \u0026 Discussion","content":"\u003cp\u003e\u003cstrong\u003eCharacteristic of Hindon river water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results obtained at the characterization of various parameters in the water samples collected from five different sites of Hindon River are summarized in Table 1. The mean values of pH in water samples from five sites, ranged from 7.25 to 7.69, were found within the permissible limits. The pH values of water samples were marginally alkaline at all five sites, showing that there is neither acid nor alkaline pollution in the water samples from Hindon river \u003cstrong\u003e(Gupta \u003cem\u003eet al.\u003c/em\u003e, 2013).\u003c/strong\u003e Electrical conductivity (EC) of water samples at all sites, where mean values varied from 1667.0 to 1916.75 \u0026mu;S/cm, were beyond the acceptable limit, where, what sample from Atali village site exhibited highest EC compared to that of other four sites. High EC was attributed to the existence of high number of dissolved ions (\u003cstrong\u003eVerma et al., 2006\u003c/strong\u003e). River water pollution by release of domestic and industrial waste rises the EC. High number of dissolved ions in water causes unpleasant taste and moreover distresses animals and plants \u003cstrong\u003e(Kumar \u003cem\u003eet al\u003c/em\u003e.,2018)\u003c/strong\u003e. Total dissolved solid (TDS) ranged from 773.25 to 1238.0 mg/L as mean value, was also beyond the permissible limit at all five sites of river. High level of TDS in river water is primarily due to the occurrence of carbonates (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), bicarbonates (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e),\u0026nbsp;chlorides (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e), phosphates (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e) and nitrate compound such as Ca (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, Mg (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, KNO\u003csub\u003e3\u003c/sub\u003e and \u0026lrm;Mn (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, organic salts, and other solid elements. Contagion of Hindon river by industrial effluent could be the responsible for increase in TDS as the dissolved ions are significantly existing in Industrial waste. The existence of high\u0026nbsp;level of TDS in river water also results in unfit for drinking. The mean values of alkalinity varied from 261.75 to 368.75 mg/L was also significantly higher than the permissible limits at the all sites of Hindon river. High concentration of dissolved ions rises the alkalinity of river water, which possibly can also be one of the factors for enlarged alkalinity of water samples at all five sites. High alkalinity of river water results in unfit for drinking as well as for irrigation\u003csup\u003e1\u003c/sup\u003e. Hardness of water samples (mean value from 319.25 to 374.50 mg/L) exceeded the permissible limit at the all five sites. Well, hardness indicates the soap forming ability of a water sample where the two cations primarily Ca\u003csup\u003e+\u003c/sup\u003e and Mg\u003csup\u003e+\u003c/sup\u003e are accountable. Existence of other ions such as sulphate (SO\u0026sup2;⁻₄), nitrate (NO⁻₃), and chloride (Cl⁻) in river water was found within the permissible limit. Sulphate is mostly natural in nature attributed to primarily by mineral sources like gypsum, etc. \u003cstrong\u003eVerma \u003cem\u003eet al\u003c/em\u003e.,2006\u003c/strong\u003e studied on high concentration sulphate in drinking water may cause several intestinal diseases. Chloride is extensively dispersed in nature in the form of salts of Na (NaCl), K (KCl) and Ca (CaCl\u003csub\u003e2\u003c/sub\u003e). COD and BOD values for Hindon river water were above the acceptable values at all five sites of river. A high BOD and COD values showed that the water has highly oxygen demanding waste attributed to the presence of organic pollutants which causes the depletion of dissolved oxygen (DO) which is a fundamental requirement for aquatic life. The high value of COD gives valuable information about the pollution potential of the Hindon river water \u003cstrong\u003e(Gupta \u003cem\u003eet al\u003c/em\u003e., 2017).\u003c/strong\u003e The heavy metals Cd, Ni and Pb were found above permissible limits for drinking water as per BIS.\u003c/p\u003e\n\u003cp\u003eThe physicochemical characterization of Hindon river water at five places revealed that the river had high levels of organic and inorganic pollution, which was most likely attributable to trash produced by companies located along the river\u0026apos;s banks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Physicochemical analysis of the water samples collected along four sites of the Hindon river (March-June 2017).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"644\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSites\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBIS/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCPCB*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.689655172413794%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.612068965517242%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS2\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.31896551724138%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.689655172413794%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS4\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.689655172413794%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS5\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn;SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e7.25\u0026plusmn;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e7.25\u0026plusmn;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e7.33\u0026plusmn;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e7.48\u0026plusmn;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e7.69\u0026plusmn;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e6.5-8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e1916.75\u0026plusmn;327.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e1901.25\u0026plusmn;85.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e1667\u0026plusmn;178.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e1767.75\u0026plusmn;332.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e1843.75\u0026plusmn;423.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026mu;S/cm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e1178.50\u0026plusmn;223.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e1238\u0026plusmn;34.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e1044\u0026plusmn;93.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e1117.25\u0026plusmn;136.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e773.25\u0026plusmn;84.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e500\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlkalinity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e344.75\u0026plusmn;21.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e368.75\u0026plusmn;33.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e327.75\u0026plusmn;56.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e348\u0026plusmn;103.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e261.75\u0026plusmn;44.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHardness\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e319.25\u0026plusmn;35.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e362.50\u0026plusmn;35.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e374.50\u0026plusmn;71.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e359.25\u0026plusmn;55.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e339\u0026plusmn;121.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e356.50\u0026plusmn;66.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e365.25\u0026plusmn;92.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e307.25\u0026plusmn;42.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e327.25\u0026plusmn;34.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e339.5\u0026plusmn;67.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBOD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e109.50\u0026plusmn;17.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e91.5\u0026plusmn;21.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e84.5\u0026plusmn;16.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e92.75\u0026plusmn;10.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e92.5\u0026plusmn;25.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le; 2*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNO⁻₃\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e40.75\u0026plusmn;10.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e48.25\u0026plusmn;4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e44\u0026plusmn;18.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e31\u0026plusmn;13.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e25\u0026plusmn;9.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSO\u0026sup2;⁻₄\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e48.25\u0026plusmn;9.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"bottom\"\u003e\n \u003cp\u003e56.00\u0026plusmn;7.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"bottom\"\u003e\n \u003cp\u003e46.75\u0026plusmn;0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e51\u0026plusmn;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"bottom\"\u003e\n \u003cp\u003e48\u0026plusmn;6.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCl⁻\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e178.25\u0026plusmn;21.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e199.25\u0026plusmn;20.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"top\"\u003e\n \u003cp\u003e168\u0026plusmn;36.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e163.75\u0026plusmn;47.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e108\u0026plusmn;25.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.022\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e0.037\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"top\"\u003e\n \u003cp\u003e0.027\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.04\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.002\u0026plusmn;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.048\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e0.021\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"top\"\u003e\n \u003cp\u003e0.144\u0026plusmn;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.068\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.012\u0026plusmn;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.111801242236025%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.052\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.130434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e0.046\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.198757763975156%\" valign=\"top\"\u003e\n \u003cp\u003e0.051\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.054\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.906832298136646%\" valign=\"top\"\u003e\n \u003cp\u003e0.016\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.385093167701863%\" valign=\"top\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.453416149068323%\" valign=\"top\"\u003e\n \u003cp\u003emg/L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present investigation total number of 32 bacteria were isolated initially from the water samples following enrichment in nutrient media containing 200 mg/L heavy metals (Cd, Ni and Pb). When these strains were further studied for resistance and degradation of higher concentrations of heavy metals, two strains, viz., strain HIB2 and HIB11 were found to utilize and grow in nutrient containing up to 500 mg/L heavy metals and were selected for further study. The morphological and biochemical characteristics attentively suggested that these isolates were \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esp\u003c/em\u003e. and \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003esp.\u003c/em\u003e (Tables 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical and Molecular characterization (16S rRNA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, the bacterial isolate B2 was found to be gram-positive, small rod-shaped and motile. This isolate was able to ferment sucrose and glucose. It was MR negative and VP positive, and showed starch hydrolysis, but did not show H\u003csub\u003e2\u003c/sub\u003eS production. It showed positive results for the catalase test, nitrate reduction, oxidase test and citrate utilization tests. However, it showed negative results for urease test and indole test. The isolate B11 was found rod-shaped, gram negative and motile. It did not produce acid with sucrose and glucose. It showed negative results of MR test, indole test, urease test, and VP test, while positive results for catalase test, oxidase test, nitrate test, and citrate test. Further did not show starch hydrolysis and H\u003csub\u003e2\u003c/sub\u003eS production. Biochemical test results are also shown in Fig. 2.\u003c/p\u003e\n\u003cp\u003eMolecular identification was performed for the confirmation of selected isolates by amplification and sequencing of the 16S rRNA gene. A pure culture of selected\u0026nbsp;isolates was grown on nutrient agar for 24h and the identification of isolates was further confirmed using 16S rRNA gene sequencing using the universal primers, the forward primer sequence\u0026nbsp;16SF (2f) 5\u0026apos;- AGAGTTTGATCMTGGCTCAG-3\u0026apos; and the reverse primer sequence was 16SR (1492r) 5\u0026apos;-TACGGYTACCTTGTTACGACTT-3\u0026apos;. The resulting sequences were compared with their closest relatives available in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the NCBI-BLAST tool. The nucleotide BLAST similarity search analysis showed that the two isolates matched with their closest relatives as follows:\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe closest sequence identity of Isolate B2 was found to be with \u003cem\u003eBacillus subtilis\u0026nbsp;\u003c/em\u003estrain based on nucleotide homology and phylogenetic analysis (\u003cstrong\u003eFig. 3a).\u003c/strong\u003e Similarly, isolate B11 had the closest sequence identity with \u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003estrain\u0026nbsp;(\u003cstrong\u003eFig. 3b).\u003c/strong\u003e Additionally, these bacterial sequences were deposited in the NCBI Gene bank and got assigned the accession numbers for further communication. Isolate B2 was named as \u003cem\u003eBacillus subtilis\u0026nbsp;\u003c/em\u003estrain HIB2\u003cem\u003e\u0026nbsp;\u003c/em\u003ewith Accession Number: MK936323.1 and isolate B11 was named as \u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003estrain HIB11\u003cem\u003e\u0026nbsp;\u003c/em\u003ewith Accession Number: MK937645.1 \u003cstrong\u003e(Table 3)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The phylogenetic tree of each strain was constructed using the neighbor-joining algorithm from MEGA-V (Version 5.0) software and their phylogenetic relationship were inferred (\u003cstrong\u003eFig. 4).\u003c/strong\u003e The approximate phylogenetic position of the strains B2 and B11 are depicted in (\u003cstrong\u003eFig 4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Morphological \u0026amp; Biochemical characteristic of the selected bacterial isolates.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"396\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBacillus Subtilis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;HIB2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;HIB11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eGram staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eShape\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003eRods\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003eRods\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eMotility\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eIndole production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eMethyl red\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eVP (Voges Proskauer)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eCitrate utilization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eUrease test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eNitrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eStarch hydrolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eSucrose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eGlucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.757575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.90909090909091%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eIdentification of effective bacterial isolates by 16S rRNA gene sequence analyses\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"456\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.473684210526315%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.8421052631579%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.19298245614035%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession no.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.49122807017544%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentity (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.473684210526315%\" valign=\"top\"\u003e\n \u003cp\u003eHIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.8421052631579%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.19298245614035%\" valign=\"top\"\u003e\n \u003cp\u003eMK936323.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.49122807017544%\" valign=\"top\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.473684210526315%\" valign=\"top\"\u003e\n \u003cp\u003eHIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.8421052631579%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeroginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.19298245614035%\" valign=\"top\"\u003e\n \u003cp\u003eMK937645.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.49122807017544%\" valign=\"top\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioremediation of Heavy metals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, metal removal was carried out using bacterial strains isolated from Hindon river water and after screening based on resistance against metal ions. Isolated bacteria in this study have unique metal accumulation characteristics and easy to cultivate. Tolerance and removal of Cd, Ni and Pb were determined in the aqueous medium. The amount of heavy metals removal was evaluated by comparing initial metal concentration and final concentration after treatment by bacterial isolates and their consortium \u003cstrong\u003e(Table 4 to 9).\u003c/strong\u003e The bacterial isolates in the medium were studied for:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(i). The reduction rate of heavy metals at different concentrations from 5 to 100 mg/L at 14 days incubation period \u003cstrong\u003e(Fig. 5 B \u0026ndash; 7B).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(ii). The reduction rate of heavy metal (10 mg/L concentration) at different time intervals of the incubation period for 14\u003csup\u003e\u0026nbsp;\u003c/sup\u003edays \u003cstrong\u003e(Fig.5 A- 7A).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeavy metal concentration was analyzed using the ICP-MS instrument. (Thermo fisher).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of Lead (Pb\u003csup\u003e2+\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results displayed in Table 4 \u0026amp; 5 shows that both isolates \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2\u003cem\u003e.\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 were well competent for removal of lead (Pb\u003csup\u003e2+\u003c/sup\u003e) from polluted water. There was significant influence on the removal efficacy of Pb\u003csup\u003e2+\u003c/sup\u003e using \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 when increased the initial concentration from 5 to 100 mg/L. The remaining values of Pb\u003csup\u003e2+\u003c/sup\u003e in the media were ranged from 1.21\u0026plusmn;0.16\u0026nbsp;to 61.49\u0026plusmn;5.65\u0026nbsp;mg/L at rising the initial concentration from 5 to 100 mg/L respectively. In case of \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2, the removal efficacy of Pb\u003csup\u003e2+\u003c/sup\u003e was also influenced at rising the initial concentration from 5 to 100 mg/L. The removal efficacy significantly varied from 78.8% to 41.9% at rising the initial concentration from 5 to 100 mg/L respectively. However, this showed that \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2 had slightly high removal efficiency of Pb\u003csup\u003e2+\u003c/sup\u003e in comparison of \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 (Fig 5). Similar findings were reported by \u003cstrong\u003eAzzam \u003cem\u003eet al.,\u003c/em\u003e (2015)\u003c/strong\u003e who found that \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003ePseudomonas\u003c/em\u003e sp. had capability of eliminating Pb\u003csup\u003e2+\u003c/sup\u003e 99.7% and 99.6%. The key process for removal of Pb\u003csup\u003e2+\u003c/sup\u003e by bacterial isolates includes primarily neutralization and adsorption.\u0026nbsp;\u003cstrong\u003eDabir \u003cem\u003eet al.,\u003c/em\u003e (2019)\u003c/strong\u003e reported \u003cem\u003eMicrobacterium oxydans\u003c/em\u003e CM3 and \u003cem\u003eRhodococcus\u003c/em\u003e sp. AM1 to reduce 39% and 58% of lead (Pb) at 400 mg/L after 72 h, respectively. \u003cstrong\u003eVimalnath \u003cem\u003eet al.,\u003c/em\u003e (2018)\u003c/strong\u003e reported \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e cells to uptake 71.7% of Pb concentration ranging from 10 to 250 mg/L. \u003cstrong\u003eMurthy \u003cem\u003eet al.,\u003c/em\u003e (2012)\u003c/strong\u003e studied the biosorption of lead ions using \u003cem\u003eBacillus cereus\u003c/em\u003e at different concentrations of lead from 100 to 500 mg/L and found decrease in removal with increase in Pb concentration\u003cstrong\u003e. Guo \u003cem\u003eet al.,\u003c/em\u003e (2010)\u003c/strong\u003e studied the \u003cem\u003eBacillus sp.\u003c/em\u003e L14 which showed the 80.48% removal of Pb under the initial concentration of 10 mg/L concentration within 24 hrs. incubation period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e The efficiency of Bioremediation of Lead (Pb\u003csup\u003e2+\u003c/sup\u003e)\u0026nbsp;at 10 mg/L at different time interval.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"357\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.565826330532213%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.43417366946778%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e7.19\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e7.15\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e28.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e5.56\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e44.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e6.47\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e35.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e4.60\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e54.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e5.01\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e49.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e3.39\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e66.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e4.14\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e58.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.606741573033707%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.213483146067414%\" valign=\"top\"\u003e\n \u003cp\u003e2.83\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e71.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.70786516853933%\" valign=\"top\"\u003e\n \u003cp\u003e2.93\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.235955056179776%\" valign=\"top\"\u003e\n \u003cp\u003e70.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u003c/strong\u003e The efficiency of Bioremediation of Lead (Pb\u003csup\u003e2+\u003c/sup\u003e) at different time interval at different concentration after 14 incubation period.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"360\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.277777777777779%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.72222222222223%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.23545706371191%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc. (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.0387811634349%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.296398891966758%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.795013850415515%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.634349030470915%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.23545706371191%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.0387811634349%\" valign=\"top\"\u003e\n \u003cp\u003e1.06\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.296398891966758%\" valign=\"top\"\u003e\n \u003cp\u003e78.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.795013850415515%\" valign=\"top\"\u003e\n \u003cp\u003e1.21\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.634349030470915%\" valign=\"top\"\u003e\n \u003cp\u003e75.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.23545706371191%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.0387811634349%\" valign=\"top\"\u003e\n \u003cp\u003e2.83\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.296398891966758%\" valign=\"top\"\u003e\n \u003cp\u003e71.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.795013850415515%\" valign=\"top\"\u003e\n \u003cp\u003e2.93\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.634349030470915%\" valign=\"top\"\u003e\n \u003cp\u003e70.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.23545706371191%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.0387811634349%\" valign=\"top\"\u003e\n \u003cp\u003e25.64\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.296398891966758%\" valign=\"top\"\u003e\n \u003cp\u003e48.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.795013850415515%\" valign=\"top\"\u003e\n \u003cp\u003e27.17\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;7.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.634349030470915%\" valign=\"top\"\u003e\n \u003cp\u003e45.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.23545706371191%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.0387811634349%\" valign=\"top\"\u003e\n \u003cp\u003e58.04\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;7.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.296398891966758%\" valign=\"top\"\u003e\n \u003cp\u003e41.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.795013850415515%\" valign=\"top\"\u003e\n \u003cp\u003e61.49\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;5.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.634349030470915%\" valign=\"top\"\u003e\n \u003cp\u003e38.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Note:\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eMean \u0026plusmn; SE; n=3, %R: Percent Removal\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eMean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p\u0026lt;0.05 significance level (Duncan\u0026rsquo;s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of Cadmium (Cd\u003csup\u003e2+\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results for Cadmium (Cd\u003csup\u003e2+\u003c/sup\u003e) exposed that \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 attained better removal efficacy in comparison to \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2\u0026nbsp;at the same initial concentrations (Table 6 \u0026amp; 7). Furthermore, the removal efficacy\u003cem\u003e\u0026nbsp;\u003c/em\u003eof Cd\u003csup\u003e2+\u003c/sup\u003e using\u003cem\u003e\u0026nbsp;Pseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 was also somewhat influenced (72.4\u0026ndash;37.9%) at mostly varied the initial concentration from 5 to 100 mg/L (Fig 6). Similarly, the removal efficacy of Cd\u003csup\u003e2+\u003c/sup\u003e using \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2 was also affected at rising the initial concentration from 5 to 100 mg/L. This revealed that both \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2, and \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 were significant competent for removal of Cd\u003csup\u003e2+\u003c/sup\u003e from polluted water. However, the remaining concentration of Cd\u003csup\u003e2+\u003c/sup\u003e after treatment were quite low using \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11\u003cem\u003e\u0026nbsp;\u003c/em\u003eas compared to \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Table 6 \u0026amp; 7). This metal biosorption efficiency of both isolates\u0026nbsp;HIB2\u0026nbsp;and HIB11 could possibly be involvement of bio-flocculant formed by these bacteria. The bacterial constituent like cell wall and extracellular polysaccharide attributed to Ion uptake, has significant roles in curbing heavy metal contamination in the treatment procedures\u003csup\u003e4\u003c/sup\u003e. Similar studies were carried. Dabir \u003cem\u003eet al.,\u003c/em\u003e (2019) informed lead reduction of 200 mg/L concentration up to 20% by \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003esp. CM4. Li \u003cem\u003eet al.,\u003c/em\u003e (2019) reported average 54.7%, 43.2% and 7.34% removal of Cd at concentrations of 0.05mg/L, 0.5mg/L and 5mg/L by \u003cem\u003eB. subtilis r\u003c/em\u003eespectively after 24 days incubation period. \u003cstrong\u003eGuo \u003cem\u003eet al.,\u003c/em\u003e (2010)\u003c/strong\u003e studied the \u003cem\u003eBacillus sp.\u003c/em\u003e L14 which showed 75.78%, removal of 10 mg/L concentration of Cd within 24 hrs. incubation period. \u003cstrong\u003eKhan \u003cem\u003eet al.,\u003c/em\u003e (2015)\u0026nbsp;\u003c/strong\u003ereported \u003cem\u003eE. coli\u003c/em\u003e P4 to reduce 18.8%, 37%, and 56% Cd\u003csup\u003e2+\u003c/sup\u003e after 48h, 96h, and 144h, respectively\u003cstrong\u003e. Zeng \u003cem\u003eet al.,\u003c/em\u003e (2009)\u003c/strong\u003e studied the removal efficiency of \u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003efor Cd and found 43.3% removal at initial concentration of 110.2 mg/L with 24 hours incubation period. \u003cstrong\u003eZiagova \u003cem\u003eet al.,\u003c/em\u003e (2007)\u003c/strong\u003e reported \u003cem\u003ePseudomonas\u003c/em\u003e sp. Removed the 75% Cd at 200 mg/L initial concentration after 5 hours of incubation\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6.\u003c/strong\u003e Bioremediation efficiency of Cadmium (Cd\u003csup\u003e2+\u003c/sup\u003e)\u0026nbsp;at 10 mg/L at different time interval.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"376\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.649867374005305%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.35013262599469%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e8.75\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e8.57\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e8.02\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e19.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e8.26\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e6.36\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e36.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e6.42\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e35.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e5.56\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e44.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e5.80\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e42.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.691489361702128%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.127659574468087%\" valign=\"top\"\u003e\n \u003cp\u003e4.61\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.638297872340425%\" valign=\"top\"\u003e\n \u003cp\u003e53.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.638297872340424%\" valign=\"top\"\u003e\n \u003cp\u003e4.24\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.904255319148936%\" valign=\"top\"\u003e\n \u003cp\u003e57.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7.\u003c/strong\u003e The efficiency of Bioremediation of Cadmium (Cd\u003csup\u003e2+\u003c/sup\u003e)\u0026nbsp;at different time interval at different concentration after 14 incubation period.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"376\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.57446808510639%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc. (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.73404255319149%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.57446808510638%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.73404255319149%\" valign=\"top\"\u003e\n \u003cp\u003e1.40\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e72.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.57446808510638%\" valign=\"top\"\u003e\n \u003cp\u003e1.38\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e72.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.73404255319149%\" valign=\"top\"\u003e\n \u003cp\u003e4.61\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e53.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.57446808510638%\" valign=\"top\"\u003e\n \u003cp\u003e4.24\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e57.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.73404255319149%\" valign=\"top\"\u003e\n \u003cp\u003e26.81\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e46.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.57446808510638%\" valign=\"top\"\u003e\n \u003cp\u003e25.04\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;2.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e49.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.425531914893616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.73404255319149%\" valign=\"top\"\u003e\n \u003cp\u003e58.86\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;13.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e41.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.57446808510638%\" valign=\"top\"\u003e\n \u003cp\u003e62.02\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;7.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.76595744680851%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e37.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eMean \u0026plusmn; SE; n=3, %R: Percent Removal\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eMean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p\u0026lt;0.05 significance level (Duncan\u0026rsquo;s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of Nickel (Ni\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilar patterns were observed for the reduction of Nickel (Ni\u003csup\u003e2+\u003c/sup\u003e) ions using \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2\u0026nbsp;and \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 as presented in Table 8 and 9. The removal in Ni\u003csup\u003e2+\u003c/sup\u003e value was considerably higher using \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2\u0026nbsp;in comparison to \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 Nonetheless, the removal efficacy of Ni\u003csup\u003e2+\u003c/sup\u003e was highly declined at rising the initial concentration from 5 to 100 mg/L. \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2 had the highest nickel removal proportion (69.4%) than \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11 (68.8%) at initial concentration of 5 mg/L. The removal efficacy of Ni\u003csup\u003e2+\u003c/sup\u003e was significantly declined from 68.8% to 33.8% at rising the initial concentration from 5 to 100 mg/L using \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003eaeruginosa\u003c/em\u003e HIB11. Similarly, in case of \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003esubtilis\u0026nbsp;\u003c/em\u003eHIB2, the removal efficacy of Ni\u003csup\u003e2+\u003c/sup\u003e was significantly declined from 69.40% to 37.36% (Fig.7). This showed that the efficacy of both isolates is initial concentration dependent.\u0026nbsp;Similar studies were reported. \u003cstrong\u003eDas \u003cem\u003eet al.,\u003c/em\u003e (2014)\u003c/strong\u003e observed the removal of Ni by \u003cem\u003eBacillus thuringiensis\u003c/em\u003e where observed a substantial percentage (82%) removal of Ni from the medium during in vitro culture. Similarly, there are other studies on the removal of Ni such as 95% Ni removal by \u003cem\u003eMicrobacterium\u0026nbsp;\u003c/em\u003esp. (Sathyavathi \u003cem\u003eet al.,\u003c/em\u003e 2014), biosorption of Ni \u0026amp; Cd, by \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003esp. \u003cstrong\u003e(Ansari \u0026amp; Malik, 2007)\u003c/strong\u003e and removal of Ni by \u003cem\u003eP\u003c/em\u003e\u003cem\u003eroteus vulgaris strain, Stenotrophomonas\u0026nbsp;\u003c/em\u003esp. and \u003cem\u003eBacillus thuringiensis\u0026nbsp;\u003c/em\u003erespectively \u003cstrong\u003e(Kumar \u003cem\u003eet al.,\u003c/em\u003e 2016)\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8.\u003c/strong\u003e Bioremediation efficiency of Nickel (Ni\u003csup\u003e2+\u003c/sup\u003e)\u0026nbsp;at 10 mg/L at different time interval.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"356\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.07042253521126%\" colspan=\"3\" valign=\"top\" style=\"width: 71.9101%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.07042253521126%\" valign=\"top\" style=\"width: 8.1605%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime (days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e8.95\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e8.10\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e7.77\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e5.56\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e44.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e7.01\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e29.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e4.96\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e50.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e6.39\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e36.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e4.47\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e55.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.929577464788732%\" valign=\"top\" style=\"width: 14.9609%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.04225352112676%\" valign=\"top\" style=\"width: 25.5695%;\"\u003e\n \u003cp\u003e3.14\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.267605633802816%\" valign=\"top\" style=\"width: 11.1527%;\"\u003e\n \u003cp\u003e68.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.929577464788736%\" valign=\"top\" style=\"width: 32.914%;\"\u003e\n \u003cp\u003e3.36\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.830985915492958%\" valign=\"top\" style=\"width: 11.6967%;\"\u003e\n \u003cp\u003e66.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 9.\u003c/strong\u003e The efficiency of Bioremediation of Nickel (Ni\u003csup\u003e2+\u003c/sup\u003e)\u0026nbsp;at different time interval at different concentration after 14 incubation period.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"365\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"84.10958904109589%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResidual values (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc. (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.753424657534246%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.876712328767123%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.97260273972603%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.506849315068493%\" valign=\"top\"\u003e\n \u003cp\u003e%R\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.753424657534246%\" valign=\"top\"\u003e\n \u003cp\u003e1.53\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.876712328767123%\" valign=\"top\"\u003e\n \u003cp\u003e69.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.97260273972603%\" valign=\"top\"\u003e\n \u003cp\u003e1.56\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.506849315068493%\" valign=\"top\"\u003e\n \u003cp\u003e68.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.753424657534246%\" valign=\"top\"\u003e\n \u003cp\u003e3.14\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.876712328767123%\" valign=\"top\"\u003e\n \u003cp\u003e68.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.97260273972603%\" valign=\"top\"\u003e\n \u003cp\u003e3.36\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.506849315068493%\" valign=\"top\"\u003e\n \u003cp\u003e66.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.753424657534246%\" valign=\"top\"\u003e\n \u003cp\u003e23.89\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;7.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.876712328767123%\" valign=\"top\"\u003e\n \u003cp\u003e52.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.97260273972603%\" valign=\"top\"\u003e\n \u003cp\u003e30.38\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;6.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.506849315068493%\" valign=\"top\"\u003e\n \u003cp\u003e39.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.89041095890411%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.753424657534246%\" valign=\"top\"\u003e\n \u003cp\u003e62.64\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;10.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.876712328767123%\" valign=\"top\"\u003e\n \u003cp\u003e37.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.97260273972603%\" valign=\"top\"\u003e\n \u003cp\u003e66.14\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;7.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.506849315068493%\" valign=\"top\"\u003e\n \u003cp\u003e33.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eMean \u0026plusmn; SE; n=3, %R: Percent Removal\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eMean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p\u0026lt;0.05 significance level (Duncan\u0026rsquo;s test).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA physicochemical examination indicates the river's dire state and recommends taking immediate and decisive measures to restore its water quality. Hindon river has heavy metal resisting bacteria pertaining to genus \u003cem\u003eBacillus\u003c/em\u003e sp., \u003cem\u003ePseudomonas\u003c/em\u003e sp., which have the competence to be utilized for bioremediation of metals from river water. The purified bacterial isolates were proficient of surviving in high concentration of Cd, Ni and Pb. \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003ePseudomonas\u003c/em\u003e sp. were previous reported to be highly competent in removal of metals Cd, Ni and Pb, which supports their utilization for cost-saving and ecofriendly removal of these metals into less toxic form from river water. Results of biodegradation experiments shown the removal competences of 53.9% for Cd\u003csup\u003e2+\u003c/sup\u003e and 68.6% for Ni\u003csup\u003e2+\u003c/sup\u003e and 71.7% for Pb\u003csup\u003e2+\u003c/sup\u003e using \u003cem\u003eBacillus subtilis\u003c/em\u003e HIB2, where 57.6% for Cd\u003csup\u003e2+\u003c/sup\u003e and 66.4% for Ni\u003csup\u003e2+\u003c/sup\u003e and 70.7% for Pb\u003csup\u003e2+\u003c/sup\u003e using \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e HIB11 after 14 days of incubation at pH 7.0 and 37\u0026deg;C. However, the removal effectiveness of these three heavy metals was similar in both isolates. Ex-situ application for the removal of metals from polluted locations requires more research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to the faculty of Amity School of Biotechnology, AUH, Gurgaon, India, for providing the support and grateful to ITC Lab, Gurgaon for providing necessary facilities for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntellectual Property\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. 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Comparative study of Cd (II) and Cr(VI) biosorption on Staphylococcus xylosus and \u003cem\u003ePseudomonas sp\u003c/em\u003e. in single and binary mixtures. \u003cem\u003eBioresource Technology\u003c/em\u003e, \u003cem\u003e98\u003c/em\u003e(15), 2859\u0026ndash;2865. https://doi.org/10.1016/j.biortech.2006.09.043\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biodegradation, Hindon river, Heavy Metals, Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11","lastPublishedDoi":"10.21203/rs.3.rs-4652732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4652732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe goal of this work was to isolate, identify, and characterize heavy metals degrading bacterial strains from the contaminated Hindon river (Yamuna's tributary) in India. Hindon river water quality was also assessed using physico-chemical and heavy metals analyses, revealing the river's degraded state. The bacterial isolates were isolated from Hindon river. Two bacterial isolates coded as HIB2 and HIB7 were screened and selected for further study based on the resistance to heavy metals (Cd, Ni, and Pb). Finally, these were identified based on morphological, biochemical tests and 16S rRNA sequencing. Biodegradation potential for heavy metals was assessed using inductively coupled plasma mass spectrometry (ICP-MS). The bacterial isolates HIB2 and HIB11 from Hindon river were identified as Bacillus subtilis and Pseudomonas aeruginosa respectively. At 10 mg/L initial concentration, results of biodegradation experiments showed the removal competences of 53.9% for Cd\u003csup\u003e2+\u003c/sup\u003e and 68.6% for Ni\u003csup\u003e2+\u003c/sup\u003e and 71.7% for Pb\u003csup\u003e2+ \u003c/sup\u003eusing Bacillus subtilis HIB2, where 57.6% for Cd\u003csup\u003e2+\u003c/sup\u003e and 66.4% for Ni\u003csup\u003e2+\u003c/sup\u003e and 70.7% for Pb\u003csup\u003e2+ \u003c/sup\u003eusing Pseudomonas aeruginosa HIB11 after 14 days of incubation at pH 7.0 and 37°C. Both isolates revealed the similarity in removal efficiency for these three heavy metals. The ability of Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 degrading heavy metals was found to be through curing experiments. The study showed that the Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 could be involved in effective degradation of heavy metals (Cd, Ni, and Pb).\u003c/p\u003e","manuscriptTitle":"Effective Bioremediation of Heavy Metals for Environmental Sustainability Approach using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 isolated from Hindon River, India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 17:57:19","doi":"10.21203/rs.3.rs-4652732/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3bd2ff6c-3673-46ec-a568-7abd35821883","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-05T11:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 17:57:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4652732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4652732","identity":"rs-4652732","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}