ISOLATION, CHARACTERIZATION, AND IDENTIFICATION OF MULTIPLE HEAVY METAL AND ANTIBIOTIC-RESISTANT BACTERIA FROM WASTEWATER
Main Article Content
Keywords
Bioremediation, heavy metals, minimum inhibitory concentration, phylogenetic tree, wastewater treatment
Abstract
Heavy metals are the most persistent pollutant in wastewater and cause several health and environmental hazards, contaminate soil, and reduced plant growth and productivity. Novel biotechnological approaches through inoculating bacterial strains might be adopted for the remediation of wastewater containing heavy metals. The study was conducted to reduce the toxicity of heavy metals in wastewater through inoculation of bacterial strains. The wastewater samples were collected under aseptic conditions from wastewater canals in Lahore. Bacterial strains were isolated by using nutrient agar media amended with 50 µg mL-1 of heavy metals e.g. Zn, Cd, Co, and Hg. The multiple heavy metal-resistant bacterial strains were screened for different biochemical and morphological characteristics. Furthermore, selected multiple-heavy metals tolerant strains were evaluated resistance for multiple antibiotics resistance under in vitro conditions. A total of eighty multiple heavy tolerant bacterial strains were isolated from industrial effluents. The biomass of these multiple heavy metals-resistant bacterial strains was recorded in terms of optical density (OD). The strains with heavy maximum OD in 50 µg mL-1 were selected for antibiotics resistance and revealed five bacterial strains were most resistant against antibiotics. The best-performing strains were identified as Klebsiella sp. strain BH49 and Salmonella sp. BH67 heaving accession numbers of MT074326 and MT074327, respectively, through 16S rRNA partial gene sequencing. Those identified strains might be used as a bioremediation agent for the efficient removal of heavy metals in contaminated wastewater.
References
1. Abbas SZ, M Rafatullah, N Ismail and J Lalung (2014). Isolation, identification, and characterization of Cd resistant Pseudomonas sp. M3 from industrial wastewater. Journal of Waste Management.
2. Abou-Shanab RAI, PV Berkum and JS Angle (2007). Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale. Chemosphere68(2):360-367.
3. Ahluwalia SS and D Goyal (2007). Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource technology98(12):2243-2257.
4. Akpor OB and M Muchie (2010). Remediation of heavy metals in drinking water and wastewater treatment systems: Processes and applications. International Journal of Physical Sciences 5(12):1807-1817.
5. Akpor OB, GO Ohiobor and TD Olaolu (2014). Heavy metal pollutants in wastewater effluents: sources, effects and remediation. Advances in Bioscience and Bioengineering 2(4):37-43.
6. Al-Garni SM (2005). Biosorption of Pb by Gram-ve capsulated and non-capsulated bacteria. Water Sa31(3):345-350.
7. Alloway BJ (2012). Heavy metals in soils: trace metals and metalloids in soils and their bioavailability. Springer Science & Business Media.
8. Anderson CK, Pederson & AM Jakobsson. (2006). Autoradiographic comparisons of radionuclide adsorption between subsurface anaerobic biofilms and granitic host rocks. Geomicrobiology Journal23(1)15–29.
9. Bååth E (1989). Effects of heavy metals in soil on microbial processes and populations. Water Air Soil Pollut47(3-4):335–379.
10. Badar U, N Ahmed, AJ Beswick, P Pattanapipitpaisal and LE Macaskie (2000). Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan. Biotechnology Letters 22(10):829-836.
11. Bartlett L, FW Rabe and WH Funk (1974). Effects of Cu, Zn and Cd on Selanastrum capricornutum. Water Research8(3):179-185.
12. Battin TJ, WT Sloan, S Kjelleberg, H Daims, IM Head, TP Curtis and L Eberl (2007). Microbial landscapes: new paths to biofilm research. Nature Reviews Microbiology5(1):76–81.
13. Belimov AA, N Hontzeas, VI Safronova, SV Demchinskaya, G Piluzza, S Bullitta and BR Glick (2005). Cd-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biology and Biochemistry 37(2):241-250.
14. Bestway EE, S Helmy, H Hussien, M Fahmy and R Amer (2013). Bioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteria. Applied water science3(1):181-192.
15. Bjarnsholt T, PØ Jensen, M Burmølle, M Hentzer, JAJ Haagensen, HP Hougen, H Calum (2005). Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151(2):373-383.
16. Boles BB, M Theondel & PK Singh (2004). Self-generated diversity produces insurance effects in biofilm communities. Proceedings of the National Academy of Sciences USA101(47):16630–16635.
17. Borriello G, E Werner, R Frank, AM Kim, D Garth, Ehrlich and SP Stewart (2004). Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother48(7):2659–2664.
18. Borsetti F, F Francia, RJ Turner and D Zannoni (2007). The thiol: disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32–) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus. J. Bacteriol189(3)851-859.
19. Çelo VD, B Babi, Baraj & A Çullaj (1999). An Assessment of heavy metal pollution in the sediments along the Albanian Coast. Water Air Soil Pollut111(1-4): 235–250.
20. Chang WC, GS Hsu, SM Chiang & MC Su (2006). Heavy metal removal from aqueous solution by wasted biomass from a combined AS‑biofilm process. Bioresource Technol97(13)1503–1508.
21. Chen M, P Xu, G Zeng, C Yang, D Huang and J Zhang (2015). Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides,
22. Chlorophenols and heavy metals by composting: applications, microbes and future research needs. Biotechnology Advances 33(6):745-755.
23. Collins CH, PM Lyne and JM Grange (1989). Microbiological Methods, 6th ed., Butterworth, London.
24. Congeevaram S, S Dhanarani, J Park, M Dexilin and K Thamaraiselvi (2007). Biosorption of Cr and Ni by heavy metal resistant fungal and bacterial isolates. Journal of hazardous materials146(1-2):270-277.
25. Cooper KE (1995). Theory of antibiotic inhibition zones in agar media. Nature176:510-511.
26. Costerton JW, PS Stewart & EP Greenberg (1999). Bacterial biofilms: a common cause of persistent infections. Science284(5418):1318–1322.
27. Davies JA, JJ Harrison, LL Marques, GR Foglia, CA Stremick, DG Storey, RJ Turner, ME Olson, H Ceri (2007). The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14. FEMS microbiology ecology 59(1):32-46.
28. Diels L, PH Spaans, RS Van, L Hooyberghs, A Ryngaert, H Wolters, E Walter, J Winters, L Macaskie, J Finlay, B Pernfuss (2003). Heavy metals removal by sand filters inoculated with metal sorbing and precipitating bacteria. Hydrometallurgy71(1-2):235-41.
29. Dixit R, D Malaviya, K Pandiyan, U Singh, A Sahu, R Shukla, B Singh, J Rai, P Sharma, H Lade and D Paul (2015). Bioremediation of heavy metals from soil and aquatic environment: an overview of principles and criteria of fundamental processes. Sustainability7(2):2189-2212.
30. Drancourt M, C Bollet, A Carlioz, R Martelin, JP Gayral and D Raoult (2000). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of clinical microbiology38(10)3623-3630.
31. Foulkes MJ, BR Sylvester, RK Scott (1998). Evidence for differences between winter wheat cultivars in acquisition of soil mineral nitrogen and uptake and utilization of applied fertilizer nitrogen. The Journal of Agricultural Science130(1):29-44.
32. Fulthorpe RR, SN Liss and DG Allen (1993). Characterization of bacteria isolated from a bleached kraft pulp mill wastewater treatment system. Canadian journal of microbiology39(1):13-24.
33. Gadd GM (1992). Metals and microorganisms: a problem of definition.FEMS Microbiology
34. Geslin C, J Llanos, D Prieur & C Jeanthon (2001). The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity. Research in microbiology152(10)901-905.
35. Gikas P (2008). Single and combined effects of Ni (Ni (II)) and Co (Co (II)) ions on activated sludge and on other aerobic microorganisms: a review. Journal of hazardous materials159(3):187-203.
36. Glick BR (2003). Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnology advances21(5):383–893.
37. Haagensen JA, M Klausen, RK Ernst, SI Miller, A Folkesson, TT Nielsen, S Molin (2007). Differentiation and distribution of colistin-and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms. Journal of bacteriology189(1):28-37.
38. Hall SL, JW Costerton & P Stoodley (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. Microbiol2(2):95–108.
39. Haq RU and AR Shakoori (1998). Microbiological treatment of industrial wastes containing toxic Cr involving successive use of bacteria, yeast and algae. World Journal of Microbiology and Biotechnology 14(4):583-585.
40. Harrison JJ, H Ceri, J Yerly, M Rabiei M, Y Hu, R Martinuzzi, RJ Turner (2007). Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms. Appl. Environ Microbiol73(15):4940-9.
41. Harrison JJ, H Ceri, NJ Roper, EA Badry, KM Sproule, RJ Turner (2005). Persister cells mediate tolerance to metal oxyanions in Escherichia coli. Microbiology151(10):3181-95.
42. Harrison JJ, M Rabiei, RJ Turner, EA Badry, KM Sproule, H Ceri (2006). Metal resistance in Candida biofilms. FEMS microbiology ecology55(3):479-91.
43. Hasegawa, M., H. Kishino and T. Yano (1985). Dating the human-ape split by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution.22: 160-174.
44. He Z, F Gao, T Sha, Y Hu and C He (2009). Isolation and characterization of a Cr (VI)-reduction Ochrobactrum sp. strain CSCr-3 from Cr landfill. Journal of hazardous materials163(3):869-873.
45. Hrynkiewicz K and C Baum (2014). Application of microorganisms in bioremediation of environment from heavy metals. In Environmental deterioration and human health Springer Dordrecht215 – 227.
46. Huang CT, KD Xu, GA McFeters & PS Stewart (1998). Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation. Appl Environ Microbiol6(4):1526–1531.
47. Hunter RC & TJ Beveridge (2005). Application of a pH sensitive fluoroprobe (C‑SNARF‑4) for Ph microenvironment analysis in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol71(5):2501–2510.
48. Igiri BE, SI Okoduwa, GO Idoko, EP Akabuogu, AO Adeyi and IK Ejiogu (2018). Toxicity and bioremediation of heavy metals contaminated ecosystem from tannery wastewater: a review. Journal of toxicology.
49. Jackson VA, AN Paulse, AA Bester, JH Neethling, S Khan and W Khan (2009). Bioremediation of metal contamination in the Plankenburg River, Western Cape, South Africa. International Biodeterioration & Biodegradation63(5):559-568.
50. Jaishankar M, T Tseten, N Anbalagan, BB Mathew and KN Beeregowda (2014). Toxicity, mechanism and health effects of some heavy metals. Interdisciplinary Toxicology 7(2):60-72.
51. Jefferson B, JE Burgess, A Pichon, J Harkness and SJ Judd (2001). Nutrient addition to enhance biological treatment of greywater. Water Research35(11):2702-2710.
52. Kelly CJ, N Tumsaroj and CA Lajoie (2004). Assessing wastewater metal toxicity with bacterial bioluminescence in a bench-scale wastewater treatment system. Water Research38(2):423-431.
53. Kelly JJ and RL Tate (1998). Effects of heavy metal contamination and remediation on soil microbial communities in the vicinity of a Zn smelter. Journal of environmental quality27(3):609-617.
54. Kessi J & KW Hanselmann (2004). Similarities between the abiotic reduction of selenite with glutathione andthe dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli. The Journal of Biological Chemistry279(49): 50662-50669.
55. Kim DW, DK Cha, J Wang and CP Huang (2002). Heavy metal removal by activated sludge: influence of Nocardia amarae. Chemosphere46(1):137-142.
56. Kumar S, G Stecher and K Tamura (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7):1870- 1874.
57. Lafleur MD, CA Kumamoto & K Lewis (2006). Candida albicans biofilms produce antifungal tolerant persister cells. Antimicrobial Agents Chemother 50(11):3839-3846.
58. Lawrence JR, MR Chenier, R Roy, D Beaumier, N Fortin, GD Swerhone, TR Neu, CW Greer (2004). Microscale and molecular assessment of impacts of Ni, nutrients, and oxygen level on structure and function of river biofilm communities. Appl Environ Microbiol70(7):4326-4339.
59. Lewis K (2007). Persister cells, dormancy and infectious disease. Nature Rev. Microbiol5(1):48-56.
60. Silva AAL, MA Carvalho, SA de Souza, PMT Dias, RGD Silva Filho, CS Saramago, CA Bento and E Hofer (2012). Heavy metal tolerance (Cr, Ag and Hg) in bacteria isolated from sewage. Brazilian Journal of Microbiology43(4):1620-1631.
61. Lohmeier VEM, S Ung & RJ Turner (2004). In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli. Appl Environ Microbiol70(12):7342-7347.
62. Malik A (2004). Metal bioremediation through growing cells. Environment International 30(2):261-278.
63. Marzan LW, M. Hossain, SA Mina, Y Akter and AMA Chowdhury (2017). Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint. The Egyptian Journal of Aquatic Research43(1):65-74.
64. Mathew M and JP Obbard (2001). Optimization of the dehydrogenase assay for measurement of indigenous microbial activity in beach sediments contaminated with petroleum. Biotechnology letters23(3):227-230.
65. Merroun ML, M Nedelkova, JJ Ojeda, T Reitz, ML Fernández, JM Arias, M Romero- González and S Selenska-Pobell (2011). Bio-precipitation of uranium by two bacterial isolates recovered from extreme environments as estimated by potentiometric titration, TEM and X-ray absorption spectroscopic analyses. Journal of Hazardous Materials19(7):1-10.
66. Moges F, M Endris, Y Belyhun and W Worku (2014). Isolation and characterization of multiple drug resistance bacterial pathogens from waste water in hospital and non- hospital environment. Northwest Ethiopia. BMC Research Notes 7(1):215.
67. Moten AM and A Rehman (1998). Study on heavy trace metal ions in industrial waste effluents in Pakistan.
68. Mumtaz MZ, M Ahmad, M Jamil and T Hussain (2017). Zn solubilizing Bacillus spp. potential candidates for biofortification in maize. Microbiological Research 20(2):51- 60.
69. Muñoz R, MT Alvarez, A Muñoz, E Terrazas, B Guieysse, B Mattiasson (2006). Sequential removal of heavy metal ions and organic pollutants using an algal-bacterial consortium. Chemosphere63(6):903-911.
70. Nameni M, MRA Moghadam and M Arami (2008). Adsorption of hexavalent Cr from aqueous solutions by wheat bran." International Journal of Environmental Science and Technology5(2):161-168.
71. Aka RJN and OO Babalola (2017). Identification and characterization of Cr-, Cd-, and Ni-tolerant bacteria isolated from mine tailings. Bioremediation journal 21(1):1-19.
72. Nweke CO, JC Okolo, CE Nwanyanwu and CS Alisi (2006). Response of planktonic bacteria of New Calabar River to Zn stress. African Journal of Biotechnology5(8):653-658.
73. Pacarynuk LA and HC Danyk (2004). Biochemical Tests. In: Principles of Microbiology. Laboratory Manual, spring, TX, USA 28-34.
74. Patel JB (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Molecular Diagnosis 6(4):313-321.
75. Petzow G (1999). Metallographic etching: techniques for metallography, ceramography, plastography. ASM international.
76. Pomposiello PJ & B Demple (2002). Global adjustment of microbial physiology during free radical stress. Adv Microb Physiol 4(6):319-341.
77. Pringault O, E Epping, R Guyoneaud, A Khalili & M Kuhl (1999). Dynamics of anoxygenic photosynthesis in an experimental green sulphur bacteria biofilm. Environ Microbiol1(4):295-305.
78. Purevdorj B, WJ Costerton & P Stoodley (2005). Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms. Microbiology151(5):1569–1576.
79. Raja CE, K Anbazhagan and GS Selvam (2006). Isolation and characterization of a metal-resistant Pseudomonas aeruginosa strain. World Journal of Microbiology and Biotechnology22(6):577-585.
80. Rajbanshi A (2008). Study on heavy metal resistant bacteria in Guheswori sewage treatment plant. Our Nature 6(1):52-57.
81. Ramage G, SP Saville, PD Thomas & JL Ribot (2005). Candida biofilms: an update. Eukaryotic Cell4(4):633-638.
82. Rani SA, B Pitts, H Beyenal, VR Aeluchamy, Z Lewandowski, WM Davison, MK Buckingham, PS Stewart (2007). Spatial patterns of DNA replication, protein synthesis and oxygen concentration within bacterial biofilms reveal diverse physiological states. J. Bacteriol189(11):4223-4233.
83. Rawlings DE & DB Johnson (2007). The microbiology of biomining: development and optimization of mineral oxidizing microbial consortia. Microbiology153(2):315–324.
84. Rehman J, HJ Zhang, PT Toth, Y Zhang, G Marsboom, Z Hong, R Salgia, AN Husain, C Wietholt and SL Archer (2012). Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. The FASEB Journal 26(5):2175-2186.
85. Roohi A, I Ahmed, M Iqbal and M Jamil (2012). Preliminary isolation and characterization of halotolerant and halophilic bacteria from salt mines of Karak, Pakistan. Pakistan Journal of Botany44(1):365-370.
86. Sahlström L (2003). A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresource Technology 87(2):161-166.
87. Saitou N and N Masatoshi (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4):406-425.
88. Selvi MS, S Sasikumar, S Gomathi, P Rajkumar, P Sasikumar and S Govindan (2014). Isolation and characterization of As resistant bacteria from agricultural soil, and their potential for As bioremediation. International Journal of Agricultural Policy and Research 2(11):393-405.
89. Shakoori AR and B Muneer (2002). Cu-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater. Folia Microbiologica 47(1):43-46.
90. Siddiquee S, K Rovina, SA Azad, L Naher, S Suryani and P Chaikaew (2015). Heavy metal contaminants removal from wastewater using the potential filamentous fungi biomass: a review. J Microb Biochem Technol7(6):384-395.
91. Singh R, D Paul & RK Jain (2006). Biofilms: implications in bioremediation. Trends Microbiol 14(9):389-397.
92. Southey PCJ, DG Davies & K Sauer (2005). Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J. Bacteriol 187(23):8114-8126.
93. Spoering A & K Lewis (2001). Biofilm and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol183(23):6746-6751.
94. Stewart PS (2002). Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol292(2):107-113.
95. Stohs SJ & D Bagchi (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med18(2):321-336.
96. Tamura K, M Nei and S Kumar (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences. 101(30):11030-11035.
97. Tchounwou PB, CG Yedjou, AK Patlolla and DJ Sutton (2012). Heavy metal toxicity and the environment in Molecular, Clinical and Environmental Toxicology. Springer Basel133-164.
98. Teitzel GM & MR Parsek. (2003). Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol69(4):2313–2320.
99. Thomson JM and RA Bonomo (2005). The threat of antibiotic resistance in Gram-negative pathogenic bacteria: β-lactams in peril. Current Opinion in Microbiology 8(5):518- 524.
100. Tremaroli V, S Fedi & D Zannoni (2007). Evidence for a tellurite-dependent generation of reactive oxygen species and absence of a tellurite-mediated adaptive response to oxidative stress in cells of Pseudomonas pseudoalcaligenes KF707. Arch. Microbiol187(2):127-135.
101. Turner RJ, Y Aharonowitz, JH Weiner & DE Taylor (2001). Glutathione is a target of tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli. J. Microbiol 47(1):33-40.
102. Valls M and VD Lorenzo (2002). Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS microbiology Reviews26(4):327-338.
103. Vilchez R, C Pozo, MA Gomez, B Rodelas & JG Lopez. (2007). Dominance of sphingomonads in a Cu exposed biofilm community for groundwater treatment. Microbiology153(2):325–337.
104. Walters MC, F Roe, A Bugnicourt, MJ Franklin & PS Stewart. (2003). Contributions of antibiotic penetration, oxygen limitation and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrobial agents and chemotherapy47(1):317–323.
105. Werner E, F Roe, A Bugnicourt, MJ Franklin, A Heydorn, S Molin, B Pitts and PS Stewart (2004). Stratified growth in Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol 70(10):6188-6196.
106. Wuana RA and FE Okieimen (2011). Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. International Scholar Research Notes.
107. Xu KD, GA McFeters & PS Stewart (2000). Biofilm resistance to antimicrobial agents. Microbiology146(3):547-549.
108. Xu KD, PS Stewart, F Xia, C Huang & GA McFeters (1998). Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl. Environ. Microbiol64(10):4035-4039.
109. Yuncu B, FD Sanin and U Yetis (2006). An investigation of heavy metal biosorption in relation to C/N ratio of activated sludge. Journal of hazardous materials137(2):990-997.
110. Zannoni D, F Borsetti, JJ Harrison & RJ Turner (2007). The bacterial response to the chalcogen metalloids Se and Te. Adv. Microb. Physiol5(3):1-71.
111. Zaved HK, MM Rahman, A Rahman and SMY Arafat (2008). Isolation and characterization of effective bacteria for solid waste degradation for organic manure. Current Applied Science and Technology 8(2):44-55.
112. Zhang GL, FG Yang, YG Zhao, WJ Zhao, JL Yang & ZT Gong (2005). Historical change of heavy metals in urban soils of Nanjing, China during the past 20 centuries. Environ. Int31(6):913-919.
113. Zouboulis AI, MX Loukidou and KA Matis (2004). Biosorption of toxic metals from aqueous solutions by bacteria strains isolated from metal-polluted soils. Process Biochemistry39(8):909-916.
2. Abou-Shanab RAI, PV Berkum and JS Angle (2007). Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale. Chemosphere68(2):360-367.
3. Ahluwalia SS and D Goyal (2007). Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource technology98(12):2243-2257.
4. Akpor OB and M Muchie (2010). Remediation of heavy metals in drinking water and wastewater treatment systems: Processes and applications. International Journal of Physical Sciences 5(12):1807-1817.
5. Akpor OB, GO Ohiobor and TD Olaolu (2014). Heavy metal pollutants in wastewater effluents: sources, effects and remediation. Advances in Bioscience and Bioengineering 2(4):37-43.
6. Al-Garni SM (2005). Biosorption of Pb by Gram-ve capsulated and non-capsulated bacteria. Water Sa31(3):345-350.
7. Alloway BJ (2012). Heavy metals in soils: trace metals and metalloids in soils and their bioavailability. Springer Science & Business Media.
8. Anderson CK, Pederson & AM Jakobsson. (2006). Autoradiographic comparisons of radionuclide adsorption between subsurface anaerobic biofilms and granitic host rocks. Geomicrobiology Journal23(1)15–29.
9. Bååth E (1989). Effects of heavy metals in soil on microbial processes and populations. Water Air Soil Pollut47(3-4):335–379.
10. Badar U, N Ahmed, AJ Beswick, P Pattanapipitpaisal and LE Macaskie (2000). Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan. Biotechnology Letters 22(10):829-836.
11. Bartlett L, FW Rabe and WH Funk (1974). Effects of Cu, Zn and Cd on Selanastrum capricornutum. Water Research8(3):179-185.
12. Battin TJ, WT Sloan, S Kjelleberg, H Daims, IM Head, TP Curtis and L Eberl (2007). Microbial landscapes: new paths to biofilm research. Nature Reviews Microbiology5(1):76–81.
13. Belimov AA, N Hontzeas, VI Safronova, SV Demchinskaya, G Piluzza, S Bullitta and BR Glick (2005). Cd-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biology and Biochemistry 37(2):241-250.
14. Bestway EE, S Helmy, H Hussien, M Fahmy and R Amer (2013). Bioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteria. Applied water science3(1):181-192.
15. Bjarnsholt T, PØ Jensen, M Burmølle, M Hentzer, JAJ Haagensen, HP Hougen, H Calum (2005). Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151(2):373-383.
16. Boles BB, M Theondel & PK Singh (2004). Self-generated diversity produces insurance effects in biofilm communities. Proceedings of the National Academy of Sciences USA101(47):16630–16635.
17. Borriello G, E Werner, R Frank, AM Kim, D Garth, Ehrlich and SP Stewart (2004). Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother48(7):2659–2664.
18. Borsetti F, F Francia, RJ Turner and D Zannoni (2007). The thiol: disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32–) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus. J. Bacteriol189(3)851-859.
19. Çelo VD, B Babi, Baraj & A Çullaj (1999). An Assessment of heavy metal pollution in the sediments along the Albanian Coast. Water Air Soil Pollut111(1-4): 235–250.
20. Chang WC, GS Hsu, SM Chiang & MC Su (2006). Heavy metal removal from aqueous solution by wasted biomass from a combined AS‑biofilm process. Bioresource Technol97(13)1503–1508.
21. Chen M, P Xu, G Zeng, C Yang, D Huang and J Zhang (2015). Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides,
22. Chlorophenols and heavy metals by composting: applications, microbes and future research needs. Biotechnology Advances 33(6):745-755.
23. Collins CH, PM Lyne and JM Grange (1989). Microbiological Methods, 6th ed., Butterworth, London.
24. Congeevaram S, S Dhanarani, J Park, M Dexilin and K Thamaraiselvi (2007). Biosorption of Cr and Ni by heavy metal resistant fungal and bacterial isolates. Journal of hazardous materials146(1-2):270-277.
25. Cooper KE (1995). Theory of antibiotic inhibition zones in agar media. Nature176:510-511.
26. Costerton JW, PS Stewart & EP Greenberg (1999). Bacterial biofilms: a common cause of persistent infections. Science284(5418):1318–1322.
27. Davies JA, JJ Harrison, LL Marques, GR Foglia, CA Stremick, DG Storey, RJ Turner, ME Olson, H Ceri (2007). The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14. FEMS microbiology ecology 59(1):32-46.
28. Diels L, PH Spaans, RS Van, L Hooyberghs, A Ryngaert, H Wolters, E Walter, J Winters, L Macaskie, J Finlay, B Pernfuss (2003). Heavy metals removal by sand filters inoculated with metal sorbing and precipitating bacteria. Hydrometallurgy71(1-2):235-41.
29. Dixit R, D Malaviya, K Pandiyan, U Singh, A Sahu, R Shukla, B Singh, J Rai, P Sharma, H Lade and D Paul (2015). Bioremediation of heavy metals from soil and aquatic environment: an overview of principles and criteria of fundamental processes. Sustainability7(2):2189-2212.
30. Drancourt M, C Bollet, A Carlioz, R Martelin, JP Gayral and D Raoult (2000). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of clinical microbiology38(10)3623-3630.
31. Foulkes MJ, BR Sylvester, RK Scott (1998). Evidence for differences between winter wheat cultivars in acquisition of soil mineral nitrogen and uptake and utilization of applied fertilizer nitrogen. The Journal of Agricultural Science130(1):29-44.
32. Fulthorpe RR, SN Liss and DG Allen (1993). Characterization of bacteria isolated from a bleached kraft pulp mill wastewater treatment system. Canadian journal of microbiology39(1):13-24.
33. Gadd GM (1992). Metals and microorganisms: a problem of definition.FEMS Microbiology
34. Geslin C, J Llanos, D Prieur & C Jeanthon (2001). The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity. Research in microbiology152(10)901-905.
35. Gikas P (2008). Single and combined effects of Ni (Ni (II)) and Co (Co (II)) ions on activated sludge and on other aerobic microorganisms: a review. Journal of hazardous materials159(3):187-203.
36. Glick BR (2003). Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnology advances21(5):383–893.
37. Haagensen JA, M Klausen, RK Ernst, SI Miller, A Folkesson, TT Nielsen, S Molin (2007). Differentiation and distribution of colistin-and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms. Journal of bacteriology189(1):28-37.
38. Hall SL, JW Costerton & P Stoodley (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. Microbiol2(2):95–108.
39. Haq RU and AR Shakoori (1998). Microbiological treatment of industrial wastes containing toxic Cr involving successive use of bacteria, yeast and algae. World Journal of Microbiology and Biotechnology 14(4):583-585.
40. Harrison JJ, H Ceri, J Yerly, M Rabiei M, Y Hu, R Martinuzzi, RJ Turner (2007). Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms. Appl. Environ Microbiol73(15):4940-9.
41. Harrison JJ, H Ceri, NJ Roper, EA Badry, KM Sproule, RJ Turner (2005). Persister cells mediate tolerance to metal oxyanions in Escherichia coli. Microbiology151(10):3181-95.
42. Harrison JJ, M Rabiei, RJ Turner, EA Badry, KM Sproule, H Ceri (2006). Metal resistance in Candida biofilms. FEMS microbiology ecology55(3):479-91.
43. Hasegawa, M., H. Kishino and T. Yano (1985). Dating the human-ape split by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution.22: 160-174.
44. He Z, F Gao, T Sha, Y Hu and C He (2009). Isolation and characterization of a Cr (VI)-reduction Ochrobactrum sp. strain CSCr-3 from Cr landfill. Journal of hazardous materials163(3):869-873.
45. Hrynkiewicz K and C Baum (2014). Application of microorganisms in bioremediation of environment from heavy metals. In Environmental deterioration and human health Springer Dordrecht215 – 227.
46. Huang CT, KD Xu, GA McFeters & PS Stewart (1998). Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation. Appl Environ Microbiol6(4):1526–1531.
47. Hunter RC & TJ Beveridge (2005). Application of a pH sensitive fluoroprobe (C‑SNARF‑4) for Ph microenvironment analysis in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol71(5):2501–2510.
48. Igiri BE, SI Okoduwa, GO Idoko, EP Akabuogu, AO Adeyi and IK Ejiogu (2018). Toxicity and bioremediation of heavy metals contaminated ecosystem from tannery wastewater: a review. Journal of toxicology.
49. Jackson VA, AN Paulse, AA Bester, JH Neethling, S Khan and W Khan (2009). Bioremediation of metal contamination in the Plankenburg River, Western Cape, South Africa. International Biodeterioration & Biodegradation63(5):559-568.
50. Jaishankar M, T Tseten, N Anbalagan, BB Mathew and KN Beeregowda (2014). Toxicity, mechanism and health effects of some heavy metals. Interdisciplinary Toxicology 7(2):60-72.
51. Jefferson B, JE Burgess, A Pichon, J Harkness and SJ Judd (2001). Nutrient addition to enhance biological treatment of greywater. Water Research35(11):2702-2710.
52. Kelly CJ, N Tumsaroj and CA Lajoie (2004). Assessing wastewater metal toxicity with bacterial bioluminescence in a bench-scale wastewater treatment system. Water Research38(2):423-431.
53. Kelly JJ and RL Tate (1998). Effects of heavy metal contamination and remediation on soil microbial communities in the vicinity of a Zn smelter. Journal of environmental quality27(3):609-617.
54. Kessi J & KW Hanselmann (2004). Similarities between the abiotic reduction of selenite with glutathione andthe dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli. The Journal of Biological Chemistry279(49): 50662-50669.
55. Kim DW, DK Cha, J Wang and CP Huang (2002). Heavy metal removal by activated sludge: influence of Nocardia amarae. Chemosphere46(1):137-142.
56. Kumar S, G Stecher and K Tamura (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7):1870- 1874.
57. Lafleur MD, CA Kumamoto & K Lewis (2006). Candida albicans biofilms produce antifungal tolerant persister cells. Antimicrobial Agents Chemother 50(11):3839-3846.
58. Lawrence JR, MR Chenier, R Roy, D Beaumier, N Fortin, GD Swerhone, TR Neu, CW Greer (2004). Microscale and molecular assessment of impacts of Ni, nutrients, and oxygen level on structure and function of river biofilm communities. Appl Environ Microbiol70(7):4326-4339.
59. Lewis K (2007). Persister cells, dormancy and infectious disease. Nature Rev. Microbiol5(1):48-56.
60. Silva AAL, MA Carvalho, SA de Souza, PMT Dias, RGD Silva Filho, CS Saramago, CA Bento and E Hofer (2012). Heavy metal tolerance (Cr, Ag and Hg) in bacteria isolated from sewage. Brazilian Journal of Microbiology43(4):1620-1631.
61. Lohmeier VEM, S Ung & RJ Turner (2004). In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli. Appl Environ Microbiol70(12):7342-7347.
62. Malik A (2004). Metal bioremediation through growing cells. Environment International 30(2):261-278.
63. Marzan LW, M. Hossain, SA Mina, Y Akter and AMA Chowdhury (2017). Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint. The Egyptian Journal of Aquatic Research43(1):65-74.
64. Mathew M and JP Obbard (2001). Optimization of the dehydrogenase assay for measurement of indigenous microbial activity in beach sediments contaminated with petroleum. Biotechnology letters23(3):227-230.
65. Merroun ML, M Nedelkova, JJ Ojeda, T Reitz, ML Fernández, JM Arias, M Romero- González and S Selenska-Pobell (2011). Bio-precipitation of uranium by two bacterial isolates recovered from extreme environments as estimated by potentiometric titration, TEM and X-ray absorption spectroscopic analyses. Journal of Hazardous Materials19(7):1-10.
66. Moges F, M Endris, Y Belyhun and W Worku (2014). Isolation and characterization of multiple drug resistance bacterial pathogens from waste water in hospital and non- hospital environment. Northwest Ethiopia. BMC Research Notes 7(1):215.
67. Moten AM and A Rehman (1998). Study on heavy trace metal ions in industrial waste effluents in Pakistan.
68. Mumtaz MZ, M Ahmad, M Jamil and T Hussain (2017). Zn solubilizing Bacillus spp. potential candidates for biofortification in maize. Microbiological Research 20(2):51- 60.
69. Muñoz R, MT Alvarez, A Muñoz, E Terrazas, B Guieysse, B Mattiasson (2006). Sequential removal of heavy metal ions and organic pollutants using an algal-bacterial consortium. Chemosphere63(6):903-911.
70. Nameni M, MRA Moghadam and M Arami (2008). Adsorption of hexavalent Cr from aqueous solutions by wheat bran." International Journal of Environmental Science and Technology5(2):161-168.
71. Aka RJN and OO Babalola (2017). Identification and characterization of Cr-, Cd-, and Ni-tolerant bacteria isolated from mine tailings. Bioremediation journal 21(1):1-19.
72. Nweke CO, JC Okolo, CE Nwanyanwu and CS Alisi (2006). Response of planktonic bacteria of New Calabar River to Zn stress. African Journal of Biotechnology5(8):653-658.
73. Pacarynuk LA and HC Danyk (2004). Biochemical Tests. In: Principles of Microbiology. Laboratory Manual, spring, TX, USA 28-34.
74. Patel JB (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Molecular Diagnosis 6(4):313-321.
75. Petzow G (1999). Metallographic etching: techniques for metallography, ceramography, plastography. ASM international.
76. Pomposiello PJ & B Demple (2002). Global adjustment of microbial physiology during free radical stress. Adv Microb Physiol 4(6):319-341.
77. Pringault O, E Epping, R Guyoneaud, A Khalili & M Kuhl (1999). Dynamics of anoxygenic photosynthesis in an experimental green sulphur bacteria biofilm. Environ Microbiol1(4):295-305.
78. Purevdorj B, WJ Costerton & P Stoodley (2005). Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms. Microbiology151(5):1569–1576.
79. Raja CE, K Anbazhagan and GS Selvam (2006). Isolation and characterization of a metal-resistant Pseudomonas aeruginosa strain. World Journal of Microbiology and Biotechnology22(6):577-585.
80. Rajbanshi A (2008). Study on heavy metal resistant bacteria in Guheswori sewage treatment plant. Our Nature 6(1):52-57.
81. Ramage G, SP Saville, PD Thomas & JL Ribot (2005). Candida biofilms: an update. Eukaryotic Cell4(4):633-638.
82. Rani SA, B Pitts, H Beyenal, VR Aeluchamy, Z Lewandowski, WM Davison, MK Buckingham, PS Stewart (2007). Spatial patterns of DNA replication, protein synthesis and oxygen concentration within bacterial biofilms reveal diverse physiological states. J. Bacteriol189(11):4223-4233.
83. Rawlings DE & DB Johnson (2007). The microbiology of biomining: development and optimization of mineral oxidizing microbial consortia. Microbiology153(2):315–324.
84. Rehman J, HJ Zhang, PT Toth, Y Zhang, G Marsboom, Z Hong, R Salgia, AN Husain, C Wietholt and SL Archer (2012). Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. The FASEB Journal 26(5):2175-2186.
85. Roohi A, I Ahmed, M Iqbal and M Jamil (2012). Preliminary isolation and characterization of halotolerant and halophilic bacteria from salt mines of Karak, Pakistan. Pakistan Journal of Botany44(1):365-370.
86. Sahlström L (2003). A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresource Technology 87(2):161-166.
87. Saitou N and N Masatoshi (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4):406-425.
88. Selvi MS, S Sasikumar, S Gomathi, P Rajkumar, P Sasikumar and S Govindan (2014). Isolation and characterization of As resistant bacteria from agricultural soil, and their potential for As bioremediation. International Journal of Agricultural Policy and Research 2(11):393-405.
89. Shakoori AR and B Muneer (2002). Cu-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater. Folia Microbiologica 47(1):43-46.
90. Siddiquee S, K Rovina, SA Azad, L Naher, S Suryani and P Chaikaew (2015). Heavy metal contaminants removal from wastewater using the potential filamentous fungi biomass: a review. J Microb Biochem Technol7(6):384-395.
91. Singh R, D Paul & RK Jain (2006). Biofilms: implications in bioremediation. Trends Microbiol 14(9):389-397.
92. Southey PCJ, DG Davies & K Sauer (2005). Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J. Bacteriol 187(23):8114-8126.
93. Spoering A & K Lewis (2001). Biofilm and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol183(23):6746-6751.
94. Stewart PS (2002). Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol292(2):107-113.
95. Stohs SJ & D Bagchi (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med18(2):321-336.
96. Tamura K, M Nei and S Kumar (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences. 101(30):11030-11035.
97. Tchounwou PB, CG Yedjou, AK Patlolla and DJ Sutton (2012). Heavy metal toxicity and the environment in Molecular, Clinical and Environmental Toxicology. Springer Basel133-164.
98. Teitzel GM & MR Parsek. (2003). Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol69(4):2313–2320.
99. Thomson JM and RA Bonomo (2005). The threat of antibiotic resistance in Gram-negative pathogenic bacteria: β-lactams in peril. Current Opinion in Microbiology 8(5):518- 524.
100. Tremaroli V, S Fedi & D Zannoni (2007). Evidence for a tellurite-dependent generation of reactive oxygen species and absence of a tellurite-mediated adaptive response to oxidative stress in cells of Pseudomonas pseudoalcaligenes KF707. Arch. Microbiol187(2):127-135.
101. Turner RJ, Y Aharonowitz, JH Weiner & DE Taylor (2001). Glutathione is a target of tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli. J. Microbiol 47(1):33-40.
102. Valls M and VD Lorenzo (2002). Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS microbiology Reviews26(4):327-338.
103. Vilchez R, C Pozo, MA Gomez, B Rodelas & JG Lopez. (2007). Dominance of sphingomonads in a Cu exposed biofilm community for groundwater treatment. Microbiology153(2):325–337.
104. Walters MC, F Roe, A Bugnicourt, MJ Franklin & PS Stewart. (2003). Contributions of antibiotic penetration, oxygen limitation and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrobial agents and chemotherapy47(1):317–323.
105. Werner E, F Roe, A Bugnicourt, MJ Franklin, A Heydorn, S Molin, B Pitts and PS Stewart (2004). Stratified growth in Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol 70(10):6188-6196.
106. Wuana RA and FE Okieimen (2011). Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. International Scholar Research Notes.
107. Xu KD, GA McFeters & PS Stewart (2000). Biofilm resistance to antimicrobial agents. Microbiology146(3):547-549.
108. Xu KD, PS Stewart, F Xia, C Huang & GA McFeters (1998). Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl. Environ. Microbiol64(10):4035-4039.
109. Yuncu B, FD Sanin and U Yetis (2006). An investigation of heavy metal biosorption in relation to C/N ratio of activated sludge. Journal of hazardous materials137(2):990-997.
110. Zannoni D, F Borsetti, JJ Harrison & RJ Turner (2007). The bacterial response to the chalcogen metalloids Se and Te. Adv. Microb. Physiol5(3):1-71.
111. Zaved HK, MM Rahman, A Rahman and SMY Arafat (2008). Isolation and characterization of effective bacteria for solid waste degradation for organic manure. Current Applied Science and Technology 8(2):44-55.
112. Zhang GL, FG Yang, YG Zhao, WJ Zhao, JL Yang & ZT Gong (2005). Historical change of heavy metals in urban soils of Nanjing, China during the past 20 centuries. Environ. Int31(6):913-919.
113. Zouboulis AI, MX Loukidou and KA Matis (2004). Biosorption of toxic metals from aqueous solutions by bacteria strains isolated from metal-polluted soils. Process Biochemistry39(8):909-916.
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Oct 23, 2023
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Muhammad Bakir Hussain, Saba Abbas, Hafiz Khawar, Muhammad Raza, Hubba Chaudhary, Hafiza Nida Shehzadi, Noor Fatima, Zeemal Seemab Amin, & Muhammad Hamza. (2023). ISOLATION, CHARACTERIZATION, AND IDENTIFICATION OF MULTIPLE HEAVY METAL AND ANTIBIOTIC-RESISTANT BACTERIA FROM WASTEWATER. Journal of Population Therapeutics and Clinical Pharmacology, 30(18), 879–897. https://doi.org/10.53555/jptcp.v30i18.3200
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Author Biographies
Muhammad Bakir Hussain
Laboratory manager, Horizon hospital Lahore
Saba Abbas
School of Medical Laboratory Technology, Faculty of Allied Health Sciences, Minhaj University Lahore
Hafiz Khawar
Institute of Industrial Biotechnology, Government College University, Lahore
Muhammad Raza
School of Medical Laboratory Technology, Faculty of Allied Health Sciences, Minhaj University Lahore
Hubba Chaudhary
Institute of Industrial Biotechnology, Government College University, Lahore
Hafiza Nida Shehzadi
School of Medical Laboratory Technology, Faculty of Allied Health Sciences, Minhaj University Lahore
Noor Fatima
University of Central Punjab, Lahore.
Zeemal Seemab Amin
School of Biochemistry, Faculty of Applied Sciences, Minhaj University Lahore.
Muhammad Hamza
Quality Assurance Officer CSH pharmaceutical Lahore.