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Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum

  • Asfa RizviEmail author
  • Bilal Ahmed
  • Almas Zaidi
  • Mohd. Saghir Khan
Article

Abstract

Heavy metal pollution destruct soil microbial compositions and functions, plant’s performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL−1 of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL−1 of Cd (cell size 1.7 µm) and 100 of µg mL−1 Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL−1 of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL−1 when grown with 100 µg mL−1 each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH3 in fatty acids and CH2 groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, 1H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.

Keywords

Heavy metal toxicity A. chroococcum Exopolysaccharides Melanin Metallothioneins Environmental management 

Notes

Acknowledgements

The funding for the research work was supported by Department of Science and Technology, New Delhi, India in the form of INSPIRE fellowship. AR is highly thankful to University Sophisticated Instruments Facility (USIF), Aligarh Muslim University, Aligarh and Macrogen Inc., Seoul, South Korea for the analyses.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. Abinaya M, Vaseeharan B, Divya M, Vijayakumar S, Govindarajan M, Alharbi NS, Khaled JM, Al-anbr MN, Benelli G (2018) Structural characterization of Bacillus licheniformis Dahb1 exopolysaccharide-antimicrobial potential and larvicidal activity on malaria and Zika virus mosquito vectors. Environ Sci Pollut Res 25:18604–18619Google Scholar
  2. Ackerman CM, Lee S, Chang CJ (2016) Analytical methods for imaging metals in biology: from transition metal metabolism to transition metal signaling. Anal Chem 89:22–41Google Scholar
  3. Adam V, Chudobova D, Tmejova K, Cihalova K, Krizkova S, Guran R, Kominkova M, Zurek M, Kremplova M, Jimenez AM, Konecna M (2014) An effect of cadmium and lead ions on Escherichia coli with the cloned gene for metallothionein (MT-3) revealed by electrochemistry. Electrochim Act 140:11–19Google Scholar
  4. Alnuaimi MM, Saeed IA, Ashraf SS (2012) Effect of various heavy metals on the enzymatic activity of E. coli alkaline phosphatase. Int J Biotechnol Biochem 8:47–59Google Scholar
  5. Ana RL, Garcia-Vazquez E (2006) A simple assay to quantify metallothionein helps to learn about bioindicators and environmental health. Biochem Mol Biol Edu 34:360–363Google Scholar
  6. Apte M, Girme G, Bankar A, RaviKumar A, Zinjarde S (2013) 3, 4-dihydroxy-L-phenylalanine-derived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures. J Nanobiotechnol 11:2Google Scholar
  7. Aravindhan R, Madhan B, Raghava Rao J, Unni Nair B, Ramasami T (2004) Bioaccumulation of chromium from the tannery waste water: an approach for chrome recovery and reuse. Environ Sci Technol 38:300–306Google Scholar
  8. Ates O (2015) Systems biology of microbial exopolysaccharides production. Front Bioengg. Biotechnol 3:200Google Scholar
  9. Ayangbenro AS, Babalola OO (2017) A new strategy for heavy metal polluted environments: a review of microbial biosorbents. J Environ Res Public Health 14:94Google Scholar
  10. Banerjee A, Supakar S, Banerjee R (2014) Melanin from the nitrogen-fixing bacterium Azotobacter chroococcum: a spectroscopic characterization. PLoS ONE 9(1):e84574Google Scholar
  11. Batool R, Marghoob U, Kalsoom A (2017) Estimation of exopolysaccharides (EPS) producing ability of Cr (VI) resistant bacterial strains from tannery effluent. J Basic Appl Sci 13:589–596Google Scholar
  12. Benit N, Roslin AS (2018) Isolation and characterization of larvicidal extracellular polysaccharide (EPS) from Pseudomonas aeruginosa B01. Int J Curr Microbiol Appl Sci 7:109–120Google Scholar
  13. Bhagat N, Vermani M, Bajwa HS (2016) Characterization of heavy metal (cadmium and nickel) tolerant Gram negative enteric bacteria from polluted Yamuna River, Delhi. Afr J Microbiol Res 10:127–137Google Scholar
  14. Chatelain M, Gasparini J, Jacquin L, Frantz A (2014) The adaptive function of melanin-based plumage coloration to trace metals. Biol Lett 10:20140164Google Scholar
  15. Chaturvedi AD, Pal D, Penta S, Kumar A (2015) Ecotoxic heavy metals transformation by bacteria and fungi in aquatic ecosystem. World J Microbiol Biotechnol 31:1595–1603Google Scholar
  16. Chen Y, Chao Y, Li Y, Lin Q, Bai J, Tang L, Qiu R (2016) Survival strategies of the plant-associated bacterium Enterobacter sp. strain EG16 under cadmium stress. Appl Environ Microbiol 82:1734–1744Google Scholar
  17. Chen CF, Foley J, Tang PC, Li A, Jiang TX, Wu P, Widelitz RB, Chuong CM (2015) Development, regeneration, and evolution of feathers. Annu Rev Anim Biosci 3:169–195Google Scholar
  18. Cordero RJ, Vij R, Casadevall A (2017) Microbial melanins for radioprotection and bioremediation. Microb Biotechnol 10:1186–1190Google Scholar
  19. Côte J, Boniface A, Blanchet S, Hendry AP, Gasparini J, Jacquin L (2018) Melanin-based coloration and host–parasite interactions under global change. Proc R Soc B 285(1879):20180285Google Scholar
  20. Dahech I, Fakhfakh J, Damak M, Belghith H, Mejdoub H, Belghith KS (2013) Structural determination and NMR characterization of a bacterial exopolysaccharide. Int J Biol Macromol 2013 59:417–422Google Scholar
  21. Das KK, Reddy RC, Bagoji IB, Das S, Bagali S, Mullur L, Khodnapur JP, Biradar MS (2018) Primary concept of nickel toxicity–an overview. J Basic Clinic Physiol Pharmacol  https://doi.org/10.1515/jbcpp-2017-0171
  22. De Guzman MLC, Arcega KSG, Cabigao JMNR, Su GLS (2016) Isolation and identification of heavy metal-tolerant bacteria from an industrial site as a possible source for bioremediation of cadmium, lead, and nickel. AdvEnviron Biol 10:10–16Google Scholar
  23. Diopan V, Shestivska V, Adam V, Macek T, Mackova M, Havel L, Kizek R (2008) Determination of content of metallothionein and low molecular mass stress peptides in transgenic tobacco plants. Plant Cell, Tissue Org Cult 94:291–298Google Scholar
  24. D’Mello SA, Finlay GJ, Baguley BC, Askarian-Amiri ME (2016) Signaling pathways in melanogenesis. Int J Mol Sci 17:1144Google Scholar
  25. Drewnowska JM, Zambrzycka M, Kalska-Szostko B, Fiedoruk K, Swiecicka I (2015) Melanin-like pigment synthesis by soil Bacillus weihenstephanensis isolates from Northeastern Poland. PLoS ONE 10:e0125428Google Scholar
  26. Durve A, Chandra N (2014) FT-IR analysis of bacterial biomass in response to heavy metal stress. Int J Biotechnol Photon 112:386–391Google Scholar
  27. Dziegiel P, Pula B, Kobierzycki C, Stasiolek M, Podhorska-Okolow M (2016) Metallothioneins: structure andfunctions. In: Sutovsky P, Clascá F, Kmiec Z, Korf HW, Singh B, Timmermans JP, Schmeisser MJ (Eds) Metallothioneins in Normal and Cancer Cells, Advances in Anatomy, Embryology and Cell Biology pp 3–20. Springer, SwitzerlandGoogle Scholar
  28. Edwards NP, vanVeelen A, Anné J, Manning PL, Bergmann U, Sellers WI, Wogelius RA (2016) Elemental characterisation of melanin in feathers via synchrotron X-ray imaging and absorption spectroscopy. Sci Rep 6:34002Google Scholar
  29. El-Naggar NE, El-Ewasy SM (2017) Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H. Sci Rep 7:42129Google Scholar
  30. Enshaei M, Khanafari A, Sepahey AA (2010) Metallothionein induction in two species of Pseudomonas exposed to cadmium and copper contamination. Iran J Environ Health Sci Engg 7:287Google Scholar
  31. Fashola MO, Ngole-Jeme VM, Babalola OO (2016) Heavy metal pollution from gold mines: Environmental effects and bacterial strategies for resistance. Int J Environ Res Public Health 13:1047Google Scholar
  32. Francisco R, Moreno A, Morais PV (2010) Different physiological responses to chromate and dichromate in the chromium resistant and reducing strain Ochrobactrum tritici 5bvl1. Biometals 23:713–725Google Scholar
  33. François F, Lombard C, Guigner JM, Soreau P, Brian-Jaisson F, Martino G, Vandervennet M, Garcia D, Molinier AL, Pignol D, Peduzzi J (2012) Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury. Appl Environ Microbiol 78:1097–1106Google Scholar
  34. Fukuyama Y, Yoshida S, Yanagisawa S, Shimizu M (1999) A study on the differences between oral squamous cell carcinomas and normal oral mucosas measured by Fourier transform infrared spectroscopy. Biospectroscopy 5:117–126Google Scholar
  35. Galván I, Solano F (2016) Bird integumentary melanins: biosynthesis, forms, function and evolution. Int J Mol Sci 17:520Google Scholar
  36. Girisha ST (2014) Lead Bioremediation with respect to mining and industrial effluents. Int Res J Environ Sci 3:58–61Google Scholar
  37. Gobi N, Vaseeharan B, Rekha R, Vijayakumar S, Faggio C (2018) Bioaccumulation, cytotoxicity and oxidative stress of the acute exposure of selenium in Oreochromis mossambicus. Ecotoxicol Environ Saf 162:147–159Google Scholar
  38. Golding CG, Lamboo LL, Beniac DR, Booth TF (2016) The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci Rep 6:26516Google Scholar
  39. Gomes LC, Mergulhão FJ (2017) SEM analysis of surface impact on biofilm antibiotic treatment. Scanning  https://doi.org/10.1155/2017/2960194
  40. Gupta P, Diwan B (2017) Bacterial exopolysaccharide mediated heavy metal removal: A review on biosynthesis, mechanism and remediation strategies. Biotechnol Rep 13:58–71Google Scholar
  41. Haferburg G, Kothe E (2010) Metallomics: lessons for metalliferous soil remediation. Appl Microbiol Biotechnol 87:1271–1280Google Scholar
  42. Hao L, Li J, Kappler A, Obst M (2013) Mapping of heavy metal ion sorption to cell-extracellular polymeric substance-mineral aggregates by using metal-selective fluorescent probes and confocal laser scanning microscopy. Appl Environ Microbiol 79:6524–6534Google Scholar
  43. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Gram negative aerobic/microaerophilic rodsand cocci. In: Holt JG (Ed) Bergey’s Manual of Determinative Bacteriology. 9th edn. Williams & Wilkins, Lippincott, pp 93–168Google Scholar
  44. Hu WL, Dai DH, Huang GR, Zhang ZD (2015) Isolation and characterization of extracellular melanin produced by Chroogomphus rutilus D447. Am J Food Technol 10:68–77Google Scholar
  45. Huleihel M, Salman A, Erukhimovich V, Ramesh J, Hammody Z, Mordechai S (2002) Novel optical method for study of viral carcinogenesis in vitro. J Biochem Biophys Methods 50:111–121Google Scholar
  46. Ianeva OD (2009) Mechanisms of bacteria resistance to heavy metals. Mikrobiolohichnyi Zh (Kiev, Ukr: 1993) 71:54–65Google Scholar
  47. Irvine GW, Stillman MJ (2017) Residue modification and mass spectrometry for the investigation of structural and metalation properties of metallothionein and cysteine-rich proteins. Int J Mol Sci 18:913Google Scholar
  48. Kimura T, Onodera A, Okumura F, Nakanishi T, Itoh N (2015) Chromium (VI)-induced transformation is enhanced by Zn deficiency in BALB/c 3T3 cells. J Toxicol Sci 40:383–387Google Scholar
  49. Lin SJ, Foley J, Jiang TX, Yeh CY, Wu P, Foley A, Yen CM, Huang YC, Cheng HC, Chen CF, Reeder B (2013a) Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge. Science 340:1442–1445Google Scholar
  50. Liang TW, Wang SL (2015) Recent advances in exopolysaccharides from Paenibacillus spp.: production, isolation, structure, and bioactivities. Mar Drugs 13:1847–1863Google Scholar
  51. Loukidou MX, Zouboulis AI, Karapantsios TD, Matis KA (2004) Equilibrium and kinetic modeling of chromium (VI) biosorption by Aeromonas caviae. Colloid Surf A Physicochem Eng Asp 242:93–104Google Scholar
  52. Lu X, Wang J, Al-Qadiri HM, Ross CF, Powers JR, Tang J, Rasco BA (2011) Determination of total phenolic content and antioxidant capacity of onion (Allium cepa) and shallot (Allium oschaninii) using infrared spectroscopy. Food Chem 12:637–644Google Scholar
  53. Ma Y, Oliveira RS, Freitas H, Zhang C (2016) Biochemical and molecular mechanisms of plant-microbe-metal interactions: Relevance for phytoremediation. Front Plant Sci 7:918Google Scholar
  54. Maru V, Gadre S (2016) Melanin pigment production studies from Azotobacter vinelandii. Int J Adv Lif Sci 9:44–49Google Scholar
  55. Marzan LW, Hossain M, Mina SA, Akter Y, Chowdhury AMA (2017) Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint. Egypt J Aquat Res 43:65–74Google Scholar
  56. Maquelin K, Kirschner C, Choo-Smith LP, van den Braak N, Endtz HP, Naumann D, Puppels GJ (2002) Identification of medically relevant microorganisms by vibrational spectroscopy. J Microb Methods 51:255–271Google Scholar
  57. Mishra A, Mishra KP (2015) Bacterial response as determinant of oxidative stress by heavy metals and antibiotic. J Innov Pharm Biol Sci 2:229–239Google Scholar
  58. Mody BR, Bindra MO, Modi VV (1989) Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies. Arch Microbiol 1:2–5Google Scholar
  59. Moghannem SA, Refaat BM, El-Sherbinya GM, El-Sayeda MH, Elsehemyb IA, Kalaba MH (2015) Characterization of heavy metal and antibiotic-resistant bacteria isolated from polluted localities in Egypt. Egypt Pharm J 14:158–165Google Scholar
  60. Movasaghi Z, Rehman S, Rehman DI (2008) Fourier transform infrared (FTIR) spectroscopy of biological tissues. Appl Spectros Rev 43:134–179Google Scholar
  61. Murthy S, Bali G, Sarangi SK (2011) Effect of lead on metallothionein concentration in lead resistant bacteria Bacillus cereus isolated from industrial effluent. Afr J Biotechnol 10:15966–15972Google Scholar
  62. Muthu M, Wu HF, Gopal J, Sivanesan I, Chun S (2017) Exploiting microbial polysaccharides for biosorption of trace elements in aqueous environments-scope for expansion via nanomaterial intervention. Polymers 9:721Google Scholar
  63. Nasti TH, Timares L (2015) MC 1R, Eumelanin and Pheomelanin: Their role in determining the susceptibility to skin cancer. Photochem Photobiol 91:188–200Google Scholar
  64. Nocelli N, Bogino PC, Banchio E, Giordano W (2016) Roles of extracellular polysaccharides and biofilm formation in heavy metal resistance of rhizobia. Materials 9:418Google Scholar
  65. Ojuederie OB, Babalola OO (2017) Microbial and plant-assisted bioremediation of heavy metal polluted environments: A review. Int J Environ Res Public Health 14:1504Google Scholar
  66. Oliveira H (2012) Chromium as an environmental pollutant: insights on induced plant toxicity. J Botany  https://doi.org/10.1155/2012/375843
  67. Oves M, Khan MS, Zaidi A (2013) Biosorption of heavy metals by Bacillus thuringiensis strain OSM29 originating from industrial effluent contaminated north Indian soil. Saudi J Biol Sci 20:121–129Google Scholar
  68. Rahimzadeh MR, Rahimzadeh MR, Kazemi S, Moghadamnia AA (2017) Cadmium toxicity and treatment: an update. Casp J Int Med 8:135Google Scholar
  69. Ramya D, Thatheyus AJ (2018) Microscopic investigations on the biosorption of heavy metals by bacterial cells: a review. Sci Int 6:11–17Google Scholar
  70. Rao N, Prabhu M, Xiao M, Li WJ (2017) Fungal and bacterial pigments: Secondary metabolites with wide applications. Front Microbiol 8:1113Google Scholar
  71. Rizvi A, Khan MS (2018) Heavy metal induced oxidative damage and root morphology alterations of maize (Zea mays L.) plants and stress mitigation by metal tolerant nitrogen fixing Azotobacter chroococcum. Ecotoxicol Environ Saf 157:9–20Google Scholar
  72. Rizvi A, Khan MS (2017) Cellular damage, plant growth promoting activity and chromium reducing ability of metal tolerant Pseudomonas aeruginosa CPSB1 recovered from metal polluted chilli (Capsicum annuum) rhizosphere. Acta Sci Agric 1:36–46Google Scholar
  73. Rubino FM (2015) Toxicity of glutathione-binding metals: a review of targets and mechanisms. Toxics 3:20–62Google Scholar
  74. Samuel J, Paul ML, Ravishankar H, Mathur A, Saha DP, Natarajan C, Mukherjee A (2013) The differential stress response of adapted chromite mine isolates Bacillus subtilis and Escherichia coli and its impact on bioremediation potential. Biodegradation 24:829–842Google Scholar
  75. Seneviratne M, Gunaratne S, Bandara T, Weerasundara L, Rajakaruna N, Seneviratne G, Vithanage M (2016) Plant growth promotion by Bradyrhizobium japonicum under heavy metal stress. South Afr J Bot 105:19–24Google Scholar
  76. Shahid M, Khan MS (2018) Glyphosate induced toxicity to chickpea plants and stress alleviation by herbicide tolerant phosphate solubilizing Burkholderia cepacia PSBB1 carrying multifarious plant growth promoting activities. 3Biotech 8:131Google Scholar
  77. Shameer S (2016) Biosorption of lead, copper and cadmium using the extracellular polysaccharides (EPS) of Bacillus sp., from solar salterns. 3Biotech 6:194Google Scholar
  78. Sharma H, Rawal N, Mathew BB (2015) The characteristics, toxicity and effects of cadmium. Int J Nanotech Nanosci 3:1–9Google Scholar
  79. Si M, Lang J (2018) The roles of metallothioneins in carcinogenesis. J Hematol Oncol 11:107Google Scholar
  80. Singh Y, Lal N (2015) Investigations on the heavy metal resistant bacterial isolates in vitro from industrial effluents. World J Pharm Pharm Sci 4:343–350Google Scholar
  81. Solano F (2014) Melanins: skin pigments and much more-types, structural models, biological functions, and formation routes. New J Sci 1:1–28Google Scholar
  82. Tarangini K, Mishra S (2013) Production, characterization and analysis of melanin from isolated marine Pseudomonas sp. using vegetable waste Res J Engg Sci 2:40–46Google Scholar
  83. Thaira H, Raval K, Manirethan V, Balakrishnan RM (2018) Melanin nano-pigments for heavy metal remediation from water. Sep Sci Technol 2:1–10Google Scholar
  84. Vignesh KS, Deepe Jr GS (2017) Metallothioneins: emerging modulators in immunity and infection. Int J Mol Sci 18:2197Google Scholar
  85. Xie Y, Fan J, Zhu W, Amombo E, Lou Y, Chen L, Fu J (2016) Effect of heavy metals pollution on soil microbial diversity and bermudagrass genetic variation. Front Plant Sci 7:755Google Scholar
  86. Zhang Z, Cai R, Zhang W, Fu Y, Jiao N (2017) A novel exopolysaccharide with metal adsorption capacity produced by a marine bacterium Alteromonas sp. JL2810. Mar Drugs 15:175Google Scholar
  87. Zimova M, Hackländer K, Good JM, Melo‐Ferreira J, Alves PC, Mills LS (2018) Function and underlying mechanisms of seasonal colour moulting in mammals and birds: what keeps them changing in a warming world? Biol Rev 93:1478–1498Google Scholar

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Authors and Affiliations

  • Asfa Rizvi
    • 1
    Email author
  • Bilal Ahmed
    • 1
  • Almas Zaidi
    • 1
  • Mohd. Saghir Khan
    • 1
  1. 1.Faculty of Agricultural Sciences, Department of Agricultural MicrobiologyAligarh Muslim UniversityAligarhIndia

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