Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in
Characterization of Heavy Metal Resitant Endophytic Fungi from Boswellia Ovalifoliolata 1 1
Aishwarya Sani , 2Venkateswarulu Nagam, 1 Vasudeva Reddy Netala , 2Vijaya Tartte*.
Department of Biotechnology, Sri Venkateswara University, Tirupati-517502, A.P,INDIA. 2 Department of Botany, Sri Venkateswara University, Tirupati-517502, A.P,INDIA.
Abstract: Heavy metal pollution is one of the major environmental problem globally. The aim of the study was to isolate endophytic fungi from Boswellia ovalifoliolata an endemic plant of Tirumal hills and to assess their ability to grow in presence of various heavy metals Cobalt Chloride(CoCl2), Zinc Chloride (ZnCl2), Copper Chloride (CuCl2) and Cadmium Chloride (CdCl2).Twelve isolates were isolated among them after screening the isolate which showed highest resistance against heavy metals was identified as Chaetomium .sp. (Co-400ppm, Zn- 400ppm, Cu200ppm and Cd-100ppm). Our results highlighted the potential endophytic fungi for treatment of heavy metal pollution. Keywords: Heavy endemic , potential.
metals,
endophytic
fungi,
Introduction Heavy metals like As, Cd, Co, Cu, Ni, Zn, and Cr are phytotoxicity either at all concentrations or above certain threshold levels. Heavy metals are continuously released into the environment due to industrial and technological development and contamination of agricultural soil with heavy metals is a major problem at industrial and defenserelated sites all over the world [1]. They damage the environment by affecting soil fertility, biomass and crop yields and ultimately human health [2]. Metal pollution has harmful effect on biological systems and does not undergo biodegradation they cannot be biodegraded but can be accumulated in living organisms, thus causing various diseases and disorders even in relatively lower concentrations [3]. Heavy metal pollution is one of the major environmental problem globally. Several conventional physico-chemical approaches have been used for the removal and treatment of heavy metal pollution sites such as electrochemical treatment, ion exchange, precipitation, reverse osmosis, evaporation and sorption [4-5]. However these methods are reported to be uneconomical and ineffective and often result in incomplete metal removal, high reagent and energy requirements and generation of toxic sludge [6]. Bioremediation on
Imperial Journal of Interdisciplinary Research (IJIR)
the other hand offers a promising and economical option to treat heavy metal in contaminated sites [7].One of the processes biosorption involves the removal of metal ions by organisms through solidliquid separation [8] and has received much attention because it has several advantages such as being highly efficient in removing heavy metal from diluted solutions [9-11]. Endophytes are organisms that inhabit plant organs and their presence is generally inconspicuous [12]. Bioaugmentation with endophytes has several benefits over traditional bioaugmentation, such as enriching the soil with a consortium of bioremediation strains. Endophytes reside in a contained environment, making them less susceptible to predation and the plant provides nutrients to the endophytes to support growth and establishment [13].There are very few on the potential use of endophytes for the treatment and removal of heavy metals. [14]Endophytic fungi can be used for phytoremediation or bioremediation at contaminated sites [15]. The aim of the present study is to isolate and screen heavy metal resistant endophytic fungi and to evaluate their efficiency to remove heavy metals under laboratory conditions. The aim of the study is to isolate heavy metal resistant endophytic fungi and screen them against Co, Zn, Cu and Cd heavy metals to assess their resistance to grow under elevated concentration. MATERIAL AND METHODS Isolation of Endophytic fungi Endophytic fungi were isolated from Boswellia ovalifoliolata an endemic medicinal plant from Eastern Ghats of Tirumala hills. The Eastern Ghats are one of the richest floristic and phytogeographical regions of India. Eastern Ghats are located between 76’ 56” and 86’ 30” E longitudes and 11’ 30” and 22’ N latitudes. They extend in a north east-south west strike in the Indian Peninsula covering an area of about 70, 000 sq km with an average width of 200 km in the north and 100 km in the south. They extend over a length of 1750 km between the rivers Mahanadi and Vaigai along the East coast. The leaves of Boswellia ovalifoliolata were surface sterilized
Page 1072
Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in 70% ethanol, 1% chlorox and rinsed with sterilized water [16]. Negative plates were prepared by spreading the on PDA plates with sterilized water used to rinse B. ovalifoliolata. The plates were incubated at 280 c for 7 days and observations were made regularly. After the observation of growth pure cultures of endophytic fungi were stored. Identification of endophytic fungi Morphological identification After 7 days of an inoculation period on PDA, a sterile loop was used to transfer the culture to the microscope slide. Lacto phenol cotton blue was used to stain the endophytic fungi for easier and better visualization. The slides were viewed under trinocular microscope and identification was based on fungal morphological keys [17-20]. Screening of fungal isolates against heavy metals Fungal isolates were screened against Co, Zn, Cu and Cd starting at concentration of 50ppm to 600ppm on PDA plates supplemented with increasing concentration of heavy metals using Cobalt Chloride (CoCl2), Zinc Chloride (ZnCl2) , Copper Chloride (CuCl2) and Cadmium Chloride (CdCl2) respectively. The isolates were transferred on to petridishes containing PDA agar supplemented with 50ppm to 600ppm concentration of specific heavy metals (Co, Zn, Cu and Cd). The medium without adding heavy metal
was served as control. Observations on growth of fungal isolates were made after 72hr of incubation. The growth of the fungal isolates was recorded as normal growth or absent growth in comparison to control. Results and Discussion Isolation of endophytic fungi After 48 hrs of inoculation growth was observed from the leaf tissue. Based on the surface sterilization protocol pure cultures were isolated by removing the epiphyte. A total of 32 endophytic fungi belonging to 12 taxa were successfully isolated from Boswellia ovalifoliolata an endemic medicinal plant. Microorganisms such as bacteria, fungi, algae and yeast have been increasingly studied due to their metal sequestering property [21]. Identification of endophytic fungi Among the twelve isolates the isolate which showed highest resistance to heavy metals was identified as Cheatomium .sp and the morphological features include Colonies are rapidly growing , cottony and white in colour (Fig1.A). The ascocarps are covered with thick walled, pale, branched hairs (Fig.1.B). Perithecia are large, fragile and globose in shape and have filamentous hair on the surface. Based on the colony morphology and microscopic identification it was identified as Chaetomium .sp.
Fig.1 (A and B) Morphological and microscopic identification of Cheatomium. sp Screening of fungal isolates against heavy metals 12 isolates were screened against Cobalt, Zinc, Copper and Cadmium. All the isolates showed resistance only up to 100ppm for Co and Zn but the Cheatomium. Sp is the only isolate that showed resistance against the four individuals. In this study Cheatomium. Sp showed resistance CoCl2 up to 400ppm, ZnCl2 up to 400ppm, CuCl2 up to 200ppm and CdCl2 up to 100ppm and further increased concentration of heavy metals inhibited the fungal growth (Table.1). As the concentration of heavy
Imperial Journal of Interdisciplinary Research (IJIR)
metals increased the diameter of the fungi was reduced when compared to control (Fig.2). Similar studies have been undertaken on filamentous fungal strains and mostly members from the genera Aspergillus, Fusarium, Humicola and Nannizzia have been reported to possess resistance against heavy metals [22-24].Recently, several studies have reported a similar trend among endophytic fungi being able to resist several heavy metals such as copper, zinc and cadmium [25-27] .
Page 1073
Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in Table .1 summary of growth observed (50-600ppm) concentration of heavy metals(Co, Zn Cu, Cd) S.No concentation CoCl2 ZnCl2 CuCl2 CdCl2 1. Control growth growth growth growth 2. 50ppm growth growth growth growth 3. 100ppm growth growth growth growth 4. 200ppm growth growth growth No growth 5. 400ppm growth growth No growth No growth 6. 600ppm No growth No growth No growth No growth
Fig.2 Screening of Cheatomium.sp against Cobalt, Zinc , Copper and Cadmium(50- 600ppm) Conclusion Twelve fungal isolates were isolated from the leaves of B.Ovalifoliolata .When the fungi were screened against four heavy metals (Co, Zn, Cu and Cd) there was decrease in no of isolates when increase in concentration of heavy metal from 50 to 600ppm. Among them the one which showed highest resistance to four individual heavy metals was identified as Cheatomium. Sp based on morphological and microscopic characters. Our preliminary findings indicated that Cheatomium.sp was resistant to CoCl2 up to 400ppm, ZnCl2 up to 400ppm, CuCl2 up to 200ppm and CdCl2 up to 100ppm. Further investigations are needed to
Imperial Journal of Interdisciplinary Research (IJIR)
determine the proteins involved in their biosorption process. Acknowledgement The authors are thankful to MOEF & CC , project , New Delhi for their financial support. Reference 1.AMINI M., YOUNESI H., BAHRAMIFAR N., AKBAR A., LORESTANI Z., GHORBANI F., DANESHI A., SHARIFZADEH M. Application of response surface methodology for optimization of lead biosorption in an aqueous solution by Aspergillus niger. Hazardous Materials 154, 694, 2008.
Page 1074
Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in 2.Mudgal V, Madaan N, Mudgal A (2010) Heavy metals in plants: phytoremediation: plants used to remediate heavy metal pollution. Agric Biol J N Am 1(1):40–46. 3. E. Pehlivan, A. M. Özkan, S. Dinç, and S. Parlayici, “Adsorption of Cu and Pb ion on dolomite powder,” Journal of Hazardous Materials, vol. 167, no. 1–3, pp. 1044–1049, 2009. 4. Kadirvelu K, Senthilkumar P, Thamaraiselvi K, Subburam V (2002) Activated carbon prepared from biomass as adsorbent: rlimination of Ni(II) from aqueous solution. Bioresource Tech 81:87-90. 5. Luo JM, Xiao X, Luo SL (2010) Biosorption of cadmium (II) from aqueous solutions byindustrial fungus Rhizopuscohnii. T Nonferr Metal Soc 20:1104-1111. 6. Hemambika B, Johncy RM, Kannan VR (2011) Biosorption of heavy metals by immobilized and deadfungal cells: a comparative assessment. J Ecol Nat Environ 3(5):168-175. 7. Iskandar NL, Zainudin NAIM, Tan SG (2011) Tolerance and biosorption of copper (Cu) and lead (Pb) by filamentous fungi isolated from a freshwater ecosystem. J Environ Sci 23(5):824830. 8. Yang HB, Tan N, Wu FJ, Liu HJ (2012) Biosorption of uranium (VI) by a mangrove endophytic fungus Fusarium sp. #ZZF51 from the South China Sea. J Radioanal Nucl Chem 292:1011-1016. 9. Kapoor A, Viraraghavan T, Cullimore DR (1999) Removal of heavy metals using the fungus Aspergillusniger. Bioresource Tech 70:95-104. 10. Cruz CC, da Costa AC, Henriques CA, Luna AS (2004) Kinetic modeling and equilibrium studies during cadmium biosorption by dead Sargassum sp. biomass. Bioresource Tech 91(3): 249-257. 11. Fan T, Liu Y, Feng B, Zeng G, Yang C, Zhou M, Zhou H, Tan Z, Wang X (2008) Biosorption of cadmium(II), zinc(II) and lead(II) by Penicillium simplicissimum: Isotherms, kinetics and thermodynamics. J Hazard Mater 160:655-661. 12. Li H, Wei D, Shen M, Zhou Z (2012a) Endophytes and their role in phytoremediation. Fungal Divers 54:11–18.
Imperial Journal of Interdisciplinary Research (IJIR)
13.Weyens N, van der Lelie D, Artois T, Smeets T, Taghavi S, Newman L, Carleer R, Vangronsveld J (2009) Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation. Environ Sci Technol 43:9413– 9418. 14. Li YH, Zhu JN, Zhai ZH, Zhang Q (2010) Endophytic bacterial diversity in roots of Phragmitesaustralis in constructed Beijing Cuihu Wetland (China). FEMS Microbiol Lett 309:84-93. 15. Yang HB, Tan N, Wu FJ, Liu HJ (2012) Biosorption of uranium (VI) by a mangrove endophytic fungus Fusarium sp. #ZZF51 from the South China Sea. J Radioanal Nucl Chem 292:1011-10. 16. El-Gendy MMA, Hassanein NM, Ibrahim HAE, El-Baky DHA (2011) Evaluation of some fungal endophytes of plant potentiality as low-cost adsorbents for heavy metals uptake from aqueous solution. Aust J Basic Appl Sci 5(7):466-473. 17. Maharachchikumbura SN, Guo L-D, Ekachai C, Ali HB, Hyde KD (2011) Pestalotiopsismorphology, phylogeny, biochemistry and diversity. Fungal Divers 50:167-187 18. Jeewon R, Liew CY, Simpson JA, Hodgkiss IJ, Hyde KD (2003) Phylogenetic significance of morphological characters in the taxonomy of Pestalotiopsis species. Mol Phylogenet Evol 27:372-383 19. Steyaert RL (1949) Contribution à l'étudemonographique de Pestalotia de Not.et Monochaetia Sacco (Truncatella gen. nov. Et Pestalotiopsis gen. nov.). Bull Jardin Bot État Bruxelles 19:285-354 20.Guba EF (1929) Monograph of the genus Pestalotia de Notaris. Part1. Phytopathology 19:191-232. 21. Wang J, Chen C (2009) Biosorbents forheavy metals removal and their future. Biotechnol Adv 27(2):195-226. 22. Iram S, Zaman A, Iqbal Z, Shabbir R (2013) Heavy metal tolerance of fungus isolated from soil contaminated with sewage and industrial wastewater. Polish J Environ Stud 22(3):691-697. 23. Ezzouhri L, Castro E, Moya M, Espinola F, Lairini K (2009) Heavy metal tolerance of
Page 1075
Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in filamentous fungi isolated from polluted sites in Tangier, Morocco. Afr J Microbiol Res 3(2):35-48. 24. Valix M, Tang J, Malik R (2001) Heavy metal tolerance of fungi. Mine Eng 14(5):499-505. 25. Hong JW, Park JY, Gadd GM (2010) Pyrene degradation and copper and zinc uptake by Fusarium solani and Hypocrealixii isolated from petrol station soil. J Appl Microbiol 108(6):20302040.
and Uptake of Copper Nanoparticles by Dead Biomass of Hypocrea lixii Isolated from the Metal Mine in the Brazilian Amazon Region. PloS ONE 8(11):e80519. doi:10.1371/ journal. pone.0080519. 27. Deng Z, Zhang R, Shi Y, Tan H, Cao L (2014) Characterization of Cd-, Pb-, Zn-resistant endophytic Lasiodiplodia sp. MXSF31 from metal accumulating Portulaca oleracea and its potential in promoting the growth of rape in metalcontaminated soils. Environ Sci Pollut Res 21(3):2346-2357.
26. Salvadori MR, Lepre LF, Ando RA, Nascimento CAO, CorrĂŞa B (2013) Biosynthesis
Imperial Journal of Interdisciplinary Research (IJIR)
Page 1076