臺灣博碩士論文加值系統

本研究由受油品與鋅/銅汙染之油泥中篩選分離兼具有效降解柴油或甲苯與耐受鋅/銅的高效降解菌，研究中共計篩選出10株高效降解菌，其中包括：甲苯降解菌T5, T6, T7, T10, T11與T12；柴油降解菌D2, D5, D6與D7，進一步利用16S rDNA鑑定各菌種，結果發現主要之菌種為Achromobacter insolitus (D2), Candida spp. (D5), Janthobacter polyaromaticivorans (D6與D12), Ochrobactrum intermedium (D7), Streptomyces caelestis (T1與T5), Gordonia amicalis (T6), Brevibacillus brevis (T10), Bacillus fumarioli (T11)與真菌(T7)。運用氣相層析火焰離子化偵檢器(gas chromatography/flame ionization detector, 簡稱GC/FID)測定高效降解菌對柴油或甲苯之降解能力，比較各菌株之降解力，結果可知T6, T7與T11具有較佳之甲苯降解能力；D2, D5與 D6則具有較佳之柴油降解能力。進一步探討各降解菌的重金屬耐受性與共代謝能力，本研究篩選之甲苯分解菌均能忍受250 ppm Zn，柴油分解菌D2, D5與D7能忍受250 ppm Zn，高Zn濃度則對多數菌種產生抑制作用，其中T7與D5可忍受高濃度(500 ppm)；菌種之生長較易受銅影響，篩選出之菌種中僅以T7與D5能耐受高濃度Cu，後續利用化學連續萃取法分析探討耐受菌種之重金屬分布型態，以探討重金屬在細胞中之累積機制。結果顯示，細胞外之吸附量(biosorption)為59.7~70.8%，細胞內累積量(bioaccumulation)則約在29.2~40.3 %，微生物對重金屬之耐受主要是將重金屬排除於細胞外，或將重金屬結合於細胞壁與細胞外聚合物。

The experiments were performed to screen and isolate the bacteria that might be suitable for bioremediation purposes to remove toluene or diesel from polluted soil in presence of Zn and Cu. Ten bacterial strains were isolated from different sources of soil slurry in Taiwan, including 6 strains of toluene-degrading bacteria (T5, T6, T7, T10, T11 and T12) and 4 strains of diesel oil-degrading bacteria (D2, D5, D6 and D7). To illustrate the result of 16S rDNA approach that the dominate bacteria are Achromobacter insolitus (D2)， Candida spp. (D5)，Xanthobacter polyaromaticivorans (D6 and T12)，Ochrobactrum intermedium (D7)，Streptomyces caelestis (T1 and T5)，Gordonia amicalis (T6)，Brevibacillus brevis (T10)，Bacillus fumarioli (T11) and Fungus (T7). Their ability to degrade toluene and diesel were analyzed by a gas chromatograph coupled to a FID detector (GC-FID) method. The observations indicated that T6, T7, T11 and D2, D5, D6 have the high performance on toluene and diesel degradation respectively. In the research, tolerant to heavy metal and the effect of different nutrients on co-metabolism system was studied. Whether show the inhibition to the growth or increase efficiency to the percent removal of the petroleum-hydrocarbon. The results showed, 250 ppm Zn-tolerant on all toluene-strains and D2, D5, D7 strains while 250 ppm Cu-tolerant revealed to T7 strain. The strains was evaluated the accumulation of heavy metal on cell mechanism by sequential chemical extraction (SCE), thus, resulting 59.7-70.8 % biosorption and 29.2-40.3 % bioaccumulation.

ABSTRACT I
ACKNOWLEDGEMENT II
LIST OF CONTENTS III
LIST OF TABLES VII
LIST OF FIGURES XI
LIST OF ABBREVIATIONS XIV
CHAPTER 1: INTRODUCTION 1
1.1 Introduction 1
1.1.1 Soil contamination 1
1.1.2 Bioremediation 2
1.2 Objectives 3
1.3 Scope of study 4
CHAPTER 2: LITERATURE REVIEW 6
2.1 Principle of bioremediation 6
2.1.1 Soil characteristics 7
2.1.2 Contaminant characteristics 8
2.1.3 Bacteria 9
2.1.4 Nutrient 13
2.1.5 Environmental requirements 14
2.2 Culture media and Cultivation of bacteria 16
2.2.1 Bacterial culture media 16
2.2.2 Cultivation of bacteria 21
2.3 Metal toxicity and microbial tolerance mechanisms 23
2.3.1 Metal tolerance mechanisms 25
2.3.2 Biosorption and bioaccumulation of heavy metals 26
2.4 Effect of adding nutrient source on growth and 28
biodegradation
CHAPTER 3: MATERIALS AND METHODS 29
3.1 Isolation of bacteria degrading strain 29
3.1.1 Soil samples 29
3.1.2 Culture Media 30
3.1.3 Growing bacteria cultures from soil (Isolation) 31
3.2 Identification bacteria strains by 16s rDNA 35
3.3 Biodegradation of hydrocarbon (toluene/diesel)
by isolated strain 35
3.4 Heavy metal (Zn/Cu) tolerance 37
3.5 Improving growth by carbon (C) anddition 38
3.6 Improving growth by nitrogen (N) addition 38
3.7 Summarize analysis method 38
3.7.1 Hydrocarbon analysis using
gas chromatography (GC-FID) 38
3.7.2 Sequential chemical extraction (SCE) with ICP 39
3.7.3 Cell concentration (OD) 39
3.7.4 pH measurement 40
3.7.5 Emulsification index (E24) 40
CHAPTER 4: RESULTS AND DISCUSSIONS 41
4.1 Isolation of bacteria degrading strains 41
4.2 Identification of isolated strain 42
4.3 Biodegradation of hydrocarbon (toluene/diesel)
by isolated strain 42
4.4 Heavy metal (Zn/Cu) tolerance 55
4.5 Improving isolated strains growth by Carbon (C) addition 64
4.6 Improving isolated strains growth by Nitrogen (N) addition 73
CHAPTER 5: CONCLUSIONS AND SUGGESTIONS 82
5.1 Conclusions 82
5.2 Suggestions 84
REFERENCE 85
APPENDIX 95









LIST OF TABLES

Table 2-1. Optimal conditions for microbial growth and hydrocarbon biodegradation 8
Table 2-2. Composition of microbial cell. 13
Table 2-3. Environmental conditions affecting degradation. 14
Table 3-1. Sampling coordinate 29
Table 3-2. Bushnell-Haas (ATCC medium 1287) 30
Table 3-3. Phosphate-Buffered Saline 31
Table 4-1. Sampling sources and the strains 41
Table 4-2. Toluene concentration declining over time 43
Table 4-3. Toluene removal efficiency on each strains 44
Table 4-4. Diesel concentration declining over time 49
Table 4-5. Diesel removal efficiency on each strains 49
Table 4-6. pH on D-strains over time 50
Table 4-7. OD on D-strains over time 50
Table 4-8. Positive/Negative growth test with Zn metal on T-strains 56
Table 4-9. Positive/Negative growth test with Zn metal on D-strains 56
Table 4-10. Positive/Negative growth test with Cu metal on T-strains 56
Table 4-11. Positive/Negative growth test with Cu metal on D-strains 57
Table 4-12. Toluene concentration declining over time with Zn metal and Cu metal tolerance on T-strains 58
Table 4-13. Diesel concentration declining over time with Zn metal and Cu metal tolerance on D-strains 58
Table 4-14. Sequential extraction procedure for metal 62
Table 4-15. Adsorption conformation of T-strains with Zn metal in BH medium 63
Table 4-16. Adsorption conformation of D-strains with Cu metal in BH medium 63
Table 4-17. Adsorption conformation of T-strain and D-strain with Cu metal in BH medium 64
Table 4-18. pH change on T-strains with glucose addition over time 64
Table 4-19. pH change on T-strains with sucrose addition over time 64
Table 4-20. pH change on D-strains with glucose addition over time 65
Table 4-21. pH change on D-strains with sucrose addition over time 65
Table 4-22. OD660 effect on T-strains with glucose addition over time 65
Table 4-23. OD660 effect on T-strains with sucrose addition over time 66
Table 4-24. OD660 effect on D-strains with glucose addition over time 66
Table 4-25. OD660 effect on D-strains with sucrose addition over time 66
Table 4-26. pH effect on T-strains with Ammonium Sulphate addition over time 73
Table 4-27. pH effect on T-strains with Sodium nitrate addition over time 73
Table 4-28. pH effect on D-strains with Ammonium Sulphate addition over time 74
Table 4-29. pH effect on D-strains with Sodium nitrate addition over time 74
Table 4-30. OD660 effect on T-strains with Ammonium Sulphate addition over time 75
Table 4-31. OD660 effect on T-strains with Sodium nitrate addition over time 75
Table 4-32. OD660 effect on D-strains with Ammonium Sulphate addition over time 76
Table 4-33. OD660 effect on D-strains with Sodium nitrate addition over time 76













LIST OF FIGURES
Figure 1-1. Summary of the scope of study 5
Figure 2-1. Bacterial colony morphology characters and descriptions. 12
Figure 3-1. Dilution soil samples 31
Figure 3-2. Pour plate hydrocarbon agar media 32
Figure 3-3. Streaking plate method 33
Figure 3-4. Incubate streaked plated 34
Figure 3-5. Preservation of strains 35
Figure 3-6. Toluene calibration curve 36
Figure 3-7. Diesel calibration curve 37
Figure 4-1. Toluene concentration and % removal over time on T5-strain 45
Figure 4-2. Toluene concentration and % removal over time on T6-strain 45
Figure 4-3. Toluene concentration and % removal over time on T7-strain 46
Figure 4-4. Toluene concentration and % removal over time on T10-strain 46
Figure 4-5. Toluene concentration and % removal over time on T11-strain 47
Figure 4-6. Toluene concentration and % removal over time on T12-strain 47
Figure 4-7. Diesel concentration and % removal over time on D2-strain 53
Figure 4-8. Diesel concentration and % removal over time on D5-strain 53
Figure 4-9. Diesel concentration and % removal over time on D6-strain 54
Figure 4-10. Diesel concentration and % removal over time on D7-strain 54
Figure 4-11. Heavy metals (Zn/Cu) test growth among different 55
Figure 4-12. Toluene degrading with Zn metal tolerance on T-strains 60
Figure 4-13. Toluene degrading with Cu metal tolerance on T-strains 60
Figure 4-14. Diesel degrading with Zn metal tolerance on D-strains 61
Figure 4-15. Diesel degrading with Cu metal tolerance on T-strains 61
Figure 4-16. Glucose addition with pH effect on T-strains 69
Figure 4-17. Sucrose addition with pH effect on T-strains 69
Figure 4-18. Glucose addition with pH effect on D-strains 70
Figure 4-19. Sucrose addition with pH effect on D-strains 70
Figure 4-20. Glucose addition with OD660 effect on T-strains 71
Figure 4-21. Sucrose addition with OD660 effect on T-strains 71
Figure 4-22. Glucose addition with OD660 effect on D-strains 72
Figure 4-23. Sucrose addition with OD660 effect on D-strains 72
Figure 4-24. Ammonium Sulphate addition with pH effect on T-strains 78
Figure 4-25. Sodium nitrate addition with pH effect on T-strains 78
Figure 4-26. Ammonium Sulphate addition with pH effect on D-strains 79
Figure 4-27. Sodium nitrate addition with pH effect on D-strains 79
Figure 4-28. Ammonium Sulphate addition with OD660 effect on T-strains 80
Figure 4-29. Sodium nitrate addition with OD660 effect on T-strains 80
Figure 4-30. Ammonium Sulphate addition with OD660 effect on D-strains 81
Figure 4-31. Sodium nitrate addition with OD660 effect on D-strains 81

[1] Wolicka, D., Suszek, A., Borkowski, A., Bielecka, A., 2009. Application of aerobic microorganisms in bioremediation in situ of soil contaminated by petroleum products. Bioresource Technology 100, 3221-3227.
[2] Chen, C.-H., 2012. The bioavailability of Petroleum Hydrocarbon Contaminants in soil and Seawater. Thesis of Master of Science. National Chen Kung University.
[3] Liu, P.-W.G., et al., Bioaugmentation efficiency investigation on soil organic matters and microbial community shift of diesel-contaminated soils, International Biodeterioration & Biodegradation (2014), http://dx.doi.org/10.1016/j.ibiod.2014.05.004
[4] Mukherjee, A.K., Bordoloi, N.K., 2011. Bioremediation and reclamation of soil contaminated with petroleum oil hydrocarbons by exogenously 379 seeded bacterial consortium: a pilot-scale study. Environ, Sci. Pollut, Res. 18, 471-478.
[5] Coulon, F., Brassington, K.J., Bazin, R., Linnet, P.E., Thomas, K.A., Mitchell, T.R., Lethbridge, G., Smith, J.W.N., Pollard, S.J.T., 2012. Effect of fertilizer formulation and bioaugmentation on biodegradation and leaching of crude oils and refined products in soils. Environ, Sci. Technol. 31, 771-775
[6] S.Y.T. Adeline, H.C.T. Carol, C.S. Aw, Hydrocarbon-degradation by isolate Pseudomonas lundensis UTAR FPE2. Malaysian Journal of Microbiology, 5(2), 2009, 104-108.
[7] Ljah, U.J.J. and Antai, S.P., (1998), Degradation and Mineralization of crude oil by bacteria, Niger. J. Biotechnology, 5, pp 79-86.
[8] Antai, S.P. and Mgbomo, E., (1993), Pattern of degradation of bonny light crude oil by Bacillus spp. and Pseudomonas spp. isolated from oil spilled site, W.A.J. boil. Appl. Chem. 38, pp 16-20.
[9] Olu-Arotiowa OA, Aremu MO, Alade AO (2007) Ex-situ bioremediation of diesel polluted waste water in tropical hot climate. Asian J Info Technol. 6(9): 961-963.
[10] Supaka N, Pinphanichakam P, Pattaragulwanit K, Thaniyavah S, Omori T, Juntonggjin K (2001) Isolation and characterization of a phenanthrene degrading Sphingomonas sp. Strain P2 and its ability to degrade Fluoranthene and Pyrene via cometabolism. Res Article Sci Asia. 27: 21-28.
[11] Tazeena Hassan Islam, Badal Ghosh, Md. Mehedi Hasan Magnet, et al., 2013. Isolation and identification of petroleum degrading bacteria from oil contaminated soil & water and assessment of their potentiality in bioremediation. IOSR Journal Of Environmental Science, Toxicology And Food Technology. 5(2): 55-58.
[12] Wen-Ta Su, Bo-Shin Wu, Wen-Jang Chen, 2011. Characterization and biodegradation of motor oil by indigenous Pseudomonas aeruginosa and optimizing medium constituents. Journal of the Taiwan Institute of Chemical Engineer. 42, pp 689-695.
[13] State of Mississippi. Department of Environmental Quality, 1998, Fundamental Principles of Bioremediation. April 1998. 27 Nov 2006 <http://www.deq.state.ms.us/MDEQ.nsf/pdf/ GARD_Bioremediation/$File/Bioremediation.pdf? Verified 12/15/2006.
[14] Adebusoye, S. A., M. O. llori, O. O. Amund, O. D. Teniola, and S. O. Olatope, “Microbial Degradation of Petroleum Hydrocarbons in a Polluted Tropical Stream,” World J. Microbiol. Biotechnol. 23, 1149 (2007).
[15] Farahat, L. A. and N. S. El-Gendy, “Biodegradation of Baleym mix crude oil in soil microcosm by some locally isolated Egyptian bacterial strains,” Soil Sediment Contam. 17, 150 (2008).
[16] National Research Council, op. cit., footnote 1, p. 17.
[17] http://www.pollutionissues.com/Na-Ph/Petroleum.html, 27-03-2011
[18] AM Mukred, A A Hamid, A Hamzah & WMW Yusoff, Development of Three Bacteria Consortium for the Bioremediation of Crude Petroleum-oil in Contaminated Water, OnLine Journal of Biological Sciences, 8 (4) (2008) 73ISSN 1608-4217.
[19] ISU Engineering Information Technology
[20] http://home.eng.iastate.edu/~tge/ce421-521/brubaker.pdf, retrieved on 27th Mar. 2011
[21] Alexander, M. (1995). How toxic are toxic chemicals in soil? Environmental Science and Technology, Vol. 29, No. 11, pp. 2713–2717, ISSN 0013-936X.
[22] Jain, P.K.; Gupta, V.K.; Guar, R.K.; Lowry, M.; Jaroli, D.P. & Chauhan, U.K. (2011). Bioremediation of petroleum contaminated soil and water. Research Journal of Environmental Toxicology, Vol. 5, No. 1, pp. 1-26, ISSN 1819-3420.
[23] Balba, M.T.; Al-Awadhi, N. & Al-Daher, R. (1998). Bioremediation of oil-contaminated soil: microbiological methods for feasibility assessment and field evaluation. Journal of Microbiological Methods, Vol. 32, No. 2, pp. 155–164, ISSN 0167-7012.
[24] Loeher, R.C.; Mc Millen, S.J. & Webster, M.T. (2001). Predictions of biotreatability and actual results: soils with petroleum hydrocarbons. Pratice periodical of hazardous, toxic, and radioactive waste management, Vol. 5, No. 2, pp. 78–87, ISSN 1090-025X.
[25] Ivančev-Tumbas, I.; Tričković, J.; Karlović, E.; Tamaš, Z.; Rončević, S.; Dalmacija, B.; Petrović, O. & Klašnja, M. (2004). GC/MS-SCAN to follow the fate of crude oil components in bioreactors set to remediate contaminated soil. International Biodeterioration and Biodegradation, Vol. 54, No. 4, pp. 311-318, ISSN 0964-8305.
[26] Brassington, K.J.; Hough, R.L.; Paton, G.I.; Semple, K.T.; Risdon, G.C.; Crossley, J.; Hay, I.; Askari, K. & Pollard, S.J.T. (2007). Weathered Hydrocarbon Wastes: A Risk Management Primer. Critical Reviews in Environmental Science and Technology, Vol. 37, No. 3, pp. 199-232, ISSN 1064-3389.
[27] Stroud, J.L.; Paton, G.I. & Semple, K.T. (2007). Microbe–aliphatic hydrocarbon interactions in soil: implications for biodegradation and bioremediation. Journal of Applied Microbiology, Vol. 102, No. 5, pp. 1239–1253, ISSN 1364-5072.
[28] Biostimulation - Wikipedia, the free encyclopedia, http://en.wikipedia.org/wiki/Biostimulation, retrieved on 28 Mar. 2011.
[29] Eugrishttp://www.eugris.info/newsdownloads / Green Remediation/ pdf /D05 AnneLouise Gimsing_Paper.pdf.
[30] Atlas, op. cit., footnote 23, p. 212.
[31] U S Congress, Office of Technology Assessment, Bioremediation for Marine Oil Spills- Background Paper, OTA-BP-0-70 (Washington, DC: U.S. Government Printing Office, May 1991).
[32] Stillman, M. j and Presta, A., Molecular Biology and Toxicology of Metals, 2000, 1: 1-33.
[33] Garbarino JR., Hayes H, Roth D et al. (1995). Contaminants in the Mississippi river. U. S. Geological Survey Circular, Virginia, U.S.A. 1133.
[34] Gadd G.M. Metals and microorganisms: A problem of definition. FEMS Microbiol. Lett. 1992;100(1–3):197–204.
[35] Hughes M.N., Poole R.K. The functions of metals in micro-organisms. In: Hughes M.N., Poole R.K., editors. Metals and microorganisms. London: Chapman and Hall; 1989. pp. 1–38.
[36] Shi, W. Becker, J. et al, Appl Environ Microbiol, 2002, 68(8): 3859-3866.
[37] Badar, U. Ahmed, N. et al, Biotech Lett, 2000, 22(10): 829-836.
[38] Koropatnick, J. Leibbrandt, M.E.I, Effects of metals on gene expression, Handbook of Experimental Pharmacology, Toxicology of Metals, Biochemical aspects, 1995, 115: 93-120.
[39] Goering, P.L. Beck, B.D. et al, Toxicol Sci,1999, 49(1): 5-14.
[40] Nies, D.H. (1999). Microbial heavy metal resistance. Appl Microbiol Biotechnol 51: 730-750.
[41] P Vasudevan; V Padmavathy; N Tewari; SC Dhingra; J Sci Ind Res; 2001;60, 112-120.
[42] A Pandey; P Nigam; D Singh; Chem Ind Digest; 2001; 14; 93–95.
[43] A Rani; R Goel; Microbial strategies for crop improvement, Springer, Berlin 2009; 105–132.
[44] M. A. Ekpo and U. S. Udofia, Fate of biodegradation of crude oil by microorganisms isolated from oil sludge environment, African Journal of Biotechnology, 7(24), Dec 2008, 4495-4499.
[45] Saadoun I. Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel. J Basic Microbiol 2002; 42:420-8.
[46] JiHyeon Song, HyeongKyu Namgung, Zubair Ahmed, 2012. Biodegradation of toluene using Candida tropicalis immobilized on polymer matrices in fluidized bed bioreactors. Journal of Hazardous Materials. 241-241, pp 316-322.
[47] Lake DL. Chemical speciation of heavy metals in sewage sludge and related matrices. In: Lester JN, editor. Heavy metals in wastewater and sludge treatment processes sources analysis and legislation, vol. 1. Florida, USA: CRC Press, 1987. P. 125-53.
[48] Stover RC, Sommer LE, Silviera DJ. Evaluation of metals in wastewater sludge. J Water Pollut Control Fed 1976; 8:2165-75.
[49] Geetha S. J., Sanket J. Joshi, Shailesh Kathrotiya, 2013. Isolation and characterization of hydrocarbon degrading bacterial isolate from oil contaminated sites. APCBEE Procedia. 5, pp. 237-241.
[50] Cooper, D. G. and Goldenberg, B. G.: Surface active agents from Bacillus species. Appl. Environ. Microbiol., 55, 224-229 (1987).
[51] Cerniglia, C. E. (1984) Microbial transformation of aromatic hydrocarbons. In Petroleum Microbiology (R. M. Atlas, ed.): 99–128. Macmillan, New York.
[52] Weber, F. J., Hage, K. C. & de Bont, J. A. M. (1995) Growth of the fungus Cladosporium sphaerospermum with toluene as the sole carbon and energy source. Applied and Environmental Microbiology 61: 3562–3566.
[53] Jackisch-Matsuura, Ani Beatriz, Santos, Leonardo Silva, Eberlin, Marcos Nogueira, Faria, Andréia Fonseca de, Matsuura, Takeshi, Grossman, Matthew James, & Durrant, Lucia Regina. (2014). Production and characterization of surface-active compounds from Gordonia amicalis. Brazilian Archives of Biology and Technology, 57(1), 138-144
[54] Arenskötter M, Broker D, Steinbuchel A. Biology of the metabolically diverse genus Gordonia. Appl Environ Microbiol. 2004; 70: 3195-3204.
[55] Mariano, A. P., Bonotto, D.M., De Angelis, D.F., 2008. Biodegradability of commercial and weathered diesel oils. Brazilian Journal of Microbiology, 39, 133–142.
[56] Ghosal, D., Chakraborty, J., Khara, P., 2010. Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD. FEMS Microbiology Letters, 313, 103–110.
[57] Yang, N., Zhang, R., Xiang, Z., 2013. Study the optimal condition of fenpropathrin degradation by Ochrobactrum anthropi based on bacteria microscopic image detection method. Advance Journal of Food Science and Technology, 5(6), 688–694.
[58] Whang L-M, Liu P-WG, Ma C-C, Cheng S-S. Application of rhamnolipid and surfactin for enhanced diesel biodegradation-Effects of pH and ammonium addition. J Hazard Mater. 2009;164:1045–1050.
[59] Xia W, Li J, Xia Y, Song Z, Zhou J. Optimization of diesel oil biodegradation in seawater using statistical experimental methodology. Water Sci Technol. 2012;66:1301–1309.
[60] Sathishkumar M, Binupriya AR, Baik S-H, Yun S-E. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas. Clean-Soil Air Water. 2008;36:92–96.
[61] Ray, S., Ray, M. K. (2009) Bioremediation of heavy metal toxicity-with special reference to chromium. Al Ameen Journal of Medical Science 2: 57-63.
[62] Abou-Shanab, R. A. I., van Berkum, P. Angle, J. S., (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. Chemosphere, 68: 360-367.
[63] Hassan, S. H. A., Abskharon, R. N. N., Gad El-Rab, S. M. F., Shoreit, A. A. M. (2008) Isolation, characterization of heavy metal resistant strain of Pseudomonas aeruginosa isolated from polluted sites in Assiut city, Egypt. Journal of Basic Microbiology 48: 168-176.
[64] Nies, D.H., 1999. Microbial heavy metal resistance. Applied Microbiol. Biotechnol., 51: 730-750.
[65] Gadd, G.M., 1992. Metals and microorganisms: A problem of definition. FEMS Microbiol. Lett.,100: 197-203.
[66] Zolgharnein, H., K. Karami, M.M. Assadi and A.D. Sohrab, 2010. Investigation of heavy metals biosorption on Pseudomonas aeruginosa strain MCCB 102 isolated from the persian gulf. Asian J. Biotechnol., 2: 99-109.
[67] Rani, A. and R. Goel, 2009. Strategies for Crop Improvement in Contaminated Soils Using Metal-Tolerant Bioinoculants. In: Microbial Strategies for Crop Improvement, Khan, M.S., A. Azidi and J. Musarrat (Eds.). Springer, Berlin, pp: 105-132.