1 / 39

ZUBAIRU UMAR DARMA, PhD

BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS USING BACTERIA ISOLATED FROM USED ENGINE OIL CONTAMINATED SOIL. ZUBAIRU UMAR DARMA, PhD. DEPARTMENT OF MICROBIOLOGY FACULTY OF NATURAL AND APPLIED SCIENCES UMARU MUSA YAR’ADUA UNIVERSITY, KATSINA. OVERVIEW. BETTER REMOVAL APPROACH. PAHs.

bferrero
Download Presentation

ZUBAIRU UMAR DARMA, PhD

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS USING BACTERIA ISOLATED FROM USED ENGINE OIL CONTAMINATED SOIL ZUBAIRU UMAR DARMA, PhD DEPARTMENT OF MICROBIOLOGY FACULTY OF NATURAL AND APPLIED SCIENCES UMARU MUSA YAR’ADUA UNIVERSITY, KATSINA

  2. OVERVIEW BETTER REMOVAL APPROACH PAHs • Incomplete combustion process • Natural & Anthropogenic sources • Wide environmental distribution Conventional removal Biodegradation SUGGESTION • Hazardous intermediates • Labour intensive • High environmental impact • Environmentally friendly • Involved microorganisms • Slow degradation limitation Identify effective bacteria from PAHs source that will perform rapid degradation Dangerous to living cells

  3. OVERVIEW PAHs REMOVAL STRATEGIES DISADVANTAGES ADVANTAGES • The process is very fast • Costly equipments are used • Partial degradation to more hazardous products • (Vela et al., 2012) PHYSICAL • Costly chemicals needed • Chemicals react with PAHs & form much complex compounds • (Shih & Lederberg, 1976) • Requires no modification of natural environment CHEMICAL • Complete PAHs degradation to less hazardous products • Less expensive & environmentally reliable • Slow degradation limitation • Not all PAHs are biodegradable • (Vidali, 2001) BIODEGRAD. • Difficult to extrapolate laboratory results to full scale field operation • Effective degradation response • Adequate energy generation for microbial effective functions AEROBIC DEGRADATION Is aerobic degradation effective enough to remove significant PAHs pollutants in 24h?

  4. THE IDEA Rapid PAHs biodegradation 2 Effective PAHs degrading bact. 1 Used engine oil cont. soil IMPROVED DEGRADATION STRATEGY ~30% PAHs

  5. QUESTION ? • Does PAHs degrading bacteria exist in used engine oil contaminated soil? • OBJECTIVE 1 • Identification of PAHs degrading bacteria from used engine oil contaminated soil OBJECTIVE 1: An attempt to identify effective PAHs degrading bacteria • HOW DO WE ACHIEVE THIS? • Enrichment of soil bacteria & screening the best isolates • Molecular identification • SIGNIFICANT OF THE STUDY • Strategizing the best PAHs biodegradation

  6. EXPERIMENT 1: Identification of PAHs degrading bacteria • Isolation • Soil sampling • Enrichment1 • Serial dilution & Plating • Bacteria purification & preservation • Parameters • Spray plate assay2 • Colorimetric assay3 • Screening • Bacteria cultured on BHA (72h) then MSM-DCPIP (24h) at 37°C, 200 rpm1 • PAHs • 500 mg/L PHN • 250 mg/L PYR • Potential degraders • Bacteria resting cells4 • Quantification of PHN & PYR degraded in 24h3 • Identification • Morphology • 16S rRNA sequencing5 • Tao et al., (2007) • Kiyoharaet al., (1982) • Hanson et al., (1993) • Sogani et al., (2012) • Kumar et al., (2016)

  7. OUTCOME 1: Isolation & Screening • Total bacteria isolated : 93 Isolates • 72h spray plate screened : 53 Isolates • 24h colorimetric rapid response : MM045 & MM087 DCPIP

  8. OUTCOME 1: PAHs quantifications Strain MM045 PHN PYR (Error bars represent ± standard deviations; n = 3)

  9. Cont. Strain MM087 PHN PYR • Both C. sakazakiiMM045 and Enterobacter sp. MM087 were found to have potentially degraded significant PAHs concentrations in 24 hours

  10. OUTCOME 1: Bacteria Identification • Conclusion • Both strains MM045 and MM087 degraded >70% PHN & >50% PYR in 24 hrs • The isolates could be potential degraders that effectively remove PAHs rapidly Can the degradation of either one or both isolate(s) be enhanced to attain 100% PAHs degradation?

  11. QUESTION ? • Can C. sakazakii MM045 & Enterobacter sp. MM087 perform 100% PAHs degradation in 24 hrs? • OBJECTIVE 2 ! • Optimization of phenanthrene and pyrene biodegradation conditions OBJECTIVE 2: Optimization of PAHs biodegradation conditions • HOW TO ACHIEVE IT ? • Single factor optimization • Response surface methodology • SIGNIFICANCE • Enhancing degradation performance ofC. sakazakii MM045 & Enterobacter sp. MM087

  12. EXPERIMENT 2: Optimization of biodegradation conditions • Strain MM045 & MM087 • Colorimetric assay3 • PHN (500 mg/L) & PYR (250 mg/L) • Parameters • Agitation • Temp. • pH • Inoc. vol. • Salinity • Single factor optimization6 • Bacteria resting cells preparation, • Culturing cells @ variable factors, • PAH quantification after 24 hours3, • Selecting significant deg. response • Response surface optimization7 • Experimental design • Modeling • Optimization • Parameters • SFO selected factors range Djekrif et al., (2006) Silver & Roberto, (2001)

  13. OUTCOME 2: Single factor optimization – C. sakazakii MM45 (n = 3) • SFO selected range • RPM: 140 – 180 • Temp.: 30 - 40 • pH: 6 – 7 • IV: 4 – 7%, v/v • NaCl: 0 – 4 g/L

  14. OUTCOME 2: SFO – Enterobacter sp. MM087 (n = 3) • SFO selected range • RPM: 140 – 180 • Temp.: 30 - 40 • pH: 6 – 7 • IV: 4 – 7%, v/v • NaCl: 0 – 5 g/L

  15. OUTCOME 2: Response Surface Optimization

  16. OUTCOME 2: Statistical summary

  17. OUTCOME 2: Surface Interactions among variables on C. sakazakii MM045 response to PHN

  18. OUTCOME 2: C. sakazakii MM045 response on PYR

  19. OUTCOME 2: C. sakazakii MM045 degrad. response • Optimum PAHs degradation is being generated by the following quadratic equations Y1 = + 99.55 - 3.13X1 + 4.12X2 - 0.85X3 + 0.66X4 - 1.56X5 - 13.20X12 - 16.19X22 - 15.48X32 - 15.46X42 - 8.32X52 - 1.42X1X2 - 0.37X1X3 - 1.89X1X4 - 3.17.44X1X5 - 2.06X2X3 - 1.86X2X4 + 1.84X2X5 + 2.17X3X4 + 4.47X3X5 - 2.63X4X5 Y2 = + 99.26 - 2.21X1 - 6.75X2 - 3.49X3 - 2.95X4 - 5.41X5 - 13.72X12 - 14.49X22 - 12.37X32 - 14.44X42 - 10.96X52 + 8.26X1X2 + 2.48X1X3 - 2.20X1X4 - 2.35X1X5 - 1.98X2X3 - 3.41X2X4 + 1.91X2X5 + 2.45X3X4 + 0.12X3X5 + 3.61X4X5 • Optimum variables & C. sakazakii MM045 maximum degradation response (Y1 = PHN deg.; Y2 = PYR deg.; X1 = RPM; X2 = Temp.; X3 = pH; X4 = IV; X5 = NaCl)

  20. OUTCOME 2: Interactions among variables for the PHN degradation by Enterobacter sp. MM87

  21. OUTCOME 2: Enterobacter sp. MM87 response on PYR

  22. Quadratic Equations Y1 = + 98.46 + 0.13X1 - 0.76X2 - 7.67X3 - 0.82X4 + 0.79X5 - 9.37X12 - 16.44X22 - 8.54X32 - 11.73X42 - 10.55X52 - 1.11 X1X2 + 7.83X1X3 + 2.64X1X4 + 8.44X1X5 + 2.41X2X3 + 2.67X2X4 - 4.98X2X5 + 0.01X3X4 + 0.16X3X5 - 4.41X4X5 Y2 = + 98.69 + 0.35X1 + 0.95X2 - 14.20X3 + 0.42X4 - 3.36X5 - 12.93 X12 - 15.90X22 - 8.94X32 - 7.42X42 - 3.44X52 + 4.93X1X2 + 1.66X1X3 + 0.76X1X4 - 1.02X1X5 + 8.74X2X3 + 5.67X2X4 - 2.03X2X5 + 2.47X3X4 - 0.21X3X5 + 4.67X4X5 • Optimum degradation response

  23. How do we confirm such effective degradation responses? Objective 2: Conclusion • RSM optimization significantly enhanced PAHs biodegradation responses of C. sakazakiiMM045 and Enterobacter sp. MM087 from < 70% degradations to 100% within 24 hours

  24. QUESTION ? • What strategy can be applied to ascertain the effective PAHs degradation by MM045 & MM087 ? • OBJECTIVE 3 ! • Identification of phenanthrene and pyrene biodegradation metabolites OBJECTIVE 3: Identification of PHN & PYR degrading intermediates • HOW TO ACHIEVE IT? • Gas chromatography-mass spectrophotometer (GC-MS) analyses • SIGNIFICANT • To establish PAHs degradation pathway followed by C. sakazakii MM045 & Enterobacter sp. MM087

  25. EXPERIMENT 3: Identific. of PHN & PYR metabolites • Metabolites Extraction8 • Deg. culture incubated for 12h • PAHs metabolites extracted using solvent separation (Liquid-Liquid extraction tech.) • Column Chromatography • Metabolites purification9 • Extracts passed through silica gel & hexane • Dried & re-dissolved in methanol • GC-MS • Metabolites identification10 • MS spectra were identified from the NIST database Yuan et al., (2001) Okuda et al., (2006) Pinyakonget al., (2003)

  26. OUTCOME 3: PAHs metabolites from C. sakazakii MS Identified metabolites GC Chromatograms • M1 • M7 • M8 • M5 • M4 • M2 • M3 • M2 • M7 • M3 • M4 • M6

  27. OUTCOME 3: PAHs metabolites from Enterobacter sp. GC Chromatograms MS Identified metabolites M1 M2 M5 M6 M3 M4 M2 M4 M5 M3

  28. OUTCOME 3: PAHs degradation pathways • Conclusion • The established degradation pathways validates the efficiency of C. sakazakii & Enterobacter sp. • Many identified metabolites were non-hazardous with commercially important applications Are these degradations affected by heavy metals co-contaminants?

  29. QUESTION ? • What impact does heavy metals co-contaminants have on the degradation efficiency of MM045 & MM087? • OBJECTIVE 4 ! • To determine the effect of heavy metals co-contaminants on PHN & PYR biodegrading consortium. OBJECTIVE 4: Effects of heavy metals on PAHs degrading consortium • HOW DO WE ACHIEVE THIS? • Formulating bacteria consortium using MM045 & MM087 • PAHs deg. effects of heavy metals on bacteria consortium • SIGNIFICANT • To confirm heavy metals resistance during PAHs degradation by C. sakazakii MM45 & Enterobacter sp. MM87

  30. EXPERIMENT 4: Metals effects on consortium • Consortium formulation • Compatibility testing11 • Bacteria grown on MSM-PAHs for 24h3 • As, Co, Cr, Cu, Zn, Mn, Cd, Ni, Pb, V, • Heavy metals selection • Sample digestion12 • ICP-OES analyses • Parameter • % PAHs deg. • Effects of selected metals13 • Consortium cultured at different CRMs conc. • 350C, 160 rpm, 24hrs Raja et al., (2006) Zulkifliet al., (2010) Varjani & Upasani, (2013)

  31. OUTCOME 4: Consortium selection • C. sakazakii MM045 (2%, v/v) + Enterobacter sp. MM087 (2%, v/v)

  32. Cont. • Heavy metals analyses from soil samples where isolates MM045 & MM087 were obtained Toxic Metals selection Pb> Cr > Ni > V > As > Cd> Zn > Mn > Cu > Co

  33. OUTCOME 4: Metals effect on PAHs biodegradation • The selected consortium was effective enough to have performed PAHs biodegradation even in the presence of high concentrations of toxic metals

  34. Conclusion • Effective PAHs degrading bacteria were identified as C. sakazakiiMM045 and Enterobacter sp. MM087 • The degradation effectiveness of both bacteria was significantly enhanced by SFO and RSM • Both bacteria degraded 100% of 500 mg/L PHN & 250 mg/L PYR in just 24h • Such degradation response was successfully validated by establishing the bacteria degradation pathway. • The bacteria responses were further complimented with heavy metals resistance during PAHs biodegradation C. Sakazakii MM045 and Enterobacter sp. MM087 could be used for effective PAHs bioremoval

  35. PUBLICATIONS Umar, Z.D., Aziz, N.A.A., Zulkifli, S.Z., & Muskhazli, M. (2016): Identification of phenanthrene and pyrene degrading bacteria from used engine oil contaminated soil. International Journal of Scientific & Engineering Research, 7, 680-686. Umar, Z.D., Aziz, N.A.A., Zulkifli, S.Z., & Muskhazli, M. (2017): Rapid biodegradation of polycyclic aromatic hydrocarbons using effective Cronobactersakazakii MM045 (KT933253). MethodsX, 4, 104-117. Umar, Z.D., Aziz, N.A.A., Zulkifli, S.Z., & Muskhazli, M. (in press): Effective phenanthrene and pyrene biodegradation using Enterobacter sp. MM087 (KT933254) isolated from used engine oil contaminated soil. Egyptian Journal of Petroleum, vol. 27. doi: 10.1016/j.ejpe.2017.06.001 Umar, Z.D., Aziz, N.A.A., Zulkifli, S.Z., & Muskhazli, M. (submitted): Efficiency of effective polycyclic aromatic hydrocarbon (PAHs) degrading consortium in resisting toxic heavy metals during PAHs biodegradation (submitted)

  36. The Research was conducted at the: • Universiti Putra Malaysia (2014 to 2017) • Under the Supervisions of • Associate Prof. Muskhazli Mustafa (Main Super.) • Associate Prof. NurAzwadyAbd. Aziz (Co Super.) • Dr SyaizwanZahmirZulkifli (Co Super.)

  37. Acknowledgements • The Management of UMYUK for the PhD Sponsorship under the Staff Development Program • The Putra Grant (GP-IPS/2016/9489800) for Funding the research

  38. References • Tao, X.Q., Lu, G.N., Dang, Z., Yang, C., & Yi, X.Y. (2007): A phenanthrene-degrading strain Sphingomonas sp. GY2B isolated from contaminated soils. Process Biochemistry, 42, 401-408. • Kiyohara, H., Nagao, K., & Yana, K. (1982): Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on agar plates. Applied & Environmental Microbiology, 43, 454-457. • Hanson, K.G., Desai, J.D., & Desai, A.J. (1993): A rapid and simple screening technique for potential crude oil degrading microorganisms. Biotechnology Techniques, 7, 745-748 • Sogani, M., Mathur, N., Sharma, P., & Bhatnagar, P. (2012): Comparison of immobilized whole resting cells in different matrices vis-a-vis free cells of Bacillus megaterium for acyltransferase activity. Journal of Environmental Research and Development, 6, 695-701. • Kumar, S., Stecher, G., & Tamura, K. (2016): MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology & Evolution, 33, 1870-1874. • Djekrif-Dakhmouche, S., Gheribi-Aoulmi, Z., Meraihi, Z., & Bennamoun, L. (2006): Application of a statistical design to the optimization of culture medium for α-amylase production by Aspergillusniger ATCC 16404 grown on orange waste powder. Journal of Food Engineering, 73, 190–197. • Silva, C.J.S.M., & Roberto, I.C., (2001): Optimization of xylitol production by Candida guilliermondii FTI 20037 using response surface methodology. Process Biochemistry, 361, 119-124. • Yuan, S.Y., Chang, J.S., Yen, J.H., & Chang, B.V. (2001): Biodegradation of phenanthrene in river sediment. Chemosphere, 43, 273-278. • Okuda, T., Naoi, D., Tenmoku, M., Tanaka, S., He, K., Ma, Y. & Zhang, D. (2006): Polycyclic aromatic hydrcarbns in the aerosol in Beijing, China, measured by aminopropylsilane chemically-bonded stationary-phase column chromatography and HPLC/fluorescence detection. Chemosphere, 65, 427-435. • Pinyakong, O., Habe, H., Yoshida, T., Nojiri, H., & Omori, T. (2003): Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2. Biochemical and Biophysical Research Communications, 301, 350-357. • Raja, P., Uma, S., Gopal, H., & Govindarajan, K. (2006): Impact of bio inoculants consortium on rice root exudates, biological nitrogen fixation and plant growth. Journal Biological Science, 6, 815-823. • Zulkifli, S.Z., Ismail, A., Mohamat-Yusuff, F., Arai, T., & Miyazaki, N. (2010): Johor Strait as a hotspot for trace elements contamination in Peninsular Malaysia. Bulletin of Environmental Contamination and Toxicology, 84, 568-573. • Varjani, S.J., & Upasani, V.N. (2013): Comparative studies on bacterial consortia for hydrocarbon degradation. International Journal of Innovative Research in Science, Engineering and Technology, 2, 10. • Vela, N., Martínez-Menchón, M., Navarro, G., Pérez-Lucas, G., & Navarro, S. (2012): Removal of polycyclic aromatic hydrocarbons from groundwater by heterogeneous photocatalysis under natural sunlight. Journal of Photochemistry and Photobiology A: Chemistry, 232, 32-40.

  39. Vidali, M. (2001): Bioremediation; an overview. Pure & Applied Chemistry,73, 1163-1172. • Shih, K.L., & Lederberg, J. (1976): Chloramine mutagenesis in Bacillus subtilis. Science, 192, 1141-1143. • Brinda, L.M., Muthukumar, K., & Velan, M. (2013): Optimization of minimal salt medium for efficient phenanthrene biodegradation by Mycoplana sp. MVMB2 isolated from petroleum contaminated soil using factorial design experiments. CLEAN–Soil, Air, Water, 41, 51-59. • Cycoń, M., Żmijowska, A., Wójcik, M., & Piotrowska-Seget, Z. (2013): Biodegradation and bioremediation potential of diazinon-degrading Serratiamarcescens to remove other organophosphorus pesticides from soils. Journal of Environmental Management, 117, 7-16. • Gillespie, S.H., & Hawkey, P.M. (2006): Principles and Practice of Clinical Bacteriology, second edition, John Wiley Publisher, UK. • Kandhai, M.C., Reij, M.W., Gorris, L.G., Guillaume-Gentil, O., & Schothorst, M. (2004): Occurrence of Enterobactersakazakii in food production environments and households. Lancet,363, 39-40.

More Related