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BY KARTHIKEYAN.B.S BI0807 UNDER THE GUIDANCE OF DR.CH. SASIKALA,

IN SILICO COMPARISON OF XENOBIOTIC DEGRADATION PATHWAYS AMONG THREE STRAINS OF PURPLE NON SULFUR BACTERIA AND CONFIRMATION OF ANILINE DEGRADATION BY RHODOBACTER SPHAEROIDES DSM 158. BY KARTHIKEYAN.B.S BI0807 UNDER THE GUIDANCE OF DR.CH. SASIKALA,

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BY KARTHIKEYAN.B.S BI0807 UNDER THE GUIDANCE OF DR.CH. SASIKALA,

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  1. IN SILICO COMPARISON OF XENOBIOTIC DEGRADATION PATHWAYS AMONG THREE STRAINS OF PURPLE NON SULFUR BACTERIA AND CONFIRMATION OF ANILINE DEGRADATION BY RHODOBACTER SPHAEROIDES DSM 158 BY KARTHIKEYAN.B.S BI0807 UNDER THE GUIDANCE OF DR.CH. SASIKALA, Associate Professor, Bacterial Discovery Laboratory, Centre For Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad

  2. PROJECT SUPERVISORS Dr. Ch .Sasikala M.Sc.,Ph.D Associate Professor, Bacterial Discovery Lab, Center for Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad. Dr.Ch.Venkata Ramana, M.Sc.,Ph.D Professor, Anoxygenic Phototrophic Bacterial Lab, Department of Plant science, School of Life Science, University of Hyderabad, Hyderabad.

  3. BIOINFORMATICS COMPARATIVE GENOMICS METABOLIC DATABASES BACTERIAL DISCOVERY LABORATORY MICROBIOLOGY METABOLOMICS XENOBIOTIC DEGRADATION

  4. OBJECTIVES OF THE STUDY • Rhodopseudomans palustris CGA 009 • Rhodobacter sphaeroides 2.4.1 • Rhodospirillum rubrum ATCC 17001 In Silico: 1. To compare ability of three strains to degrade xenobiotic compounds 2. To select the efficient strain out of three strains for efficient Xenobiotic degradation 3. To select the substrate or xenobiotic compound for a single selected strain to carry out in vivo experiments In Vivo: 4. To study the effect of the selected substrate on the growth of the selected strain 5. To determine the capability of the selected strain to degrade the selected substrate 6. To determine the capability of the selected strain to transform the selected substrate into different metabolites

  5. WHOLE GENOMES OF APB (Rhodopseudonomanas palustris CGA009, Rhodobacter sphaeroides DSM 158, Rhodospirillum rubrum ATCC 11170) GENOME WIDE COMPARISONS (IMG JGI) SEQUENCE BASED COMPARISONS (KEGG, METACYC) COMPARISON BETWEEN SPECIES % OF XENOBIOTIC DEGRADATION CODING GENES IN ALL THE SELECTED STRAINS % OF ANNOTATED GENES CODING FOR XENOBIOTIC DEGRADATION RESULT IN SILICO HIERARCHY OF WORK CARRIED OUT

  6. IN SILICO RESULT SELECTION OF STRAIN AND SUBSTRATE ACCORDING TO AVAILABILITY OF STRAIN AND CHEMICAL REVIVING OF THE CULTURE & CHECKING THE PURITY GROWTH WITH CHEMICAL AS C OR N SOURCE (OD 660) GROWTH YIELD (OD 660) DISAPPEARANCE OF THE CHEMICAL (UV SPECTROPHOTOMETER) ANALYSIS OF THE RESULTS DEGRADATION OF CHEMICAL AT OPTIMUM CONCENTRATION USING HPLC OPTIMUM CONCENTRATION SELECTED IN VIVO QUALITATIVE ANALYSIS OF PRODUCTS OF DEGRADATION THROUGH HPLC ESTIMATION OF INDOLE Cont..

  7. ANOXYGENIC PHOTOTROPHIC BACTERIA • XENOBIOTIC COMPOUNDS & ITS PROPERTY - man made chemicals,, hazardous to living beings. • BIODEGRADATION & ITS ROLE - effective, minimally hazardous, and economical. • MICROBES AND ITS EFFICIENCY - microorganisms exist billions of years - survived with variety of organic compounds for energy etc., • BIOINFORMATICS FOR BIOREMEDIATION - microbial degradation pathways is so incomplete - explore new catabolic pathways • GENOMICS FOR BIOREMEDIATION - microbial genes to evolve mechanisms to degrade synthetic organic structures

  8. Cont.. - potential metabolic activity of the microbial community - isolated organisms were important in bioremediation or not ? • NEED FOR SYSTEMS APPROACH - complex interactions between cellular reactions from a genomic and proteomic level - system biology approach is necessary to predict the functioning of an organism in a complex environment and to describe the outcome of the thousands of individual reactions that are simultaneously taking place in a microbial cell. • ADVANTAGES OF USING BACTERIA - many completed whole genomes are available - most numerous and obvious microbial components of the earth - easy to culture - ease to integrate in silico and in vivo approaches

  9. Cont.. • ANOXYGENIC PHOTOTROPHIC BACTERIA (APB) - photosynthetic prokaryotes -anaerobic conditions by photosynthesis with out oxygen liberation - lack photo system-II and carryout anoxygenic photosynthesis • Purple Non-Sulfur Bacteria - metabolize wide range of aliphatic organic compounds - used in sewage treatment • Rhodopseudomonas palustris - extraordinary metabolic versatile and successful metabolic opportunist - photoautotrophic, photo heterotrophic, chemo heterotrophic , chemoautotrophic metabolism - encodes four distinct oxygenase-dependent ring cleavage pathways - well studied for aromatic compound degradation. • Rhodobacter sphaeroides -metal reduction, nitrogen fixation, hydrogen production - microaerophilic conditions, chemotropic and phototrophic growth • Rhodospirillum rubrum - production of biological plastic (PHB poly-hydroxy-butric-acid) , nitrogen fixation, biofuel production.

  10. MATERIALS AND METHODS IN SILICO • GENOME INFORMATION - Integrated Microbial Genomes (IMG) system and KEGG database • WHOLE GENOME SEQUENCES • WHOLE GENOME COMPARISONS - xenobiotic degrading genes present in the genome - comparison between the other strains • GLOBAL MAP OF ENTIRE METABOLISM - KEGG pathway database • XENOBIOTIC PATHWAYS - KEEG - METACYC • SEQUENCE COMPARISON OF XENOBIOTIC DEGRADING GENES • SELECTION OF THE STRAIN FOR DEEPER ANALYSIS • PUTATIVE ENZYMES OF RHODOBACTER SPHAEROIDES DSM 158 • SELECTION OF SUBSTRATE AND PATHWAY FOR DEGRADATION • MINING OF NAPHTHALENE AND ANTHRACENE DEGRADATION

  11. MATERIALS AND METHODS IN VIVO • PURIFICATION • PREPARATION OF MEDIA

  12. Cont.. • DETERMINATION OF GROWTH AND WHOLE CELL ABSORPTION SPECTRUM • EFFECT OF ANILINE ON NORMAL GROWTH • GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF ANILINE AS CARBON AND NITROGEN SOURCE (ANAEROBIC) • GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF ANILINE AS NITROGEN SOURCE (AEROBIC) • STUDIES ON THEPHOTODEGRADATION OF ANILINE • STUDIES ON THE PHOTOBIODEGRADATION OF ANILINE AS THE SOLE CARBON AND NITROGEN SOURCE (ANAEROBIC) • STUDIES ON THE PHOTOBIODEGRADATION OF ANILINE AS THE SOLE NITROGEN SOURCE (AEROBIC) • PERCENTAGE OF ANILINE DEGRADATION • QUANTIFICATION OF INDOLE • HPLC ANALYSIS

  13. RESULTS AND DISCUSSION • IN SILICO • PERCENTAGE OF XENOBIOTIC DEGRADING GENES In Rhodopseudomonas palustris CGA009 = 15.92% In Rhodobacter sphaeroides DSM 158 = 9.37% In Rhodospirillum rubrum ATCC 11170 = 9.89%

  14. CONT.. • GLOBAL MAP OF ENTIRE METABOLISM FOR Rhodopseudomonas palustris CGA 009

  15. CONT.. • FOR Rhodobacter sphaeroides DSM 158

  16. CONT.. • FOR Rhodospirillum rubrum ATCC 11170

  17. CONT.. • XENOBIOTIC DEGRADATION PATHWAYS KEGG - Total number of available annotated pathways for, Rhodopseudomonas palustris CGA009 = 19 Rhodobacter sphaeroides DSM 158 = 20 Rhodospirillum rubrum ATCC 11170 = 12 METACYC - Total number of available annotated pathways for, Rhodopseudomonas palustris CGA009 = 32 Rhodobacter sphaeroides DSM 158 = 19 Rhodospirillum rubrum ATCC 11170 = 13

  18. CONT.. • SEQUENCE COMPARISON OF XENOBIOTIC DEGRADING GENES

  19. CONT.. • PUTATIVE ENZYMES OF RHODOBACTER SPHAEROIDES DSM 158 PATHWAYS- 8 SUBSTRATE- 44 PRODUCT- 44 PUTATIVE ENZYMES- 44

  20. CONT.. • SELECTION OF SUBSTRATE AND PATHWAY FOR DEGRADATION • MINING OF NAPHTHALENE AND ANTHRACENE DEGRADATION

  21. CONT.. • INFORMATION ABOUT PUTATIVE FATTY ACID BETA HYDROXYLASE (CYTOCHROME P450) (EC: 1.14.-.-)

  22. CONT.. • SEQUENCE SIMILARITY OF PUTATIVE FATTY ACID BETA HYDROXYLASE (CYTOCHROME P450) (EC: 1.14.-.-) RSP_2378 WITH OTHER TWO STRAINS - NO SIMILARITY - BUT SHOWS SIMILARITY OF ~ 85 TO 98% WITH OTHER STRAINS OF RHODOBACTER SPHAEROIDES.

  23. IN VIVO • GROWTH OF RHODOBACTER SPHAEORIDES DSM 158 Table : Growth of Rhodobacter sphaeroides DSM 158 with all supplements

  24. CONT.. • EFFECT OF ANILINE ON GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 Fig : Growth of Rhodobacter sphaeroides DSM 158 in the presence of Aniline as an additional supplement at 0.5 and 1.0mM concentration.

  25. CONT.. • GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF ANILINE AS SOLE CARBON SOURCE (ANAEROBIC) Fig : Growth of Rhodobacter sphaeroides DSM 158 in presence of Aniline as sole carbon source at 0.5mM and 1.0mM concentration.

  26. CONT.. • GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF ANILINE AS SOLE NITROGEN SOURCE (ANAEROBIC) Fig : Growth of Rhodobacter sphaeroides DSM 158 in presence of Aniline as sole nitrogen source at 0.5mM and 1.0mM concentration.

  27. CONT.. • GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF ANILINE AS NITROGEN SOURCE (AEROBIC)

  28. CONT.. WHOLE CELL ABSOPRTION SPECTRUM • Fig : The whole cell absorption spectrum of Rhodobactersphaeroides DSM 158 which is grown with Pyruvate as carbon source and NH4Cl as nitrogen source.

  29. CONT.. Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is grown with Aniline as carbon source and NH4Cl as nitrogen source at 0.5mM concentration Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is grown with Aniline as carbon source and NH4Cl as nitrogen source at 1.0mM concentration

  30. CONT.. Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is grown with pyruvate as carbon source and Aniline as nitrogen source at 0.5mM concentration Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is grown with pyruvate as carbon source and Aniline as nitrogen source at 1.0mM concentration

  31. SPECTROSCOPIC ANALYSIS OF ANILINE DEGRADATION

  32. CONT..

  33. Absorption spectrum of culture supernatants • Initial absorbance of Aniline (Carbon source) at 0.5mM concentration • Overlay of 24, 48, 72 hours absorbance of Aniline (Carbon source) at 0.5mM concentration • Final (96 hours) absorbance of Aniline (Carbon source) at 0.5mM concentration

  34. Absorption spectrum of culture supernatants.. CONT.. • Initial absorbance of Aniline (Carbon source) at 1.0mM concentration • Overlay of 24, 48, 72 hours absorbance of Aniline (Carbon source) at 1.0mM concentration • Final (96 hours) absorbance of Aniline (Carbon source) at 1.0mM concentration

  35. Absorption spectrum of culture supernatants.. CONT.. • Initial absorbance of Aniline (nitrogen source) at 0.5mM concentration • Overlay of 24, 48, 72 hours absorbance of Aniline (nitrogen source) at 0.5mM concentration • Final (96 hours) absorbance of Aniline (nitrogen source) at 0.5mM concentration

  36. Absorption spectrum of culture supernatants.. CONT.. • Initial absorbance of Aniline (nitrogen source) at 1.0mM concentration. • Overlay of 24, 48, 72 hours absorbance of Aniline (nitrogen source) at 1.0mM concentration. • Final (96 hours) absorbance of Aniline (nitrogen source) at 1.0mM concentration.

  37. PHOTO BIODEGRADATION OF ANILINE BY RHODOBACTER SPHAEROIDES DSM 158 UNDER AEROBIC DARK CONDITIONS • QUANTIFICATION OF INDOLE • Culture supernatants were collected and added with double the amount of freshly prepared salpers reagent. The absorbance was read at 535 nm against reagent blank. • From the standard graph, 0.1 OD of Absorbance = 13µg/ml of total indole. • Amount of total indole produced at 0.5mM concentration of Aniline = 2.6µg/ml • Amount of total indole produced at 1.0mM concentration of Aniline = 5.72µg/ml

  38. PERCENTAGE OF ANILINE DEGRADATION • Degradation of Aniline (as carbon source) at 0.5mM concentration = 29.06 % • Degradation of Aniline (as carbon source) at 1.0mM concentration = 19.38% • Degradation of Aniline (as nitrogen source) at 0.5mM concentration = 21.88% • Degradation of Aniline (as nitrogen source) at 1.0mM concentration = 6.08%

  39. HPLC ANALYSIS FOR PHOTOBIODEGRADATION OF NITROBENZENE BY RHODOBACTER SPHAEROIDES DSM 158 Rhodobacter sphaeroides DSM 158 Centrifugation at 10,000 rpm for 15 minutes. Culture supernatant Ethyl Acetate Extraction (Thrice) Separate organic layer Condensation by Flash Rotary evaporator After Dryness Redissolved in methanol (1 ml) Filtration Injection sample 20 μL

  40. PHOTO BIODEGRADATION OF ANILINE Fig: HPLC chromatogram of culture supernatants of Rhodobacter sphaeroides DSM 158 grown under anaerobic dark condition with Aniline as nitrogen source

  41. PHOTO BIOTRANSFORMATION OF ANILINE Fig: HPLC chromatogram of culture supernatant of Rhodobacter sphaeroides DSM 158 grown under anaerobic dark condition with Aniline as nitrogen source

  42. SIMILAR PEAKS FORMED BETWEEN CONTROL AND PHOTO BIODEGRADATION FINAL

  43. DISSIMILAR PEAKS FORMED BETWEEN CONTROL AND PHOTO BIODEGRADATION FINAL

  44. COMPARISON OF SIMILAR PEAKS WITH PHOTO DEGRADATION FINAL

  45. IDENTIFIED PEAKS

  46. UNIDENTIFIED PEAKS

  47. FINAL RESULTS OF HPLC ANALYSIS Fig : HPLC chromatograms showing formation of new intermediates or compounds. The peaks represented by boxes were metabolites formed unique, obtained only with the presence of Aniline and with the presence of the strain. The peaks represented by pink color boxes are identified peaks (Table). The boxes represented by blue color are unidentified peaks.

  48. CONCLUSION • Novel study of INTEGRATING IN SILICO AND IN VIVO APPROACHES for knowing the xenobiotic degradative capability of the strains • Through in silico approaches the hierarchy of xenobiotic degradation capability of the strain was concluded as Rhodopseudomonas palustris CGA 009 Rhodobacter sphaeroides DSM 158 Rhodospirillum rubrum ATCC 11170. • Use of in silico approaches SAVED LOTS OF TIME without wasting in doing trial and error experiments • KNOWLEDGE BASED SUBSTRATE SELECTION through metabolic databases was demonstrated than blind selection of the substrate • To prove the presence or EXPRESSION OF THE GENE RSP 2378 coding for putative fatty acid beta hydroxylase for aniline degradation was initiated with microbiological and metabolomics study

  49. Cont.. • Though aniline was NOT TOXIC to growth of rhodobacter sphaeroides dsm 158, but it did not support the growth of strain. hence it cannot serve as either as CARBON SOURCE OR NITROGEN SOURCE • It was reflected in the PRODUCTION OF BACTERIOCHOLROPHYLL. it was heavily affected when aniline used as carbon or nitrogen source at 0.5mm and 1.0mm concentration. the use of lesser concentration of aniline starting from 0.1mm concentration to minimum lethal concentration for degradation study might help in knowing extensively the degradative capability of the strain • TheDISAPPEARANCE OF ANILINE measured using uv spectrophotometer RAISED CHAOS in concluding the percentage of degradation which was solved to some extent USING HPLC analysis. The COMPLETE DISAPPEARANCE OF ANILINEwas observed when used as nitrogen source at 0.5mm concentration. it was concluded that the strain utilized aniline as nitrogen source.

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