Production and characterization of colored metabolites and pigments of microbial isolates By SALMA GUL SHAH Department of Microbiology Faculty of Biological Sciences Quaid-i-Azam University, Islamabad 2015 Production and characterization of colored metabolites and pigments of microbial isolates A thesis submitted in the partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN MICROBIOLOGY By SALMA GUL SHAH Supervised by DR. NAEEM ALI Department of Microbiology Faculty of Biological Sciences Quaid-i-Azam University, Islamabad 2015 IN THE NAME OF ALLAH THE MOST MERCIFUL AND MIGHTY O my Lord! Open for me my chest (Grant me Self-confidence, Contentment and boldness) And ease my task for me, And loose the knot from my tongue that they understand my speech (words) Surah 20, Ta Ha (Al-Quran) DECLARATION The material present in this thesis is my original work and I have not presented any part of this thesis/work elsewhere for any other degree. SALMA GUL SHAH DEDICATED TO HAZRAT MUHAMMAD (P.B.U.H) MY LOVING PARENTS HUSBAND MUDASSAR BROTHER HARIS SISTERS BUSHRA AND HUMA CERTIFICATE This thesis by Salma Gul Shah is accepted in its present form by the Department of Microbiology, Quaid-i-Azam University, Islamabad, as satisfying the thesis requirement for the degree of Doctor of Philosophy in Microbiology. Internal examiner ___________________ (Ass. Professor. Dr. Naeem Ali) External examiner ___________________ (------------------) External examiner ___________________ (------------------) Chairman ___________________ (Prof. Dr. Fariha Hasan) Dated: ______________ CONTENTS Sr. # Title Page # 1. List of Abbreviations i 2. List of Figures iii 3. List of Tables xiii 4. List of Appendix xv 5. Acknowledgements xix 6. Summary 1 7. Aims and Objectives 5 8. Review of Literature 7 9. Material and Methods 46 10. Results 65 11. Chapter 4 65 12. Chapter 5 103 13. Discussion 176 14. Conclusions and future prospects 200 15. References 203 16. Appendix 263 List of Abbreviations g Gram µ Micro ml Milli liter L Liter mg Milligram cfu Colony forming units °C Degree centigrade ITS Internal transcribed spacer region PCR Polymerase chain reaction BLAST Basic Local Alignment Search Tool NH4NO3 Ammonium nitrate MgSO4.7H2O Magnesium sulphate heptahydrate KH2P04 Pottassium dihydrogen phosphate NaCl Sodium chloride YMPGB yeast extract malt extract broth PDB Potato dextrose broth SDB Sabouraud dextrose broth MSM Minimal salt media 3(x) 3 times λmax Lambda maximum nm Nano meter ATCC American type culture collection SDA Sabouraud dextrose agar MEA Malt extract agar hrs Hours DMSO Dimethyl sulphoxide n Number ppm Parts per million DPPH 2, 2-diphenyl-1-picrylhydrazyl. mM Milli molar FeCl3 Ferric chloride H2SO4 H2SO4 Conc Concentration HCl Hydrochloric acid NaOH Sodium hydroxide UV Ultra violet FTIR Fourrier transform infra red spectrophotometer cm Centimeter OD Optical density SS Solvent system SP Stationary phase SST Solvent system for TLC TLC Thin layer chromatography LCMS Liquid chromatography mass spectrometry LCMS/MS Liquid chromatography mass spectrometry/ mass spectrometry CHCl3 Chloroform Results 3D XRD 3 Dimensional X ray crystallography ESI Electro spray ionization m/z Mass to charge ratio KA31T Virally transformed cancerous cell line NIH3T3 cells Mouse embryonic fibroblasts HSCT6 cells Rat hepatic stellate cell line HEK293 Cell line derived from human embryonic kidney cells grown in tissue culture MDCK Line Madin-Darby Canine Kidney Epithelial Cells NMR Nuclear magnetic resonance 1D 1 Dimensional r RNA Ribosomal Ribonucleic acid sp Species C: N Carbon nitrogen ratio AU Absorbance unit EC Effective concentration IC Inhibitory concentration M+ Molecular ion peak LD Lethal dose DAD Diode array detector Results List of Figures Sr.No Title Sr.No Fig 2.1 Structures of Bacterial pigments/ colored metabolites 15 Fig 2.2 Structures of some fungal pigments 23 Fig 2.3 Biosynthesis of aromatic polyketides 44 Fig 2.4 Dieckmann condensation 45 Fig 3.1 Schematic representation of the pigment extraction from fungi 50 Fig 3.2 Schematic flow sheet representing Column chromatography of 59 Penicillium verruculosum and ultimate fate of the different selected fractions Fig 3.3 Schematic diagram representing Column chromatography of 60 Chaetmium strumarium and ultimate fate of the different selected fractions Fig 3.4 Schematic diagram representing Column chromatography of 61 Aspergillus fumigatus and ultimate fate of the different selected fractions Fig 4.1 Extracellular pigment production in five different media by 66 Penicillium sp SG. Fig 4.2 Extracellular pigment production in five different media by 67 Aspergillus sp SG4. Fig 4.3 Extracellular pigment production in five different media by 68 Chaetomium sp SG1. Fig 4.4 Extracellular pigment production in five different media by 69 Penicillium sp SG2 Fig 4.5 Extracellular pigment production in five different media by 70 Epicoccum sp SG3. Fig 4.6 Phylogenetic tree of Penicillium verruculosum (18srRNA) 72 Fig 4.7 Phylogenetic tree of Penicillium verruculosum (28srRNA) 72 Fig 4.8 Phylogenetic tree of Penicillium verruculosum (28srRNA, D2 73 region) Fig 4.9 Phylogenetic tree of Aspergillus fumigatus SG4 (JX863917) 74 Fig 4.10 Phylogenetic tree of Chaetomium strumarium SG1 (JX863914) 75 Fig UV/Vis spectroscopy of methanolic extract of pigments (colored 76 Results 4.11(a) metabolites) by Penicillium verruculosum Fig UV/Vis spectroscopy of methanolic extract of pigments (colored 76 4.11(b) metabolites) by Chaetomium strumarium Fig 4.11(c) UV/Vis spectroscopy of methanolic extract of pigments (colored 77 metabolites) by Aspergillus fumigatus Fig 4.12 Pigments concentration (AU) at varying pH (3-9) by three 78 pigment producing fungi Fig 4.13 Pigments concentration at varying temperatures (15-37) by three 78 pigment producing fungi Fig 4.14 Pigments concentration at varying carbon sources by three 79 pigment producing fungi Fig 4.15 Pigments concentration at varying glucose concentration by three 80 pigment producing fungi Fig 4.16 Pigments concentration at varying nitrogen sources by three 81 pigment producing fungi Fig 4.17 Pigments concentration at varying C: N ratio by three pigment 82 producing fungi Fig 4.18 Extracellular pigment production by three fungi in liquid state at 82 static and optimizied conditions Fig 4.19 FTIR pattern of Penicillium verruculosum (a), Chaetomium 84 strumarium (b) and Aspergillus fumigatus (c) showing presence of different functional groups in PDA media. Fig 4.20 UV/vis absorbance of the Penicillium verruculosum (a), 86 Chaetomium strumarium (b) and Aspergillus fumigatus (c) after treatment at different pH for 6 hrs. Fig 4.21 FTIR pattern of Penicillium verruculosum (a), Chaetomium 87 strumarium (b) and Aspergillus fumigatus (c) showing presence of different functional groups in control in comparison to pH treated PDA media after 6 hrs. Fig 4.22 UV/Vis spectroscopy of the pigmented filtrates of Penicillium 88 verruculosum (a), Chaetomium strumarium (b) and Aspergillus fumigatus (c), after treatment at different temperatures for 6 hrs Fig 4.23 FTIR pattern of Penicillium verruculosum (a), Chaetomium 89 Results strumarium (b) and Aspergillus fumigatus (c) showing presence of different functional groups in control in comparison to temperatures treated PDA media after 6 hrs. Fig 4.24 Total flavonoid content (mg RTE/g extracts) of Penicillium 91 verruculosum (a), Chaetomium strumarium (b) and Aspergillus fumigatus (c) Fig 4.25 Total Phenolic content (GAE mg/g) of Penicillium verruculosum 92 (a), Chaetomium strumarium (b) and Aspergillus fumigatus (c) Fig 4.26 Total antioxidant (a) and DPPH free radical scavenging (b) 98 activity (%) of the three fungi Fig 4.27 Total ABTS scavenging activity (a) and reducing power (%) (b) 99 of the three pigment producing fungi Fig 5.1 Preparatory TLC plate showing colored metabolites in 10 days 106 old culture filtrate of P. verruculosum SG (Fraction Be.7-Be.12) Fig 5.2 Fragmentation pattern of Monascin (c) showing presence of 107 molecular ion peak 359 m/z and a major fragment at 196 m/z predicting its chemical structure following LCMS (a) and LCMS/MS (b) Fig 5.3 Fragmentation pattern of Monascorubrine (b) [(showing the 108 presence of molecular ion peaks at 382 m/z and fragments at 338 and 256 m/z) (a)] predicting its chemical structure and absorbance pattern following LCMS (a). Fig 5.4 Fragmentation pattern of Glutamyl Monascorubrine [(showing 109 the presence of molecular ion peak483 and fragments 442 and 425 m/z) (Fig. 5.4b)] predicting its chemical structure following LCDADMS (Fig. 5.4a). Fig 5.5 Analogue of Monascorubrine having the same molecular ion peak 110 m/z 383 and the fragments at 338 and 256 as monascorubrin but different wavelength pattern. Fig 5.6 Fragmentation pattern (b) of Pyripyropene (Fraction Fc.9-Fc.12) 111 (showing the presence of molecular ion peak 564 m/z and a major fragment at 462 and 326 m/z) predicting its chemical structure following LCMSMS (a). Results Fig 5.7 Fragmentation pattern of Orevactaene (b) (Fraction Fc.4-Fc.8) 112 [showing the presence of molecular ion peak 613 m/z and loss of water by presence of m/z 595 in LCDAD/MS (a)] predicting its chemical structure Fig 5.8 Fragmentation pattern of Citrinadin (a) (Fraction E.c) [showing 113 the presence of molecular ion peak 625 m/z and fragments of 526, 481 m/z in LCDADMS (b)] predicting its chemical structure. Fig 5.9 Fragmentation pattern of Calcimycin (c) (Fraction B.d)[ showing 115 the presence of molecular ion peak 283 m/z in LCMS (a) and fragments of 247, 184 m/z in LCMSMS (b)] predicting its chemical structure. Fig 5.10 Fragmentation pattern (b) of verrucine A (Fraction B.d) [showing 116 the presence of molecular ion peak 377 m/z and fragments of 360, 331 m/z in LCDADMS (a)] predicting its chemical structure. Fig 5.11 Fragmentation pattern of Scirpentriol (b) (Fraction B.d) 117 [(showing the presence of molecular ion peak 283 m/z and fragments of 247, 184 m/z in LCMSMS) (a)] predicting its chemical structure.