356 Kumar Naveen Krishnamurthy Department

BIODEGRADATION OF AROMATIC AMINES BY ALKALOPHILIC BACTERIA • Thesis submitted to the GOA UNIVERSITY for the degree of DOCTOR OF PHILOSOPHY in 576 MICROBIOLOGY by - 356 Kumar Naveen Krishnamurthy Department of Microbiology, Goa University, Goa ria dey,„ 2007 --3 S 6 CERTIFICATE This is to certify that Mr. Kumar Naveen Krishnamurthy has worked on the thesis entitled "BIODEGRADATION OF AROMATIC AMINES BY ALKALOPHILIC BACTERIA" under my guidance and supervision. This thesis, being submitted to the Goa University, Taleigao Plateau Goa for the award of the degree of Doctor of Philosophy in Microbiology is an original record of the work carried out by the candidate himself and has not been submitted for the award of any other degree or diploma of Ipiky other university in India or abroad. \ _si t '1 Mr. Kumar Naveen Krishnamurthy 2_0(0„,‘;_. gA°‘- Candidate 1 '9 L '*'?e4 dc:1°‘°CP ok cSr4 Dr. Saroj : C‘'k 014. Research uid&& Head, Department of Microbiology , Goa University STATEMENT I hereby state that this thesis for the Ph.D. degree on "Biodegradation of Aromatic Amines by Alkalophilic Bacteria" is my original contribution and that the thesis and any part of it have not been previously submitted for the award of any degree/diploma of any University or Institute. To the best of my knowledge, the present study is the first comprehensive work of its kind from this area. Kumar Naveen Krishnamurthy Department of Microbiology Goa University, Goa. Acknowledgement The successful completion of thoughtful search endeavour is due to combined encouragement of numerous individuals who have been constantly inspired and motivated me throughout this study. With deep gratitude, I acknowledge the great debt I owe to my Guide, Dr. (Mrs.) Saroj Bhosle, for her admirable endurance, guidance, patience and encouragement given to me during the entire period of research. Her scientific experience and vast knowledge of the subject, innovative ideas and constructive criticisms have contributed immensely to my research work. I am thankful to Dr. P.R.Pednekar (Syngenta, Goa), VC's ' nominee, for his immense help, support, suggestion and providing the industrial effluent to this work. I am also thankful to Prof. G.N.Nayak, Dean, Faculty of Life Sciences for'his critical comments. I am also grateful to the entire teaching and non-teaching staff of Department of Microbiology and Marine Biotechnology for their unconditional help and co-operation. My sincere gratitude to Prof. S. Mavinkurve, Ex. H.O.D. Microbiology, Dr Sandeep Garg and Prof. U. M. X. Sangodkar for their immense assistance, valuable criticism which helped me to gather thorough knowledge of the subject. I acknowledge the financial support provided by Dept. of Biotechnology and CSIR, New Delhi, to complete the project. A special thanks goes to Prof. Y. Souche and Dr. Upal Roy for 16s rRNA sequencing of the bacterial strains. Special thanks also goes to Dr. Balsubramaniam and Dr. (Mrs.) Vijaylakshmi, Thavasi, Muthezhilan (Annamalai University, Perangipettai), for assistance in FAME analysis using MIS system. I also thank Dr. A.S.A.R. Deshmukh and Aarif Sheik (NCL, Pune), Dr. S. Paknikar (Goa Universtiy), Dr. Solemabi, Dr N.B. Bhosle and Rajesh Parvathkar (NIO), for help me in identification of the product. I am highly obliged for the zeal, enthusiasm and encouragement provided by Dr. Chanda Parulekar, Dr. Trupti Rawte, Dr Judith Braganca, Deepa Nair, Aureen Godhino, Madhan Raghavan, Christina DeSouza and Rohan Fernandes throughout my study. I am immensely grateful to Dr. Sandeep and Meenu Garg for their unconditional help and endless support. I am also grateful to Dr. Samir, Varada, Rasika, Donna, Vidya, Saieesh, Dr. Deepa, and Dr. Mohandas for being there whenever I needed them. It is very difficult to forget the attention and care I received from them. Words seem to be inadequate to express my gratitude to certain people who have been instrumental in helping me at every stage of my research. They are Nimali, Celisa, Lakshangy, Marielou, Joel, Anju, Neelam, Brahmachari, Krishnammthy, Girija, Beena, Meenal, Vimal, Bhaskar, Sandeep, Raghu, Geeta, Ana, Swaraswati, Anant Gawde, Suneeta B., Sunita K. and many more. I dedicate this work to my parents, because what I achieved till the date is only due to their love and blessings. They are my constant inspiration. Naveen Dedicated to my' Patents. ABBREVIATIONS a alpha 1-19 Microgram Abs Absorbance Microlitre APS Ammonium per sulfate Micromolar Beta Micron b.p. Boiling point NaCI Sodium chloride BCA Bicinchoninic Acid Na' Sodium on °C Degree celcius NH4NO3 Ammonium nitrate cfu Colony forming unit NH4CI Ammonium chloride D/W Double distilled Water NA Nutrient Agar EDTA Ethylene diamine - NaOH Sodium hydroxide - tetra acetic acid nm Nanometer EPS Exopolymeric substances N MR Nuclear Magnetic Resonance FAME Fatty Acid Methyl Esters NND N,N-Dimethyl,1-Naphthylamine FP Fluorescent product O.D. Optical Density Fig. Figure ONGC Oil and natural gas commission gm Gram(s) o Ortho GC Gas chromatography P Para Glu Glucose PAGE Poly-acrylamide gel electrophoresis hr(s) H our(s) PPYG Polypeptone yeast extract glucose agar HCI Hydrochloric acid PPY Polypeptone yeast extract medium H2SO4 Sulphuric acid without glucose IR ' Infra Red Rf Resolution factor KDa • Kilo Dalton rpm Revolutions per minute Kbps Kilo base pairs RT Room temperature KNO3 Potassium nitrate Rt Retension time L Litre SOS Sodium dodecyl sulfate lbs Pounds sec. Second(s) M Molar sp. Species mg Milli gram(s) TEMED Tetra methyl ethylene diamine MSM Mineral Salt Medium TLC Thin layer chromatography min(s) Minute(s) TP2 Transformed product 2 MIS Microbial Identification System UV-Vis Ultra violet - Visible ml Milli litre V Volts m Meta v/v Volume / Volume mg Milligram w/v Weight/Volume mM Milli molar 0/0 Percentage MS Mass spectrometry Lambda Lists of figures Fig. 2.1: Ortho- and meta- cleavage of catechol Fig. 2.2: Ortho- and meta- cleavage of protocatechuate Fig. 2.3: Gentisate/homogentisate pathway Fig. 2.4: Degradation pathway of nitrobenzene Fig. 2.5: Degradation pathway of nitrophenols Fig. 2.6: Degradation pathway of nitrotoluenes Fig. 2.7: Degradation pathway of dinitrotoluenes Fig. 2.8: Degradation pathway of nitrobenzoates Fig. 2.9: Interaction of aniline in environment Fig. 2.10: Degradation pathway of aminophenols Fig. 2.11: Degradation pathway of aminobenzoates Fig. 2.12: Degradation pathway of p-chloroaniline Fig. 2.13: Degradation pathway of m-chloroaniline Fig. 2.14: Classification of various oxidizing enzymes Fig. 2.15: Action of monooxygenases on toluene and xylenes Fig. 2.16: Styrene/ethylbenzene and benzo[a]pyrene oxidation by enzyme Monooxygenases Fig. 2.17: Monooxygenation by cytochrome P450 and enzyme monooxygenase Fig. 2.18: The monoxygenase cycle by cytochrome P450 Fig. 2.19: Mechanism of the naphthalene dioxygenase enzyme Fig. 2.20: Degradation pathway of 4-hydroxybenzoate/m-hydroxyphenol Fig. 2.21: Degradation pathway of benzo[a]pyrene Fig. 2.22: Degradation pathway of phenylalanine/tyrosine Fig. 2.23: Degradation pathway of tryptophan Fig. 2.24: Degradation pathway of naphthalene Fig. 2.25: Examples of typical laccase catalysed reactions Fig. 2.26: Polymerization reactions by laccases Fig. 2.27: Exapmles of typical PPO catalysed reactions Fig. 2.28: Catalytic mechanisms of peroxidases Fig. 2.29: Substrates and products of enzyme peroxidases Fig. 2.30: Anaerobic degradation pathway of the benzene ring Fig. 2.31: Anaerobic degradation pathway of naphthalene/methyl-naphthalene Fig. 3.1 a: Sampling sites at the Bombay High region (marine ecosystem) Fig. 3. lb: Sampling sites at the mangrove ecosystem in Goa Fig. 3.2a: Percentage distribution of obligate alkalophiles (ONGC) Fig. 3.2b: Percentage distribution of obligate alkalophiles (Mangrove ecosystem) Fig. 3.5: Dendogram showing the isolates NRS-01 (nrs) &NK2 (nk2) closely related to Halomonas sp. Fig. 4.1: Protocol showing enrichment of consortium/isolates towards effluent Fig. 4.2: Plasmid isolation by alkaline lysis method Fig. 4.5: Effect of pH on the growth of isolate NRS-01 Fig. 4.6: Effect of aniline concentration on the growth of isolate NRS-01 Fig. 5.3a: Growth curve of NRS-01 in glucose in the presence and absence of aniline Fig. 5.3b: Growth curve of NRS-01 in acetate in the presence and absence of aniline Fig. 5.4: Growth pattern of NRS-01 in MSM in the presence and absence of added nitrogen Fig. 5.5a&b: Graphs showing growth of NRS-01 at various aniline concentrations Fig. 5.7: Spectrophotometric scans of supernatant (various aniline concentrations) at various incubation periods Fig. 5.8a: Carbohydrate utilization and protein production compared with growth of NRS-01 Fig. 5.8b: Aniline utilization compared with growth of aniline Fig. 5.9a: Carb-ohydrate utilization and protein production compared with growth of NRS-01 Fig. 5.9b: Aniline utilization compared with growth of aniline Fig. 5.14: Spectrofluorophotometric scan of culture broth (MSM + Glu + 11mM Aniline) Fig. 5.15: Spectrofluorophotometric scan of culture broth (MSM + Glu) Fig. 5.17: Graph showing oxygen uptake by NRS-01 in presence of glucose Fig. 5.18: Graph showing oxygen uptake by NRS-01 in presence of aniline Fig. 6.1: Spectrofluorimetric scan of fluorescent product FP Fig. 6.2: Spectrophotometric scan of fluorescent product FP Fig. 6.4: Spectrophotometric scan of TP2 Fig. 6.5: IR spectrum of TP2 Fig. 6.6a: 1H-NMR spectrum of TP2 Fig. 6.6b,c: 13C-NMR spectra of TP2 Fig. 6.7a,b,c,d: GC/MS profile of TP2 Fig. 6.8: Pathway showing complex formation with aniline Fig. 6.9a-r: Various addition reactions of aniline Fig. 6.10: Proposed structures of TP2 List of Tables Table 3.1a: Total viable counts for the water samples from ONGC Table 3.1b: Total viable counts for the sediment samples from ONGC Table 3.2: Total viable counts obtained from mangrove sediment.

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