MICROBIAL ECOLOGY, DIVERSITY, AND BIOCHEMICAL POTENTIAL WITHIN BASALTIC INDIAN HOT SPRING Thesis submitted to AcSIR for the award of the degree of DOCTOR OF PHILOSOPHY In Biological Sciences BY GAJANAN T. MEHETRE (10BB13J26033) Dr. MAHESH S. DHARNE Dr. SYED G. DASTAGER (Research Supervisor) (Research Co-supervisor) NCIM RESOURCE CENTRE DIVISION OF BIOCHEMICAL SCIENCES CSIR-NATIONAL CHEMICAL LABORATORY PUNE 411008, Maharashtra, INDIA SEPTEMBER 2018 Dedicated to My beloved parents PhD Thesis AcSIR - CSIR - NCL Table of contents Table of contents.......................................................................................................................I Acknowledgements...............................................................................................................VIII Abbreviations...........................................................................................................................X List of Tables.........................................................................................................................XII List of Figures.......................................................................................................................XIV Abstract..................................................................................................................................XX CHAPTER 1.............................................................................................................................1 1.1: Introduction.......................................................................................................................2 1.2: Review of literature...........................................................................................................4 1.2.1: Terrestrial hot spring ecosystems.........................................................................4 1.2.1.1: Physico-chemical aspects of hot springs...............................................5 1.2.2.2: Microbiological aspects of hot springs.................................................5 1.2.2: Approaches and methods for microbial ecology studies......................................6 1.2.2.1: Traditional approaches: culture-dependent methods.............................6 1.2.2.2: Molecular approaches: culture-independent methods...........................7 1.2.2.3: Changing paradigm of microbial analyses..........................................11 1.2.3: Microbial taxonomy and diversity.....................................................................13 1.2.3.1: Culture-dependent approaches............................................................13 1.2.3.2: Culture-independent approaches (Metagenomics)..............................16 1.2.4.: Biotechnological applications...........................................................................16 1.2.4.1: Thermo-stable enzymes and industrial microbiology.........................16 1.2.4.2: Environmental applications................................................................17 1.2.4.2.1: Heavy metal resistance and toxicity.....................................17 1.2.4.2.2: Degradation of complex aromatic compounds.....................18 1.2.4.3: Function driven approaches...............................................................19 1.2.5: Indian hot springs...............................................................................................19 1.2.5.1: Microbiological studies.......................................................................20 1.2.5.2: Scope and formulating a research problem.........................................21 1.3: Aims and objectives........................................................................................................22 1.4: References.......................................................................................................................23 G. T. Mehetre I PhD Thesis AcSIR - CSIR - NCL CHAPTER 2...........................................................................................................................39 Abstract...................................................................................................................................40 Introduction............................................................................................................................41 2.2 Materials and methods.....................................................................................................44 2.2.1: Chemicals and reagents......................................................................................44 2.2.2 Description of the site.........................................................................................44 2.2.3: Microbial diversity analyses: an experimental plan...........................................46 2.2.4: Sampling and physicochemical analyses...........................................................46 2.2.5: Metagenomic DNA isolation and sequencing....................................................48 2.2.5.1: Isolation of total community DNA.....................................................48 2.2.5.2: Quantitative and qualitative analysis of DNA.....................................48 2.2.5.3: Sequencing methods and library preparations....................................49 2.2.5.3.1: 16S rRNA amplicon sequencing..........................................50 2.2.6: Bioinformatics analysis......................................................................................50 2.2.6.1: Fastq quality checking and preprocessing of reads............................50 2.2.6.2: OTUs and taxonomy classification.....................................................51 2.2.6.3: Analysis of alpha diversity..................................................................52 2.2.6.4: Analysis of beta diversity....................................................................52 2.2.6.5: Statistical analyses..............................................................................52 2.2.6.6: Mining for metabolic potentials..........................................................53 2.2.7: Whole metagenome sequencing analyses..........................................................53 2.2.7.1: De novo metagenome assembly..........................................................53 2.2.7.2: Taxonomy analysis..............................................................................54 2.2.7.3: Functional annotation…......................................................................54 2.2.7.4: Metabolic pathway annotation............................................................54 2.2.7.5: Statistical analyses..............................................................................55 2.2.8: Nanopore MinION sequencing..........................................................................55 2.2.9: Comparative metagenomic analyses..................................................................55 2.2.10: Submission of raw sequences in the database..................................................56 2.3: Results and discussions...................................................................................................56 2.3.1: Physicochemical analysis of water samples.......................................................56 2.3.2: Metagenomic DNA isolation and library preparation........................................58 2.3.3: Amplicon (16S rRNA) based microbial community analyses...........................59 2.3.3.1: Insights into main hot spring water.....................................................59 G. T. Mehetre II PhD Thesis AcSIR - CSIR - NCL 2.3.3.2: Profiling of water, sediment and mat of main hot spring....................60 2.3.3.3: Comparative profiling of natural hot spring and borehole sites..........66 2.3.3.3.1: Composition and diversity of Archaea.................................67 2.3.3.3.2: Composition and diversity of Bacteria.................................67 2.3.3.3.3: Alpha and beta diversity patterns.........................................72 2.3.3.3.4: Correlation with physicochemical factors............................75 2.3.3.4: Mining for functional metabolic potentials.........................................77 2.3.3.5: Comparison with other Indian hot springs..........................................82 2.3.4: Whole metagenome (shotgun) sequencing analyses..........................................86 2.3.4.1: Insights into structure and functions of water....................................86 2.3.4.2: Insights into structure and functions of sediments..............................88 2.3.4.3: Profiling using Illumina and Nanopore sequencing............................91 2.3.4.3.1: Microbial diversity...............................................................91 2.3.4.3.2: Functional analysis...............................................................92 2.3.5: Aanthropogenic impact on natural hot spring diversity.....................................93 2.3.5.1: Profiling of water samples..................................................................93 2.3.5.2: Profiling of sediment samples.............................................................96 2.4: General discussion..........................................................................................................99 2.5: Conclusion.....................................................................................................................109 2.6: References.....................................................................................................................111