Pinaki Sar Department of Biotechnology

1. Exploring microbial diversity and function within the granitic-basaltic deep crustal system of Koyna-Warna (India) region Indian Institute of Technology Kharagpur India Pinaki Sar Department of Biotechnology Collaborators Sufia K Kazy, National Institute of Technology Durgapur, India Sukanto Roy, National Geophysical Research Institute, Hyderabad, India

2. Deep biosphere within basaltic – granitic (igneous rocks) systems • Igneous rocks constitute ~95% of the Earth’s crust • Deep crustal system represents anExtremeHabitat for Life • Aphotic • Devoid of Org C • Subjected to high temperature/pressure at some point in their history • Oligotrophic Basalt Granite Image source : http://en.wikipedia.org/wiki/File:Igneous_rock_eng_text.jpg#file

3. Microbiology of deep, igneous crust seems more intriguing, though relatively less studied Biogeochemical importance; Limits of life ? • Newly generated (annually) and recycled (~ 60 M yrs) • Upper (500 m), subseafloor basalts are significantly porous and permeable, hydrologically active • Largest potential microbial habitat Who are they ? What are their function Microbiology of basaltic/grantic deep subsurface (marine/terrestrial) are less studied and mostly unexplored Some more reports for ocean crust than terrestrial habitats Unlike deep oceanic subsurface which may be partially dependent on organic C and energy derived from photosynthetic process, life within terrestrial crystalline rocks are independent to photosynthesis

4. What remained largely unexplored and poorly understood : • Distribution and diversity of microbes in terrestrial igneous rocks • Knowledge on their metabolic functions and their impact on global C and nutrient cycles Bacterial communities in different (sub-)sea floor habitats, demonstrating that subsurface crustal bacteria are distinct from the bacteria in other deep-sea environments; Wang et al 2013; Edward et al 2011

5. What powers deep microbiome ? Extent of microbial catabolic potential within deep igneous crust • Abiogenic H2 driven metabolic pathways ? • Role in C/N/nutrient cycling • Rock weathering and climate change

6. X In igneous rock systems ? Acetogenic –Methanogenic metabolism with abiogenic H2 Geomicrobial processes at a subsurface shale-sandstone interface; Fredrickson and Balkwill, 2006

7. H2 driven system Small Org comp Denitrification/NH4 oxidation Anaerobic heterotrophic metabolism N2 fixation Anaerobic lithoautotrophic metabolism SLiMEs (?) Abiotic processes Temperature Methanogen Abiotic geogenic H2 Abiotic diagenetic formation of low mw compounds H2 • Radiolytic decomposition of water • Water-rock interaction • Diffusion from deeper levels

8. The Deccan Traps • The Deccan Traps are a large igneous province, on the Deccan Plateau (west-central India (between 17–24N, 73–74E) • One of the largest volcanic features on Earth • Consist of multiple layers of solidified flood basalt [together >2,000 m thick and cover an area of 500,000 km2 and a volume of 512,000 km3 (123,000 cu mi)] • formed between 60 and 68 million years ago [end of the Cretaceous period] linked to the Cretaceous–Paleogene extinction event

9. Seismic activity in deccan Trap at Koyna-Warna region Reservoir triggered seismicity (RTS) record in past 38 years: >10 earthquakes of Mz5; >150 earthquakes of Mz4 >100,000 earthquakes of Mz0 soon after the impoundment of the ShivajiSagar Lake created by Koyna Dam in Western India in 1962

10. Drilling site at Koyna Drilling is proposed up to nearly 7 KM, so far ~1.5KM drilling is done • Cores recovered so far revealed : • Flood basalt pile with numbers of lava flow • Each flow has vesicular / amygdaloidal layer unde lined by massive basalt • Microbial presence (successful extraction of DNA and amplification of 16 S rRNA gene regions) from samples of 1300 M depth • Low C environment Core samples from borehole KBH-1 showing (a) massive basalt, (b) vesicular and amygdaloidal basalt with large vugs filled with quartz and/or calcite JOUR.GEOL.SOC.INDIA, VOL.81, FEB. 2013

11. Major aim of the proposed work • Delineating the environmental limit of life within the terrestrial baslatic/granitic system • Understanding the processes that potentially define diversity /distribution of life in deep terrestrial crustal system • Possible modes of microbial interactions within such environment affecting C and nutrient cycle, rock weathering etc.

12. Objectives • Analysis of microbial diversity and composition within the basaltic-, granitic- and transition zones from deep subsurface environment of Koyna region: Combination of metagenome based sequencing techniques and enrichment/isolation of bacteria (include virus and fungi as well after this meeting ) • Metabolic function and microbial role in biogeochemical cycling of carbon, rock microbiome interaction (weathering); effect –response of seismic activities: Metagenome and metatranscriptome analysis, WGS analysis of predominant isolates, metabolic modeling, getting ideas of novel metabolic routes running the biogeochemical reactions • Integration of geochemical/environmental data and comparative metagenomic analysis of deep basaltic-granitic biosphere with and without seismic activities: Assessment of the extent of microbial distribution and diversity, potential involvement in C cycle

13. Work flow: implementaion Elucidation of effect of seismic activity and crustal properties on microbial diversity and activity Analysis of microbial function Analysis of microbial diversity, community structure, abundance Obj . II Obj . I Obj . III 0 Time scale (year) 5 Drilling, sample collection and analysis Molecular genomic analysis Data integration and modeling

14. Deliverables • Deep carbon observatory goals : • Elucidation of microbial diversity/distribution within carbon limited, dark, deep terrestrial crust • Better insight in understanding on survival strategies and role under deep subsurface igneous rocks • Delineation of limits for microbial deep life and their interaction with critical nutrient cycling Global significance : Global primer site of RTS within basaltic/granitic crust Microbial role in rock weathering Nutrient cycling, CO2 sequestration and other aspects of climate change Biomineralization; Bioremediation, Bioprospecting (Access of novel microbes and enzymes for industrial application)

15. Budget Details (five years) PDF: post doc fellow; RF: Research fellew /Ph D, RA: Research assistant

16. Thank You

17. Deep subsurface : the hidden and unexplored habitat for microbes • The largest potential ecosystem on Earth, estimated to harbour half of all the biomass; and 2/3 of all microbial biomass on Earth (2.5-3.5 X 1030) • Depth of distribution: Functionally and taxonomically diverse populations extending several kilometres underground • Adaptation :temperature limit 121oC, pressures of up to 1.6 Gpa • Function: fundamental role in global biogeochemical cycles over short and long time scale (Itavaara et al., FEMS Microbiol Ecol 77 2012) Edwards et al., Annu. Rev. Earth Planet. Sci. 2012

18. The deep biosphere : an extreme habitat for microbes With increasing depth there are several constrains that affect composition, extent, life habitats, and the living conditions in deep subsurface • Increasing temperature and pressure • Nutrient limitation, limited porosity and permeability • Decreasing available carbon and energy sources Rates of microbial activity in deep subsurface is slow (orders of magnitude over that in surface environments) With average generation times of hundreds to thousands of years …and therefore defies our current understanding of the limits of life

19. The deep biosphere • The huge size • Largely unexplored biogeochemcial process driving the deep biosphere • “Investigation of the extent and dynamics of subsurface microbial ecosystems an intriguing and relatively new topic in today’s geoscience research” ICDP, 2010

20. Widely disseminated deep biosphere pose fundamental questions : • IODP Nankai Trough Seismogenic Zone Experiment (NanTroSEIZE) • Natural Earthquake Laboratory at Focal Depth (DAFSAM-NELSAM) • Taiwan Chelungpu Drilling Project (TCDP) • Lomonosov Ridge in the Central Arctic Basin • Outokumpu deep borehole, Fennoscandian Shield kind of microorganisms ? populate the deep subsurface? their extension and limits? metabolic processes ? carbon and energy sources ? survival strategies? link to early life on Earth? biological alteration of rock impact on the global -biogeochemical cycle and -climate? Nature of microbial communities and their function in active seismogenic zone Effect of fracturing (during earthquake) on microbial communities Interrelation between geochemistry, microbiology and nature/location of fracture zones ICDP 2010

21. Requirements of microbes in deep biosphere • Porosity • Permeability • Tectonostratigraphic setting • Electron donor • Electron acceptor • Carbon source • thermodynamic potential of chemical reactions

22. Microbial metabolism within deep subsurface

23. Scheme visualizing potential carbon and energy sources of deep microbial ecosystems OM = organic matter, mw = molecular weight CH4 Acetate, CO2 and H2 Organic acids and alcohols Soluble monomers (sugar and amino acids) Complex polymers (CH2 O, proteins) Methanogen Organic matter deposition Syntrophic fermentation Fermentation Biotic processes e acceptor limited Anaerobic microbial metabolism Thermal activation Abiotic processes Temperature Preserved OM (Kerogen, Bitumen, Humics Abiotic diagenetic formation of low mw compounds Independent from primary microbial degradation processes

24. What have we learned? All Observations are consistent with the laws of physics • Extended known biosphere to 3 km, not limited by energy • Revealed biomass, biodiversity, unusual traits & microbes with indications of autotrophic ecosystems • Slow rates of deep subsurface microbial activity but linked with geological interfaces • Deep subsurface biosphere not linked to the surface (?) • Deep anaerobic communities fueled by subsurface abiotic energy sources (?)(Likely)

25. Objectives • Analysis of microbial abundance, diversity and composition within the deep subsurface environment of the seismic zone of Koyna-Warna region • Elucidation of functional role of indigenous microorganisms within the seismic zone • The effect of seismic activity on microbial community and function

26. Work flow Elucidation of effect of seismic activity and crustal properties on microbial diversity and activity Analysis of microbial function Analysis of microbial diversity, community structure, abundance Obj . I Obj . II Obj . III 0 Time scale (year) 3 Sample collection and analysis Molecular analysis Data integration and modeling

27. Work Plan Objective 1.: Analysis of microbial abundance, diversity and composition Metagenome extraction Sample collection from cores Direct microscopic count Geochemical analysis Amplification of 16S rRNA gene DGGE analysis Enumeration of cell counts Analysis of community composition MPN count (Tot, Sox / red & Feox / red , methanogenic and hydrogen utilizing bacteria) Library preparation Elemental analysis (XRF, ICP) Sanger sequencing TOC, TC, TS, TP analysis Sequencing [NGS] Sequence analysis Plate count Anion analysis EPMA analysis Community diversity and composition

28. Objective 2 Analysis of microbial communities’ function Total community Analysis of genes related to S, Fe, C, N cycles Analysis of metabolic diversity NG sequencing of complete metagenome S cycle: dsr PM - Biolog system Fe cycle: Fur C cycle: mcrA, RuBisCO N cycle: nif, nirK, amoR

29. Objective 3 Effect of seismic activity on microbial community and function • Comparison of community structure across depth • Comparison of community function across depth • Integration of microbiological data with geochemical and other relevant data on seismic activity within the samples from various depths

30. Expected out come Understanding the deep terrestrial biosphere with seismogenic activity Distribution, extent and composition of deep microbial communities within the basaltic-granitic subsurface Impact of seismic activity and subsurface CO2, N2, and H2 production on microbial community structure and function, existence of SLiMEs? Correlation of microbial activity, geochemistry/rock systems and seismic activity within the zone of RTS

31. Recurring

32. Thank You

33. Justification of Equipment

35. Justification of Manpower

36. Justification of Consumables Consumables will be essential for carrying out culture independent RNA dependent and metagenomic analysis of microbial communities. Cost for RNA/DNA extraction kits, cDNA preparation, real time PCR reagents, primers, vectors and restriction enzymes, plasmid isolation kits, gel extraction and sequencing kits are all included. For real time based transcriptomic studies, cDNA kits and other reagents related to real time PCR (TaqMan probes, Syber green dye, etc.), nucleic acid quantification kits (pico green), etc. will be needed. For fluorescent microscopy and FISH analysis dedicated kite are required. Sequencing reagents, kits and other charges are included under this head. For all routine works general chemicals, glass and plastic ware are necessary. Bacterial type strains will be procured from National or international culture collection.

37. Justification of travel

38. Justification of Contingency

39. Extra slides

40. Expedition to deep biosphere

41. Map of DSDP, ODP, and IODP Legs (indicated by their numbers) considering microbial or deep microbial scientific objectives. b. Map showing completed and planned ICDP projects containing biogeochemical objectives. Black dots indicate ICDP projects where no biogeochemical objectives were included.

43. Microbial cells : the main biogeochemical engines of Earth • Microbes: the janitors of Earth • The most ubiquitous, abundant, most diverse live form on this planet • Occupy even most inhospitable niches • Vast metabolic and genetic repertoire • Responsible for many geobiochemical processes that take place deep in the Earth’s crust

44. Global prokaryotic biomass distribution, given in cell numbers (after Whitman et al. 1998).

45. Environmental parameters defining the dimensions of living space • Tectonostratigraphic setting • Distribution patterns, degree of sorting, lithology, etc. • Porosity and permeability • Subsidence, uplift and deformation of the basin fill control pressure (lithostatic, hydrostatic), • Modification in porosity and permeability of lithotypes. • Basin style and evolution control temperature gradient

46. Living space Pore space; pore types and degree of interconnection are important factor controlling deep biosphere microorganisms occupy only about one millionth of available porosity An adequate flux of liquids or gases through rock pores is required to sustain life and this is governed by pore throat dimensions. Permeability that regulates the pressure-driven transport of electron donors, electron acceptors, and nutrients to sustain living cells [Quartz arenites retain permeability to great depths and offer perhaps the most stable living accommodation for microorganisms while high reactivity of unstable volcanogenic sandstones and their mechanical weakness make them susceptible to rapid porosity and permeability loss, in some cases at relatively low temperatures] Fractures are orders of magnitude more permeable than pore systems and often allow microbial growth and activity

47. Supply of food Provision of food (electron donors) and oxidants (electron acceptor, e.g., O2) is controlled by the thermodynamic potential of chemical reactions, both organic and inorganic The rate of microbially catalysed reactions can be up to 106 times higher compared to abiological rates Depends on the rate of supply and removal of substrates and products, the concentration (above minimum thresholds and below toxic levels) and bioavailability of reactants and environmental conditions.

48. Microbial distribution in geospheres Greatest biomass inhabits within the surface/near surface lithosphere and shallow hydrosphere: reliance on photosynthesis / derived food chain Microorganism make the major component of biosphere because they can grow under diverse conditions and have different metabolic pathways Anaerobic organisms are dominant inhabitants of lithosphere .. generally decrease with increasing depth Because, organic matters are too recalcitrant to be degraded or water, nutrients and TEAs can not be supplied or temporaries are too high Surprisingly large bacterial populations with considerable diversity are present at depths near and over 1000m Extension of the biosphere on Earth

49. Out come of deep borehole studies by ICDP and/or IODP To be added in end The lower depth limit of the biosphere has not been reached in any borehole studies and the factors that control the abundance and activities of microbes at depth and the lower depth limit of life are still poorly understood. The largely unexplored deep biosphere must play fundamental role in global biogeochemical cycles over both short and longer time scales

50. Potential limiting factors for microbes in deep biosphere • The original chemical composition of the sediment • Response of microbes and its organic and inorganic components to increasing temperature • Availability of liquid water Increasing pressure during burial may not be a major limitation as some microorganisms can cope well with high pressure (>100 Mpa) and there is some evidence for metabolic activity at GPa pressures.