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

Deep biosphere within basaltic – granitic (igneous rocks) systems Igneous rocks constitute ~95% of the Earth’s crust Deep crustal system represents an Extreme Habitat 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

Biogeochemical importance; 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

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

What powers deep microbiome 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

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

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

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

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 Shivaji Sagar Lake created by Koyna Dam in Western India in 1962

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

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.

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

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 . I Obj . II Obj . III Time scale (year) 5 Drilling, sample collection and analysis Molecular genomic analysis Data integration and modeling

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)

Budget Details (five years) Particulars Cost in USD (approx) Equipment (NG Sequencer) 3,20,000 Accessory equipment 65,000 Drilling 1,50,000 Chemicals/Consumables, contingency 2,00,000 Staff (01 PDF, 02 RF, 01 RA) 1,20,000 International/domestic travel, material transport 45,000 Total 11,00,000 PDF: post doc fellow; RF: Research fellew /Ph D, RA: Research assistant

Thank You

Deep subsurface : the hidden and unexplored habitat for microbes Edwards et al., Annu. Rev. Earth Planet. Sci. 2012 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 With the discovery of deep microbial ecosystems in sedimentary basins−as well as microbial life in granites, deep gold mines, and oil reservoirs—the view of the scientific community was opened to a hidden and largely unexplored inhabited realm on our planet. (Itavaara et al., FEMS Microbiol Ecol 77 2012)

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

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

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

Requirements of microbes in deep biosphere ‘Living space’ Liquid water Energy and nutrients Porosity Permeability Tectonostratigraphic setting Electron donor Electron acceptor Carbon source thermodynamic potential of chemical reactions

Microbial metabolism within deep subsurface Little org C Lithoautotrophic metabolism Small org molecules are microbially synthesized from H2 and CO2 Independent of surface photosynthetically derived mater Igneous rocks Buried org C is the main C and e source Heterotrohic metabolism Depends on surface photosynthetically derived mater Sedimentary rocks

Abiotic diagenetic formation of low mw compounds 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) Fermentation Syntrophic fermentation Methanogen Organic matter deposition Biotic processes Preserved OM (Kerogen, Bitumen, Humics Thermal activation Abiotic diagenetic formation of low mw compounds Anaerobic microbial metabolism Abiotic processes Temperature e acceptor limited Independent from primary microbial degradation processes

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)

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

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 Time scale (year) 3 Sample collection and analysis Molecular analysis Data integration and modeling

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

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

Effect of seismic activity on microbial community and function 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

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

Recurring Particulars 1st Year (Rs) 2nd Year (Rs) 3rd Year (Rs) Total (Rs) Manpower Senior Research Fellow (01) 216000 648000 Technical Assistant (01) 144000 432000 Sub-Total 360000 1080000 Consumables 600000 800000 2000000 Travel 200000 100000 500000 Contingency 300000 Overhead 752000 292000 232000 1276000 Sub-Total of Recurring 2012000 1752000 1392000 5156000 Grand Total (Non-Recurring + Recurring) 4512000 7656000

Thank You

Justification of Equipment Fluorescence Microscope The fluorescent microscope is required for all microscopic enumeration of bacteria, cell counts, FISHT etc. This equipment is the major requirement for microbiological analysis related to the project. Incubator shaker The temperature controlled shaker will be used for molecular biology work. Gel electrophoresis system with accessories The gel electrophoresis apparatus will be used for all routine DNA work. Work station for bioinformatics with accessories The computer will be required for all bioinformatics data analysis Ultra deep fridge Ultra deep fridge will be used to store the samples from cores and other microbiological samples. Ion selective electrodes will be required for the Orion multiparameter meter to be used in the field.

Real time PCR machine For all quantitative determination of rRNA and other genes; monitoring of expression levels of various functional genes this instrument is absolutely essential. In the present work transcriptional analysis of selected biogeochemical cycle relevant genes, abundance of specific microbial groups, -dynamics will be studied using this equipment. The proposed model is versatile and highly efficient. For this project this equipment is extremely essential

Justification of Manpower Senior Research Fellow One dedicated senior research fellow will be essential to assist the PI and co PI for carrying out the research work Technical Assistant One TA will be essential or field work, sample collection, sample processing and other relevant activities of the project.

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.

Justification of travel Field sampling and analysis; Project meeting Several visits to fields and analytical labs for analysis; Project meeting, if any Field work and project presentation; Seminar participation Field work and project presentation at DBT, if any; Seminar participation Travel to fields Several visits to fields for survey and sample collection Travel to other laboratories Sample analysis

Justification of Contingency DNA sequencing, fatty acid analysis, GC content determination, Conference and meetings Field expenditures, photocopy, computational works, cost of gas for AAS, anaerobic station Expenditures related to sample collections and other field work, cost of field labors, porters, gases for anaerobic workstation (N2 and mix gas), computational work, photocopying; charges for PLFA analysis, type strains and genomic DNA samples (from DSMZ or ATCC or MTCC), sequencing etc. and any unforeseen expenditures Sample collection related costs, Conference and meeting related expenditures; DNA sequencing Sample collection related costs, Conference and meeting related expenditures; Visit to other labs for analysis and data verification; Cost of DNA sequencing

Extra slides

Expedition to deep biosphere

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.

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

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

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

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

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.

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 Given the remarkable capacity of microorganisms to utilize a wide range of energy sources, including light, organic matter and inorganic materials, and their broad distribution across the surface of the planet, it should come as no surprise that the deep terrestrial subsurface harbors diverse microbial populations. Many such environments contain all of the requirements for prokaryotic life including water-filled space in pores and fractures, energy in the form of buried organic matter (kerogen), gases such as methane or H2, and reduced inorganic ions (multiple sulfur species and metals such as Fe and Mn), and various essential inorganic elements including carbon, nitrogen, phosphorous, and sulfur. Extension of the biosphere on Earth

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. Given the remarkable capacity of microorganisms to utilize a wide range of energy sources, including light, organic matter and inorganic materials, and their broad distribution across the surface of the planet, it should come as no surprise that the deep terrestrial subsurface harbors diverse microbial populations. Many such environments contain all of the requirements for prokaryotic life including water-filled space in pores and fractures, energy in the form of buried organic matter (kerogen), gases such as methane or H2,and reduced inorganic ions (multiple sulfur species and metals such as Fe and Mn), and various essential inorganic elements including carbon, nitrogen, phosphorous, and sulfur. The largely unexplored deep biosphere must play fundamental role in global biogeochemical cycles over both short and longer time scales

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.

Microbiology of seismic zones Molecular hydrogen, H2, is the key component to link the inorganic lithosphere with the subsurface biosphere. Geochemical and microbiological characterizations of natural hydrothermal fields strongly suggested that H2 is an important energy source in subsurface microbial ecosystems because of its metabolic versatility. One of the possible sources of H2 has been considered as earthquakes: mechanoradical reactions on fault surfaces generate H2 during earthquake faulting. However it is unclear whether faulting can generate abundant H2 to sustain subsurface chemolithoautotrophic microorganisms, such as methanogens.

Wanger et al 2007

Culture dependent analysis Isolation of pure culture bacteria (different enrichment cond., aerobic and anaerobic cond.) Identification (16S rRNA gene, FAME, API, etc.) Metal resistance and transformation studies Metabolic Characterization

* * What have we learned? Novel indigenous microbes and communities Novel and unusual deeply branched sequences may be indicative of ancestral linkages, (early life?), Novel products for biomed and biotech applications 1 mm image courtesy of Gordon Southam Novel Bacterial lineages unique to the SA deep-subsurface: South Africa Subsurface Firmicutes Groups (SASFiG) SASFiG-6 SASFiG-5 * SASFiG-4 SASFiG-7 SASFiG-3 SASFiG-9 * SASFiG-8 SASFiG-1 *SASFiG-9 (isolated) Detected within a water-bearing dyke/fracture at 3.2 Km depth. strictly anaerobic; iron-reducer optimal growth temperature = 60 oC virgin rock temp = ~ 45 oC SASFiG-2

Key Experiments: Culture-Independent Evidence for Deep Life Genomic advancements Sequencing of a microbe required ~18 months in mid 90’s Currently >150 microbes have been sequenced In 2004 TIGR discovers 1.2 million new bacteria/archea genes in the Sargasso Sea By 2005 JGI could sequence 400 microbes per year Could early life in the subsurface have survived the Hadean bombardment?

Earth’s subsurface microbial ecology The biosphere extends deep into the subsurface Limited by geothermal gradient and nutrient flux Biomass generally low relative to the surface Distribution is very patchy and hetergenous Rates of community metabolism very low Volumetrically largest part of the biosphere

Subsurface lithoautotrophic microbial ecosystems (SLiMEs)

Basalt:  - Forms on the surface of the earth  - Because it forms on the surface it cools quickly and has a fine texture (mineral grains are too fine to see with the naked eye).  - The source of this rock comes from partially melted material in the mantle.  - It usually leaves the mantle at mid-ocean ridges, where new seafloor is being formed. That's why most of the ocean crust consists of basalt or gabbro (the intrusive version of basalt).  - Because basalt comes from a mantle source, it's very mafic and consists of dark, dense minerals rich in iron and manganese (usually olivine and pyroxene).  Granite:  - Forms underneath the surface of the earth.  - Because it forms under the surface the magma cools slowly, grains have time to grow and therefore it has a coarse grained texture. Grains can be easily seen with the naked eye.  - Granite forms when a part of the continental crust melts to form magma and solidifies again. The heat needed for this to happen can come from different sources, for example magma from the mantle which causes the crust to melt.  - Because of the above granite will be found on the continental crust (mostly at least).  - The crust consists of lighter minerals than deeper parts of the earth, and that is why the minerals you will find in granite will be lighter, less dense and richer in SiO2 than those found in basalt (granite is therefore a much more felsic rock). Minerals you will typically find is quartz, orthoclase and plagioclase

Extent, Million Years Ago Eon Era Period Extent, Million Years Ago Phanerozoic Cenozoic Quaternary (Pleistocene/Holocene) 2.588 - 0 Neogene (Miocene/Pliocene) 23.03 - 2.588 Paleogene (Paleocene/Eocene/Oligocene) 65.0 - 23.03 Mesozoic Cretaceous 145.5 - 65.0 Jurassic 201.3 - 145.0 Triassic 252.17 - 201.3 Paleozoic Permian 298.9 - 252.17 Carboniferous (Mississippian/Pennsylvanian) 358.9 - 298.9 Devonian 419.2 - 358.9 Silurian 443.4 - 419.2 Ordovician 485.4 - 443.4 Cambrian 541.0 - 485.4 Proterozoic Neoproterozoic Ediacaran 635.0 - 541.0 Cryogenian 850 - 635 Tonian 1000 - 850 Mesoproterozoic Stenian 1200 - 1000 Ectasian 1400 - 1200 Calymmian 1600 - 1400 Paleoproterozoic Statherian 1800 - 1600 Orosirian 2050 - 1800 Rhyacian 2300 - 2050 Siderian 2500 - 2300

History[edit] The Deccan Traps formed between 60 and 68 million years ago,[2] at the end of the Cretaceous period. The bulk of the volcanic eruption occurred at the Western Ghats (near Mumbai) some 66 million years ago. This series of eruptions may have lasted less than 30,000 years in total.[3] The original area covered by the lava flows is estimated to have been as large as 1.5 million km², approximately half the size of modern India. The Deccan Traps region was reduced to its current size by erosion and plate tectonics; the present area of directly observable lava flows is around 512,000 km2 (197,684 sq mi). Effect on climate and contemporary life[edit] The release of volcanic gases, particularly sulfur dioxide, during the formation of the traps contributed to contemporaryclimate change. Data points to an average drop in temperature of 2 °C in this period.[4] Because of its magnitude, scientists formerly speculated that the gases released during the formation of the Deccan Traps played a role in the Cretaceous–Paleogene extinction event (also known as the K–Pg extinction), which included theextinction of the non-avian dinosaurs. Sudden cooling due to sulfurous volcanic gases released by the formation of the traps and localised gas concentrations may have contributed significantly to mass extinctions. However, the current consensus among the scientific community is that the extinction was triggered by the Chicxulub impact event in Central America (which would have produced a sunlight-blocking dust cloud that killed much of the plant life and reduced global temperature, called an impact winter).[5]

Core samples from borehole KBH-1 showing (a) massive basalt, (b) vesicular and amygdaloidal basalt with large vugs filled with quartz and/or calcite, (c) flow-top breccia, (d) red bole bed and overlying massive basalt, (e) vugs filled with zeolite, and (f) basement granite at 951 m depth.