Presentation on theme: "Pinaki Sar Department of Biotechnology"— Presentation transcript:
1Pinaki Sar Department of Biotechnology Exploring microbial diversity and function within the granitic-basaltic deep crustal system of Koyna-Warna (India) regionPinaki SarDepartment of BiotechnologyIndian Institute of Technology KharagpurIndiaCollaboratorsSufia K Kazy, National Institute of Technology Durgapur, IndiaSukanto Roy, National Geophysical Research Institute, Hyderabad, India
2Deep biosphere within basaltic – granitic (igneous rocks) systems Igneous rocks constitute ~95% of the Earth’s crustDeep crustal system represents an Extreme Habitat for LifeAphoticDevoid of Org CSubjected to high temperature/pressure at some point in their historyOligotrophicBasaltGraniteImage source :
3Biogeochemical importance; Microbiology of deep, igneous crust seems more intriguing, though relatively less studiedBiogeochemical importance;Limits of life ?Newly generated (annually) and recycled (~ 60 M yrs)Upper (500 m), subseafloor basalts are significantly porous and permeable, hydrologically activeLargest potential microbial habitatWho are they ?What are their functionMicrobiology of basaltic/grantic deep subsurface (marine/terrestrial) are less studied and mostly unexploredSome more reports for ocean crust than terrestrial habitatsUnlike 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
4What remained largely unexplored and poorly understood : Distribution and diversity of microbes in terrestrial igneous rocksKnowledge on their metabolic functions and their impact on global C and nutrient cyclesBacterial 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
5What powers deep microbiome What powers deep microbiome ? Extent of microbial catabolic potential within deep igneous crustAbiogenic H2 driven metabolic pathways ?Role in C/N/nutrient cyclingRock weathering and climate change
6Acetogenic –Methanogenic metabolism with abiogenic H2 XIn igneous rock systems?Acetogenic –Methanogenic metabolism with abiogenic H2Geomicrobial processes at a subsurface shale-sandstone interface; Fredrickson and Balkwill, 2006
7H2 driven system Abiotic diagenetic formation of low mw compounds Anaerobic lithoautotrophic metabolismSLiMEs (?)Small Org compMethanogenAbiotic processesTemperatureAbiotic geogenic H2Anaerobic heterotrophic metabolismH2N2 fixationDenitrification/NH4 oxidationRadiolytic decomposition of waterWater-rock interactionDiffusion from deeper levels
8The Deccan TrapsThe 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 EarthConsist 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
9Seismic 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 Mz0soon after the impoundment of the Shivaji Sagar Lake created byKoyna Dam in Western India in 1962
10Drilling site at KoynaDrilling is proposed up to nearly 7 KM, so far ~1.5KM drilling is doneCores recovered so far revealed :Flood basalt pile with numbers of lava flowEach flow has vesicular / amygdaloidal layer unde lined by massive basaltMicrobial presence (successful extraction of DNA and amplification of 16 S rRNA gene regions) from samples of 1300 M depthLow C environmentCore samples from borehole KBH-1 showing (a) massive basalt, (b) vesicular and amygdaloidal basalt with large vugs filled with quartz and/or calciteJOUR.GEOL.SOC.INDIA, VOL.81, FEB. 2013
11Major aim of the proposed work Delineating the environmental limit of life within the terrestrial baslatic/granitic systemUnderstanding the processes that potentially define diversity /distribution of life in deep terrestrial crustal systemPossible modes of microbial interactions within such environment affecting C and nutrient cycle, rock weathering etc.
12ObjectivesAnalysis 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 reactionsIntegration 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
13Work flow: implementaion Elucidation of effect of seismic activity and crustal properties on microbial diversity and activityAnalysis of microbial functionAnalysis of microbial diversity, community structure, abundanceObj . IObj . IIObj . IIITime scale (year)5Drilling, sample collection and analysisMolecular genomic analysisData integration and modeling
14Deliverables Deep carbon observatory goals : Elucidation of microbial diversity/distribution within carbon limited, dark, deep terrestrial crustBetter insight in understanding on survival strategies and role under deep subsurface igneous rocksDelineation of limits for microbial deep life and their interaction with critical nutrient cyclingGlobal significance : Global primer site of RTS within basaltic/granitic crustMicrobial role in rock weatheringNutrient cycling, CO2 sequestration and other aspects of climate changeBiomineralization; Bioremediation, Bioprospecting (Access of novel microbes and enzymes for industrial application)
15Budget Details (five years) ParticularsCost in USD (approx)Equipment (NG Sequencer)3,20,000Accessory equipment65,000Drilling1,50,000Chemicals/Consumables, contingency2,00,000Staff (01 PDF, 02 RF, 01 RA)1,20,000International/domestic travel, material transport45,000Total11,00,000PDF: post doc fellow; RF: Research fellew /Ph D, RA: Research assistant
17Deep subsurface : the hidden and unexplored habitat for microbes Edwards et al., Annu. Rev. Earth Planet. Sci. 2012The largest potential ecosystem on Earth, estimated to harbour half of all the biomass; and 2/3 of all microbial biomass on Earth ( X 1030)Depth of distribution: Functionally and taxonomically diverse populations extending several kilometres undergroundAdaptation : temperature limit 121oC, pressures of up to 1.6 GpaFunction: fundamental role in global biogeochemical cycles over short and long time scaleWith 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 )
18The 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 subsurfaceIncreasing temperature and pressureNutrient limitation, limited porosity and permeabilityDecreasing available carbon and energy sourcesRates 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
19The 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
20Widely 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 BasinOutokumpu deep borehole, Fennoscandian Shieldkind 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 rockimpact on the global -biogeochemical cycle and -climate?Nature of microbial communities and their function in active seismogenic zoneEffect of fracturing (during earthquake) on microbial communitiesInterrelation between geochemistry, microbiology and nature/location of fracture zonesICDP 2010
21Requirements of microbes in deep biosphere ‘Living space’Liquid waterEnergy and nutrientsPorosityPermeabilityTectonostratigraphic settingElectron donorElectron acceptorCarbon sourcethermodynamic potential of chemical reactions
22Microbial metabolism within deep subsurface Little org CLithoautotrophic metabolismSmall org molecules are microbially synthesized from H2 and CO2Independent of surface photosynthetically derived materIgneous rocksBuried org C is the main C and e sourceHeterotrohic metabolismDepends on surface photosynthetically derived materSedimentary rocks
23Abiotic diagenetic formation of low mw compounds Scheme visualizing potential carbon and energy sources of deep microbial ecosystems OM = organic matter, mw = molecular weightCH4Acetate, CO2 and H2Organic acids and alcoholsSoluble monomers (sugar and amino acids)Complex polymers (CH2 O, proteins)FermentationSyntrophic fermentationMethanogenOrganic matter depositionBiotic processesPreserved OM (Kerogen, Bitumen, HumicsThermal activationAbiotic diagenetic formation of low mw compoundsAnaerobic microbial metabolismAbiotic processesTemperaturee acceptor limitedIndependent from primary microbial degradation processes
24What have we learned? All Observations are consistent with the laws of physics Extended known biosphere to 3 km, not limited by energyRevealed biomass, biodiversity, unusual traits & microbes with indications of autotrophic ecosystemsSlow rates of deep subsurface microbial activity but linked with geological interfacesDeep subsurface biosphere not linked to the surface (?)Deep anaerobic communities fueled by subsurface abiotic energy sources (?)(Likely)
25ObjectivesAnalysis of microbial abundance, diversity and composition within the deep subsurface environment of the seismic zone of Koyna-Warna regionElucidation of functional role of indigenous microorganisms within the seismic zoneThe effect of seismic activity on microbial community and function
26Work flowElucidation of effect of seismic activity and crustal properties on microbial diversity and activityAnalysis of microbial functionAnalysis of microbial diversity, community structure, abundanceObj . IObj . IIObj . IIITime scale (year)3Sample collection and analysisMolecular analysisData integration and modeling
27Work PlanObjective 1.:Analysis of microbial abundance, diversity and compositionMetagenome extractionAmplification of 16S rRNA geneLibrary preparationSequencing [NGS]Sequence analysisDGGE analysisSanger sequencingCommunity diversity and compositionSample collection from coresDirect microscopic countMPN count (Tot, Sox / red & Feox / red , methanogenic and hydrogen utilizing bacteria)Plate countGeochemical analysisEnumeration of cell countsAnalysis of community compositionElemental analysis (XRF, ICP)TOC, TC, TS, TP analysisAnion analysisEPMA analysis
28Objective 2 Analysis of microbial communities’ function Total communityAnalysis of metabolic diversityPM - Biolog systemAnalysis of genes related to S, Fe, C, N cyclesS cycle: dsrFe cycle: FurC cycle: mcrA, RuBisCON cycle: nif, nirK, amoRNG sequencing of complete metagenome
29Effect of seismic activity on microbial community and function Objective 3Effect of seismic activity on microbial community and functionComparison of community structure across depthComparison of community function across depthIntegration of microbiological data with geochemical and other relevant data on seismic activity within the samples from various depths
30Expected out comeUnderstanding the deep terrestrial biosphere with seismogenic activityDistribution, extent and composition of deep microbial communities within the basaltic-granitic subsurfaceImpact 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
31Recurring Particulars 1st Year (Rs) 2nd Year (Rs) 3rd Year (Rs) Total (Rs)ManpowerSenior Research Fellow (01)216000648000Technical Assistant (01)144000432000Sub-Total360000Consumables600000800000Travel200000100000500000Contingency300000Overhead752000292000232000Sub-Total of RecurringGrand Total(Non-Recurring + Recurring)
33Justification of Equipment Fluorescence MicroscopeThe 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 shakerThe temperature controlled shaker will be used for molecular biology work.Gel electrophoresis systemwith accessoriesThe gel electrophoresis apparatus will be used for all routine DNA work.Work station for bioinformatics with accessoriesThe computer will be required for all bioinformatics data analysisUltra deep fridgeUltra 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.
34Real time PCR machineFor 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
35Justification of Manpower Senior Research FellowOne dedicated senior research fellow will be essential to assist the PI and co PI for carrying out the research workTechnical AssistantOne TA will be essential or field work, sample collection, sample processing and other relevant activities of the project.
36Justification 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.
37Justification of travel Field sampling and analysis; Project meetingSeveral visits to fields and analytical labs for analysis; Project meeting, if anyField work and project presentation; Seminar participationField work and project presentation at DBT, if any; Seminar participationTravel to fieldsSeveral visits to fields for survey and sample collectionTravel to other laboratoriesSample analysis
38Justification of Contingency DNA sequencing, fatty acid analysis, GC content determination, Conference and meetingsField expenditures, photocopy, computational works, cost of gas for AAS, anaerobic stationExpenditures 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 expendituresSample collection related costs, Conference and meeting related expenditures; DNA sequencingSample collection related costs, Conference and meeting related expenditures; Visit to other labs for analysis and data verification; Cost of DNA sequencing
41Map 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.
43Microbial cells : the main biogeochemical engines of Earth Microbes: the janitors of EarthThe most ubiquitous, abundant, most diverse live form on this planetOccupy even most inhospitable nichesVast metabolic and genetic repertoireResponsible for many geobiochemical processes that take place deep in the Earth’s crust
44Global prokaryotic biomass distribution, given in cell numbers (after Whitman et al. 1998).
45Environmental parameters defining the dimensions of living space Tectonostratigraphic settingDistribution patterns, degree of sorting, lithology, etc.Porosity and permeabilitySubsidence, 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
46Living spacePore space; pore types and degree of interconnection are important factor controlling deep biospheremicroorganisms occupy only about one millionth of available porosityAn 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
47Supply of foodProvision of food (electron donors) and oxidants (electron acceptor, e.g., O2) is controlled by the thermodynamic potential of chemical reactions, both organic and inorganicThe rate of microbially catalysed reactions can be up to 106 times higher compared to abiological ratesDepends 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.
48Microbial distribution in geospheres Greatest biomass inhabits within the surface/near surface lithosphere and shallow hydrosphere: reliance on photosynthesis / derived food chainMicroorganism make the major component of biosphere because they can grow under diverse conditions and have different metabolic pathwaysAnaerobic organisms are dominant inhabitants of lithosphere .. generally decrease with increasing depthBecause, organic matters are too recalcitrant to be degraded or water, nutrients and TEAs can not be supplied or temporaries are too highSurprisingly large bacterial populations with considerable diversity are present at depths near and over 1000mGiven the remarkablecapacity of microorganisms to utilize a wide range of energysources, including light, organic matter and inorganic materials,and their broad distribution across the surface of the planet, itshould come as no surprise that the deep terrestrial subsurfaceharbors diverse microbial populations. Many such environmentscontain all of the requirements for prokaryotic life includingwater-filled space in pores and fractures, energy in the form ofburied organic matter (kerogen), gases such as methane or H2,and reduced inorganic ions (multiple sulfur species and metalssuch as Fe and Mn), and various essential inorganic elementsincluding carbon, nitrogen, phosphorous, and sulfur.Extension of the biosphere on Earth
49Out come of deep borehole studies by ICDP and/or IODP To be added in endThe lower depth limit of the biosphere has not been reached in any borehole studiesand 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 includingwater-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
50Potential limiting factors for microbes in deep biosphere The original chemical composition of the sedimentResponse of microbes and its organic and inorganic components to increasing temperatureAvailability of liquid waterIncreasing 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.
52Microbiology of seismic zones Molecular hydrogen, H2, is the key component to linkthe inorganic lithosphere with the subsurface biosphere.Geochemical and microbiological characterizations of naturalhydrothermal fields strongly suggested that H2 is an importantenergy source in subsurface microbial ecosystems because ofits metabolic versatility. One of the possible sources of H2has been considered as earthquakes: mechanoradical reactionson fault surfaces generate H2 during earthquake faulting.However it is unclear whether faulting can generateabundant H2 to sustain subsurface chemolithoautotrophicmicroorganisms, such as methanogens.
55Culture 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 studiesMetabolic Characterization
56* * 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 applications1 mmimage courtesy of Gordon SouthamNovel Bacterial lineages unique to the SA deep-subsurface:South Africa Subsurface Firmicutes Groups (SASFiG)SASFiG-6SASFiG-5*SASFiG-4SASFiG-7SASFiG-3SASFiG-9*SASFiG-8SASFiG-1*SASFiG-9 (isolated)Detected within a water-bearing dyke/fracture at 3.2 Km depth.strictly anaerobic; iron-reduceroptimal growth temperature = 60 oCvirgin rock temp = ~ 45 oCSASFiG-2
57Key Experiments: Culture-Independent Evidence for Deep Life Genomic advancementsSequencing of a microbe required ~18 months in mid 90’sCurrently >150 microbes have been sequencedIn 2004 TIGR discovers 1.2 million new bacteria/archea genes in the Sargasso SeaBy 2005 JGI could sequence 400 microbes per yearCould early life in the subsurface havesurvived the Hadean bombardment?
58Earth’s subsurface microbial ecology The biosphere extends deep into the subsurfaceLimited by geothermal gradient and nutrient fluxBiomass generally low relative to the surfaceDistribution is very patchy and hetergenousRates of community metabolism very lowVolumetrically largest part of the biosphere
60Basalt: - 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
61Extent, Million Years Ago EonEraPeriodExtent, Million Years AgoPhanerozoicCenozoicQuaternary (Pleistocene/Holocene)Neogene (Miocene/Pliocene)Paleogene (Paleocene/Eocene/Oligocene)MesozoicCretaceousJurassicTriassicPaleozoicPermianCarboniferous (Mississippian/Pennsylvanian)DevonianSilurianOrdovicianCambrianProterozoicNeoproterozoicEdiacaranCryogenianTonianMesoproterozoicStenianEctasianCalymmianPaleoproterozoicStatherianOrosirianRhyacianSiderian
62HistoryThe Deccan Traps formed between 60 and 68 million years ago, 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.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 lifeThe 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.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).
64Core 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.