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
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 - PowerPoint PPT Presentation
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Exploring microbial diversity and function within the granitic-basaltic deep crustal system of Koyna-Warna (India) region
Pinaki SarDepartment of Biotechnology
Indian Institute of Technology KharagpurIndia
CollaboratorsSufia 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
Microbiology of deep, igneous crust seems more intriguing, though relatively less studied
Microbiology of basaltic/grantic deep subsurface (marine/terrestrial) are less studied and mostly unexplored
Some more reports for ocean crust than terrestrial habitats
Who are they ?What are their function
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
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
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 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
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
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
Abiogenic H2 driven metabolic pathways ? Role in C/N/nutrient cycling Rock weathering and climate change
Geomicrobial processes at a subsurface shale-sandstone interface; Fredrickson and Balkwill, 2006
XX
??Acetogenic –Methanogenic metabolism with
abiogenic H2
Acetogenic –Methanogenic metabolism with abiogenic H2
In igneous rock systemsIn igneous rock systems
H2 driven system
Abiotic diagenetic formation of low mw compounds
Anaerobic lithoautotrophic metabolismSLiMEs (?)
Anaerobic lithoautotrophic metabolismSLiMEs (?)
Small Org compSmall Org compMethanogenMethanogenAbio
tic p
roce
sses
Tem
pera
ture
Abiotic geogenic H2
Anaerobic heterotrophic metabolism
Anaerobic heterotrophic metabolism
H2
N2 fixationN2 fixation
Denitrification/NH4 oxidation
Denitrification/NH4 oxidation
Radiolytic decomposition of waterWater-rock interactionDiffusion from deeper levels
Radiolytic decomposition of waterWater-rock interactionDiffusion 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 byKoyna Dam in Western India in 1962
Drilling site at Koyna
JOUR.GEOL.SOC.INDIA, VOL.81, FEB. 2013
Drilling is proposed up to nearly 7 KM, so far ~1.5KM drilling is doneDrilling 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
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
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.
ObjectivesAnalysis 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
Drilling, sample collection and
analysis
Drilling, sample collection and
analysis
Analysis of microbial diversity, community structure, abundanceAnalysis of microbial diversity, community structure, abundance
Analysis of microbial functionAnalysis of microbial function
Elucidation of effect of seismic activity and crustal properties on microbial diversity and activity
Elucidation of effect of seismic activity and crustal properties on microbial diversity and activity
Data integration and modelingData integration and modeling
Time scale (year)0 5
Obj . I Obj . II Obj . III
Molecular genomic analysis
Molecular genomic analysis
DeliverablesDeep 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 weatheringNutrient cycling, CO2 sequestration and other aspects of
climate changeBiomineralization; Bioremediation, Bioprospecting (Access of novel microbes and enzymes for industrial application)
Global significance : Global primer site of RTS within basaltic/granitic crust
Microbial role in rock weatheringNutrient cycling, CO2 sequestration and other aspects of
climate changeBiomineralization; 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 65,000Drilling 1,50,000
Chemicals/Consumables, contingency 2,00,000
Staff (01 PDF, 02 RF, 01 RA) 1,20,000International/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
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)
The deep biosphere : an extreme habitat for microbes
Increasing temperature and pressureNutrient limitation, limited porosity and permeabilityDecreasing available carbon and energy sources
With increasing depth there are several constrains that affect composition, extent, life habitats, and the living conditions in deep subsurface
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 :
1. kind of microorganisms ? populate the deep subsurface?
2. their extension and limits? 3. metabolic processes ? carbon and energy sources ? 4. survival strategies? link to early life on Earth? 5. biological alteration of rock 6. impact on the global -biogeochemical cycle and -
climate?
ICDP 2010
1. Nature of microbial communities and their function in active seismogenic zone
2. Effect of fracturing (during earthquake) on microbial communities
3. Interrelation between geochemistry, microbiology and nature/location of fracture zones
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
Requirements of microbes in deep biosphere
•Electron donor•Electron acceptor•Carbon source•thermodynamic potential of chemical reactions
•Electron donor•Electron acceptor•Carbon source•thermodynamic potential of chemical reactions
Sub-Total of Recurring 2012000 1752000 1392000 5156000
Grand Total (Non-Recurring + Recurring)
4512000 1752000 1392000 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 systemwith 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
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).
•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
Environmental parameters defining the dimensions of living space
Living spacePore 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
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.
Supply of food
Microbial distribution in geospheres
Extension of the biosphere on Earth
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
Out come of deep borehole studies by ICDP and/or IODP
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
To be added in end
The original chemical composition of the sediment
Response of microbes and its organic and inorganic components to increasing temperature
Availability of liquid water
Potential limiting factors for microbes in deep biosphere
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.
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.
Microbiology of seismic zones
Wanger et al 2007
Isolation of pure culture bacteria
(different enrichment cond., aerobic and anaerobic cond.)
Isolation of pure culture bacteria
(different enrichment cond., aerobic and anaerobic cond.)
Metabolic Characterization
Metabolic Characterization
Metal resistance and transformation
studies
Metal resistance and transformation
studies
Identification (16S rRNA gene, FAME, API, etc.)
Identification (16S rRNA gene, FAME, API, etc.)
Culture dependent analysis
*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-1
SASFiG-2
SASFiG-3SASFiG-4
SASFiG-5
SASFiG-6
SASFiG-7SASFiG-9
SASFiG-8
*
image courtesy of Gordon Southam
What have we learned?Novel indigenous microbes and communitiesNovel and unusual deeply branched sequences may be indicative of ancestral linkages, (early life?), Novel products for biomed and biotech applications
Novel Bacterial lineages unique to the SA deep-subsurface:South Africa Subsurface Firmicutes Groups (SASFiG)
1 m
Key Experiments: Culture-Independent Evidence for Deep Life
Could early life in the subsurface have survived the Hadean bombardment?
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
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
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
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.