KYT seminar 18.3kyt2014.vtt.fi/.../Itavaara_KYT2014_loppuseminaari.pdf · 2015-05-06 · -...
Transcript of KYT seminar 18.3kyt2014.vtt.fi/.../Itavaara_KYT2014_loppuseminaari.pdf · 2015-05-06 · -...
Microbiology research in KYT2014
Merja ItävaaraVTT Technical Research Centre of Finlandwww.vtt.fi
KYT seminar 18.3.2015
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ContentsIntroduction
- Microbiology in repositories – why microorganisms and their functionsshould be studied
Microbiology research in KYT2014 (2010-2014)
GEOMICRO VTTSALAMI GTK (2011-2014)GEOBIOINFO Aalto Deep BIOSPHERE/GEOSPHERE
BOA microbes in bentonite, VTT (2011-2014) BUFFER
KKK-koe (Gas generation of low radioactive waste) (2013-2014)VTT
REMIC (Corrosion of demolition waste) (2012-2014) VTT LLW
Conclusions Merja Itävaara, VTT
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IGD-TP Implementing Geological Disposal ofRadioactive Waste Technology Platformhttp://www.igdtp.eu/
Euratom/Mind project (2015-2019)to support the implementation of planned geological disposalprojects for higher-level radioactive wastes across the EU.“high urgency” and “high importance” topics identified in themost recent IGDTP Strategic Research Agenda, focusingspecifically on the influence of microbial processes on wasteforms and their behavior, and the technical feasibility and long-term performance of repository components.15 European groups working on the impact of microbialprocesses on safety cases for geological repositories across theEU, focusing on key questions posed by waste managementorganisations
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IntroductionWhat microbes can do in the repositories?
Microbes can- degrade organic C and can disintegrate constructionmaterials- utilize and generate gases- migrate radionuclides- change geochemistry- oxidize and reduce metals- form hydrogen sulphides which are corrosive for steel
and copper- change redox state of radionuclides
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Isolate (pure culture)
Genomics
Microbial community
Metagenomics
Metagenomics to study uncultivableOnly 1-5% of microbes are cultivable
Courtesy of Susannah Green Tringe, DOE JGI
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Opening the genetic code - Sequencing
Reading the nucleotide structure (A, G, T, C) of thegenome or metagenome
Sequencing technologies- Sanger sequencing- Next generation sequencing (high throughputsequencing):
454 pyrosequencing,
Illumina sequencing; HiSeq,MiSeq. Next Seq
Ion Torrent PGM,
PacBio RS
Oxford Nanopore(Life technologies)
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Bioinformatics for highthroughput data analysisFast development of tools ongoing
What species are there?Bacteria and Archaea, 16S rRNA,Fungal diversity, ITS region
Blast, Mothur, Geneious, Green genes,Bionumerics, Giime pipeline for moreautomatic large data analysis
What are they doing?Total sequencing of the whole community tostudy metabolic pathways:
IMG/M, MG-RAST, Megan, Uniprot,KEGG,etcNew pipeline developed recently in HUMAnNmicrobiome project (Abubucker, S et al. 2012, Plos Computationalbiology 8, 6)
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Future research is focusing on
OMIC’s approach in Systems Biology of livingorganisms
Metatranscriptome
Metagenome
Metaproteome
Metabolome
Metabolism
Identification of enzymesinvolvedin metabolite transformation
Accurateannotation ofORFs
Link with cognatemetabolic pathways
Final cellularoutput
Maria-eugenia Guazzaroni and Manuel Ferrer 2011. Handbook of Molecular Microbial Ecology, Vol. IMetagenic approaches in systems biology, Chapter 54
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QUANTIFICATION
qPCR
16S rRNA
Functional genes
FUNCTION
DIVERSITY
DGGE,functionalgenes,
454 pyrosequencing
Radiolabelledsubstrates, SIP,
Geomicrobiology
VTT
INTERACTION
Total sequencing,
Metabolic pathways’
Competences developed in the microbiology projects
Geosphere/biosphereDeep biosphere consortium
SALAMI, GEOMICRO, GEOBIOINFO 2011-2014
GEOMICRO M.Itävaara/VTT-Microbial sampling-Microbial diversity anjd function,highthrouhput sequencingi-Anaerobic methane oxidation-Chemical small molecules ingroundwaters
SALAMI GTK Ilmo Kukkonen, LAhonen-Deep groundwater samplingtechniques-Geochemistry, gases, isotopes-Online gas analysis
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Scientists involved at VTTMicrobial diversity
Malin BombergMari NyyssönenLotta PurkamoMaija Nuppunen-Puputti,Pauliina Rajala,Mari RaulioLeea OjalaHanna MiettinenMinna VikmanElina Sohlberg
Metapathway analysis andenzymes
Heikki SalavirtaKaisa MarjamaaPeter BlombergAntti NyyssöläMikko ArvasMerja OjaFahad Syed
Funding:
Finnish National Research Program for nuclearwaste disposal (KYT2010, KYT 2014)
Finnish Academy; Projects 1. Deep Life, 2. Deepmetapathway
Finnish Natural Science FoundationPh.D grant
Posiva Ltd.TVO Ltd.
Collaboration:GTK, Prof. Ilmo Kukkonen (HY),Lasse Ahonen, Riikka Kietäväinen
Aalto University Prof. JuhoRousu,
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Deep biosphere and major processes
Bacteria, archaea, fungi, viruses, nematodes
Chemolithotrophic processesIron and sulphur oxidation and reductionMethane production, methane oxidationHydrogen use and productionNitrogen fixation, ammonia oxidation, denitrificationHeterotrophic, organic compound degradationCarbon dioxide fixation
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GoalsTo Characterize microbialdiversity of FennoscandianShield by highthroughputsequencing
Combine geochemistryand geology to microbialdataTo estimate the majormetabolic functions basedon whole genomesequencing and metabolicnetwork analysis
Merja Itävaara, VTT
1430/04/2015Sampling sites (2006-2014)To provide knowledge about microbial diversity andmetabolism in Finnish bedrock aquifers
Palmottu
Olkiluoto300-1000m
Outokumpu2.5km
Pyhäsalmicave, 2 km Kuhmo
600m
Metapeltic rocks (mica gneiss) representingoriginal marine clay deposition (sulphidespresent), metamorphosed and associatedwith intrusives (granite, tonalite)
Major sitesOutokumpu deep borehole (2.5 km) Cu-Zn-Ni-Co-sulphide ore province,2.5 km, Geolaboratory, research boreholeOlkiluoto: Nuclear waste disposal site, severaldrillholes 300-1000m, 15 boreholes
Itävaara et al. 2011. . DOI:10.1111/j.1574-6941.2011.01111.x
Kietäväinen, R., et al. 2013 and 2014. doi.org/10.1016 j.apgeochem.22012.10.013
Purkamo et al. 2013 and 2014 DOI: 10.1111/1574-6941.12126 and doi 10.1007/s00248-014-0490-6
Rajala, P. et al. 2015. doi:10.3390/microorganisms3010017
Outokumpu deep borehole studies
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Anaerobic sampling in Outokumpu (deep borehole 2.5km)Sampling every summer 2007-2012
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Development of sampling techniques in KYT2014
Tube sampling, samples from 50 tai 100mPumping water samplesPressure samplesBiotrapsFiltration of large water samples fromPyhäsalmi cave, decrease in pressuresystems
Outokumpu: On-line monitoring (pH, EC,T, O2, Eh)Measurement of gas evolution
from the head space of the borehole30l/day, major gas methane
Sites:Outokumpu deep borehole 2.5 kmKuhmo, Romuvaara 600mPyhäsalmi cave ca 2 km
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PyhäsalmiSEM & EDX analysis
Very small number of cells observedA lot of crystals which plugged the filterHigh pressure problems in sampling
Kuvat Mari Raulio
Saline fluids, gases and microbes incrystalline bedrock -SALAMI
• Sampling for representative gas and microbiological samples
Pressurisedcylinder
Pressuregauge
Vacuumpump
Ultrasonicbath
Sampling in the Pyhäsalmi mine(-1430 m)
Gas separation in a vacuum line
Sampling at the Outokumpu Deep Drill Hole (2.5 kmdeep)
KYT2014 18.3.2015 Riikka Kietäväinen
Modified after Ballentine & Burnard (2002) and Ballentine et al. (2002)
SALAMI: Residence times of deep groundwaters
• Concentration of crustalnoble gas nuclides
• Concentration of U, Thand K + physicalproperties of thebedrock
accumulation rate• Noble gas components
• 3He/4He 1.5 · 10-8
No mantlecomponent
Mantle •In Outokumpu the averageresidence time 30 million years(Kietäväinen et al. 2014, GCA)
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Terminal electron acceptors in anaerobic methane oxidation
a) Mangane CH4 + 4MnO2 + 7H+ HCO3- + 4Mn2 + 5 H2O
b) Iron CH4 + 8 Fe(OH) 3 + 15H+ HCO3- + 8Fe2+ + 21 H2O
c) Nitrite: 3 CH4 + 8NO2- + 8H+ 3CO2 + 4N2 + 10 H2O
d) Nitrate: 5 CH4 + 8NO 3- + 8H+ 5CO2 + 4N2 + 14 H2O
e) Sulphate: CH4 + SO42- HS- + HCO3
- + H2O
Anaerobic methane oxidation- risk for nulcear waste safety at the disposal site- may occur at sulphate methane transition zone- until now has been observed in sediments at sea bottom- in our studies we suggest that is connected to connection of methylotrophic,
and sulphate reducing microbial community interactions
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Outokumpu deep borehole research
Merja Itävaara, VTT
What has been done- the diversity of microbial communities each 100m depths- pathways (3 depths)- borehole water and fracture microbiology
**
**
*
**
1 2 3 4
**
**
*
**
1 2 3 4
0 % 10 % 20 % 30 % 40 % 50 % 60 % 70 % 80 % 90 % 100 %
OUTOII 150-250 m
OUTOII 550-650m m
OUTOII 950-1050 m
OUTOII 1050-1150 m
OUTOII 1450-1550 m
OUTOII 1850-1950 m
OUTOII 2250-2350 m
Flavobacteria
Gammaproteobacteria
Actinobacteria
Alphaproteobacteria
Mollicutes
Clostridia
Betaproteobacteria
unclassified_Bacteria
Deltaproteobacteria
Erysipelotrichi
Anaerolineae
Sphingobacteria
0 % 10 % 20 % 30 % 40 % 50 % 60 % 70 % 80 % 90 % 100 %
OUTOII 150-250 m
OUTOII 550-650 m
OUTOII 950-1050 m
OUTOII 1050-1150 m
OUTOII 1850-1950 m
OUTOII 2250-2350 m
MethanolobusMethanomethylovoransMethanobacterium
Methanosarcinaunclassified_Methanobacteriaceae
unclassified_Euryarchaeotaunclassified_Archaea
unclassified_Methanosarcinaceaeunclassified_Methanomicrobia
unclassified_Methanobacteriales
Bacteria
Archaea
DGGE and 454pyrosequencing
Whole genomesequencing andpathways (MG/Rast)
Deep metapathways:
- Energy metabolism based onmetagenomic data
- What are the major metabolicpathways driving the community
Itävaara, M., Nyyssönen, M., Kapanen, A.,Nousiainen, A.,Ahonen, L., Kukkonen, I. 2011. Characterization of bacterialdiversity down to a depth of 1500 m of the Outokumpu deepborehole. FEMS Microbiology 2011, 1-15,.DOI:10.1111/j.1574-6941.2011.01111.x
Nyyssönen, M. Hultman, J., Ahonen, L., Kukkonen, I., Paulin, L., Laine, P., Itävaara, M. andAuvinen, P. 2014.. Taxonomically and functionally diverse microbial communities in deepcrystalline rocks of the Fennoscandian shield" - ISME J, 8: 126–138;doi:10.1038/ismej.2013.125.
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Statistical analysis of Sulphate reducers,geochemistry and geophysics
Viivi Uurtio graduationthesis
Supervised by Prof.Juho Rousu/Aalto Yliopisto
Sites compared:
Olkiluoto,Outokumpu,Nummi-Pusula
Merja Itävaara, VTT
KKK-koeBiodegradation of low-level
radioactive waste ingeological disposal
Merja Itävaara, Minna Vikman, Kaisa MarjamaaVTT Technical Research Centre of Finland Ltd
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Operating wastegenerated during operation and maintenance
Low level:Paper, cardboard, cottonFire-protected fabricsPlastic wrappings and protectiveclothingMachinery parts and pipes
59% cellulose-based material
Silo for low level radioactive waste
Photo: Posiva Oy
Picture: Small et al., 2008Merja Itävaara, VTT
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Microbiological risks in geological disposal ofLLW
Methane evolutionOrganic C causeaccelerated biocorrosionand increased activity of
microbesMicrobial metabolites
Migration ofradionuclides
Low-level waste:Cellulose-based waste:paper, cardboard, cotton,fabrics etc.
Others:PVC, PE, rubber, metals etc..
radiation
Environmentalconditions (pH)
microbes
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Results
Anaerobic biodegradation of LLW waste is ongoingConstant biogas formationMethanogens detectedMicrobial groups related to the hydrolysis of cellulosic materials
Biodegradability of LLW was below 5% in 2013The heterogenity in environmental conditions in different parts ofthe tank can also be seen in microbial activity and microbialdiversity
highest microbial activity in the drum containing mostly biodegradablewasteCorrosion of steel plates was proceeding more rapidly in the capsulescontaing biodegradable waste
Merja Itävaara, VTT
Mikrobiologisen korroosionriskit Suomen loppusijoitus-olosuhteissa (REMIC)Leena Carpén, Pauliina Rajala, MalinBombergKYT2014 Loppuseminaari18.3.2015
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TavoitteetHankkeen tavoitteena oli arvioida biofilmien muodostumista ja mikrobiologisenkorroosion riskiä metallisille materiaaleille (purkujätemetallit) Suomen loppusijoitus-olosuhteissa sekä kehittää luotettava lopusijoitusolosuhteita simuloiva koejärjestely.
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Mikrobiologiset tulokset – betonin ja ravinteet
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
A
B
A
B
Concrete A
Concrete B
Glucose A
Glucose B
Methane A
Methane B
Abio
ticAb
iotic
Biot
icBi
otic
Biot
icBi
otic
Bio
ticBi
otic
Biot
icBi
otic
Alphaproteobacteria Deltaproteobacteria BetaproteobacteriaSphingobacteriia Ignavibacteria CytophagiaGammaproteobacteria Actinobacteria Acidobacteria_Gp6Zetaproteobacteria Clostridia VerrucomicrobiaeArmatimonadetes_gp4 SR1_genera_incertae_sedis AnaerolineaeCaldilineae Flavobacteriia ParcubacteriaEpsilonproteobacteria Negativicutes BacteroidiaPlanctomycetia Subdivision3 Acidobacteria_Gp16Anaerolineae Chlorobia HolophagaeNitrospira Spirochaetia AquificaeAnaerolineae Bacilli Bacteroidetes_incertae_sedisErysipelotrichia Mollicutes
Mikrobit korroosiokuopassaKuva: Mari Raulio
• Ravinteiden lisäys muutti bakteeriyhteisönkoostumusta ja kiihdytti hiiliteräksenkorroosiota
• Betoni inhiboi biofilmin muodostumista jahidasti korroosiota, mutta aiheuttivoimakkaammin korroosion paikallistumista
Betaproteobacteria
Deltaproteobacteria
Alphaproteobacteria
Bacilli
Bacilli
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Yhteenveto tuloksista
Mikrobilajistot hyvin erilaisia paikkakohtaisesti:Rakovyöhykkeissä erilainen lajisto kuin kairareiässä (Purkamo et al.2013)Sienilajistoja runsaasti, niiden toiminta on huonosti tunnettu geologisissaolosuhteissa, monet happoja tuottavia
Mikrobien aktiivisuus on erittäin hidas geologisessa ympäristössä.kuitenkin muutokset elektroniakseptorien saannissa voivat aiheuttaanopeita aktiivisuuden muutoksia esim. metaani pulssi maan kuoresta
Ihmisen muokkaama geologinen ympäristö altistuu mikrobien lisääntyvälletoiminnalle ja vaikutukset huonosti tunnettuOrgaanisen hiilen esiintyminen edistää biokorroosiotaMikrobit voi muuttaa ympäristönsä kemiaa, pH:ta ja lämpötilaaGeologia, geokemia, paine, syvyys, lt, geokaasut
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