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

Merja Itävaara, VTT

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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

Merja Itävaara, VTT

5

Isolate (pure culture)

Genomics

Microbial community

Metagenomics

Metagenomics to study uncultivableOnly 1-5% of microbes are cultivable

Courtesy of Susannah Green Tringe, DOE JGI

6

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)

Merja Itävaara, VTT

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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,

Merja Itävaara, VTT

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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

Merja Itävaara

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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

Merja Itävaara

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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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