VTT TECHNICAL RESEARCH CENTRE OF FINLAND LTD

Microbiology

KYT2018 seminar 7.4.2017Minna Vikman, Hanna Miettinen,Leena Carpén, VTTRiikka Kietäväinen, GTK

2205/04/2017

Projects

§ Microbial sulphur cycle in final nuclear wasterepository conditions - Geobiokierto (Miettinen, VTT)

§ Microbiology related to geological disposal of low- andintermediate level waste - MAKERI (Vikman, VTT)

§ Microbially induced corrosion of low and intermediatelevel radioactive waste - CORLINE (Carpén, Rajala,VTT)

§ Nutrients, energy and gases in bedrock biosphere -RENGAS (Kietäväinen, GTK)

§ Microbiology is also studied in Coordinated projectCapsule (BASUCA, MICOR) Figure: Posiva

Figure:TVO

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Bacteria, archaea and fungi

§ have been around 3.8 billions years(long before plants and animals)§ can be found ‘everywhere’

§ deep subsurface environments to adepth of ~ 4 km

§ have adapted to very extreme anddiverse conditions§ temperature, pressure, radiation,

high salinity, pH…..§microbiological activity leaves

traces in the environment (water,gases, minerals)§ geochemistry§ isotopes

• Man-introduced microbes• Indigenous microbes

Image: Wikimedia commons

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Adaptation to extreme conditions

Deinococcus radiodurans:§ was isolated in 1956 from canned

meat that was sterilized by X-raysbut spoiled§ extremely resistant to ionizing

radiation, UV light, hydrogenperoxide etc.§ withstand an acute dose of

5 000 Gy (5 Gy kill a human)

Methanopyrus kandleri:§ was found at the bottom of a

“black smoker” chimney2,000 m deep, the Gulf ofCalifornia§ archaeon§ it can survive in

temperatures up to +120°C

Image: www.yale.edu

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What microbes need to grow?

Requirements:§Water (solvent of

biological reactions)§ Carbon source§Other essential elements:

N, O, H, P, S§ Trace elements etc.

(metals)§ Energyà electron

donors and acceptors§ Space to grow

Energy sources

Organic compounds(or other organisms)

Light

Organic chemicals Inorganic chemicalsGlucose, acetate H2, H2S, Fe2+, NH4

+

energy

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Gases at Outokumpu

Riikka Kietäväinen Mod

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• Up to 1.1 L gas / 1L ofwater

• Methane, nitrogen,hydrogen, helium,ethane, argon

•From water-rockinteraction, radioactivedecay and microbialprocesses

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Influence of microbes in final repositories

§ Produce gas (methane, CO2)§ Produce corrosive components (e.g. sulphide,

acetate)§ Enhance corrosion process§ Produce metabolites that form complexants

with radionuclides§ Change geochemical environment in

repository§ Change redox state of the radionuclides§ Can affect the performance of

engineered barrier materials§ Can influence the solubility, the

sorption and the mobility ofradionuclides

HLW:

Copper capsuleBentoniteConcrete

LLW and ILW:

DrumsWasteBitumen(Immobilization)Concrete

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Toolbox to study microbial function and activity

§ Sampling methods§ Facilities for aerobic and anaerobic

work in the laboratory§ Microscopic techniques§ Cell activation with specific substrates

and harvesting of activated cells§ Molecular techniques and

bioinformatics§ Population, group, genus,

species and strain levelidentification§ Specific functional groups and

functional genes§ Genomics, phylogenetics,

metagenomics andtranscriptomics

§What microbes are there?§ Bacteria§ Archaea§ Fungi

§What are they doing?

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Development of the safety case knowledge baseabout the influence of microbial processes on

geological disposal of radioactive wastes (MIND)2015-2018

• Swedish Nuclear Fuel and Waste• Management Co, SKB• Microbial Analytics Sweden AB, MICANS• Belgian Nuclear Research Centre, SCK•CEN• Helmholtz-Zentrum Dresden-Rossendorf e.V.,

HZDR• National Nuclear Laboratory Limited, NNL• Ecole Polytechinique Federale De Lausanne,

EPFL• Techniscka Univerzita, The Czech Rebublic• Centrum Vyzkumu REZ, The Czech Rebublic• University of Machester, UK• Universidad De Granada, Spain• TVO• Posiva• Geologian tutkimuskeskus• VTT

§ WP1: low and intermediatelevel wastes

§ WP2: high level waste andspent fuel

§ WP3: Integration,communication andDissemination

§ WP4: Project management

VTT TECHNICAL RESEARCH CENTRE OF FINLAND LTD

KYT 2018 GeobiokiertoMicrobial sulphur cycle in finalnuclear waste repository conditions

Hanna MiettinenMerja Itävaara

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Aims:§ To study microbiological mechanisms related to sulphide

formation in final nuclear waste repository conditions§ To evaluate the influence of microbes and their

metabolites on fysical structure of bentonite and itsperformace

Main achievements:§ Litterature review: 35S-tracer method for analyzing microbial

sulfur compound cycling in oligotrophic anoxic groundwaterhabitathttp://www.vtt.fi/inf/pdf/technology/2016/T249.pdf§ 35S tracer method for analyzing microbial sulphate

reduction§ Long-term lab-scale bentonite experiment

Geobiokierto

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35S tracer method for analyzing microbialsulphate reduction

§ 35S tracer method is very sensitive§ 300 x more sensitive than

chemical method§ Sulphate reduction started in

ground waters with lowconcentration of sulphate§ The tracer method can be used to

identify factors influencing sulphiteformation processes to find meansto control it

3.4 0.81 0.2 0.02mM/SO4

SRB sulphur oxidizersSO4

2- à H2S à SO42-

FeS, FeS2

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Microbial influence on bentonite(see poster)

Can microbes and theirmetabolites influence bentonitestructure in favorableconditions?Are these changes significant forthe bentonite stability in long-term scale?

Worst case scenario experiment• Bentonite (MX-80) slurry; not

compacted• Nutrients, crushed Olkiluoto graphite• Temperature 30/37°C• Aerobic (MIND) and anaerobic

conditions (KYT)• Analyses:

• Atomic force microscopy)• HAAD-STEM microscopy

(High-angle annular dark-fieldscanning transmission electronmicroscopy)

• Microbiology• Gas composition

VTT TECHNICAL RESEARCH CENTRE OF FINLAND LTD

Microbiology related to geologicaldisposal of low- and intermediatelevel waste (MAKERI)

Minna Vikman, Merja ItävaaraHanna Miettinen, Malin Bomberg, AtteMikkelson, Kaisa Marjamaa, Irina Tsitko,Mirva PyrhönenValokuva: Posiva

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Makeri

Aims:§ To evaluate the influence of environmental

conditions on microbiological degradation ofLLW/ILW, gas generation and theperformance of barrier materials

Research environments:§Gas Generation Experiment (GGE)§ Simulation experiments in the laboratory

§ Operational waste LLW§ Bitumen ILW

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Gas Generation Experiment (GGE)(Poster)

§ Located in TVO’s VLJ repository, inOlkiluoto

§ 16 waste drums (200 dm3) + concrete box +gas tight tank of acid proof steel (20 m2)

§ The tank was filled with river water§ Temperature is maintained in the level +8°C

-+11°C§ Monitored for quantity and composition of

generated gas, water chemistry andmicrobiology.

Gas production:§ potential to transport gaseous

radionuclides such as 14C to thebiosphere§ Pressurisation can influence

engineered barrier system of therepository and may increasegroundwater flow ratesà enhancedmigration of radionuclides

Small et al., 2008

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The Generalized Repository Model –Microbiological results

§ Most relevant microbial groups relatedto gas generation studied:§ Cellulose/hemicellulose

degraders§ Methanogens§ Microbes competing with

methanogens

§ The consistency of themicrobiological results withGRM model is evaluated(MIND)§Microbial community in

different compartmentsanalyzed§ Water (inside drums,

bottom of the tank, lid ofthe tank)§ LLW (waste capsules)§ Steel plates (waste

capsules)

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Corrosion of steel platesGGE capsules

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Tank Drum 1 Drum 2 Drum 3

Corrosion of steel plate

94% of cellulose-basedmaterial

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The influence of pH on microbial activity andgas generation

Laboratory experiment Large-scale gas generationexperiment

Extreme alkaline pH (pH > 12.5)is considered to limit microbialprocesses in repository conditions.

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Biodegradation of bitumen in final repositoryconditions

§Microbial degradation ofbitumen in finalrepository conditions ?§ The role of sulphate

reducing bacteria ?§ The influence of

concrete on microbialactivity?

Laboratory simulation experiment§ Bitumen Nynas 50/70§Groundwater from Olkiluoto§ Temperature +15°C§ Concrete§ Analyses

§ Microbial activity, ATP§ Gas formation§ Volatile fatty acids§ Microbial communities,

especially SRBs

VTT TECHNICAL RESEARCH CENTRE OF FINLAND LTD

Microbially induced corrosion of lowand intermediate level radioactive wasteCORLINE

KYT2018 7.4.2017Leena Carpén, Pauliina RajalaVTT

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Microbially induced corrosion of low and intermediatelevel radioactive wasteCORLINE

§ Technical goals:§ To create real-time measurement method for corrosion and in-situ

monitoring of water chemistry in drillholes§ To determine the effect of microbial activity on the corrosion rate

and mechanisms of decommisioning waste under Finnishrepository conditions§ To link formed corrosion products and gas composition to

corrosion phenomena

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CORLINEIn-situ corrosion monitoring

§ In-situ corrosion probes and water chemistrymonitoring devices were installed in two drillholes inTVO´s VLJ-cave 4/2016§ Planned monitoring time: ~2 years§ Studied materials:

§ Carbon steel, two stainless steels (EN1.4301, 1.4432)

§ Monitored parameter:§ Open circuit potentials§ Redox-potential (platinum)§ Corrosion rate (Linear polarization

resistance method)§ Water chemistry (anions, cations, total

metals, organic compounds)

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CORLINEIn-situ corrosion monitoring (Poster)

• The corrosion probes were designed and manufactured within thisproject at VTT

• The water samplers were designed in collaboration with TRL inc.

Corrosionprobes

Water samplers

Corrosionprobes

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CORLINEIn-situ corrosion monitoring

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CORLINELaboratory experiments

1. Long term corrosion experiment ingroundwater (electrochemistry, weight loss,microbiology)§ micro-organisms has role in corrosion

2. Gas formation related to microbial corrosion§ gas composition depends on steel material

3. Effect of growth media on corrosion§ experiment ongoing

4. Microbial electroactivity and its effects oncorrosion§ experiment ongoing

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CORLINEResults, long term laboratory experiment

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CORLINESummary

§ Successful design and installation of in-situ monitoring devices§ First measurements show differences in corrosion rate between

materials and also between different drillholes

§ Laboratory studies targeted to the effects of different microbialgroups on corrosion show that there are differences regarding ofthe microbial species composition

§Gas formation experiment indicate role of metanogenic archaeaon corrosion of carbon steel

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RENGAS – Nutrients,energy and gases inbedrock biosphereRiikka KietäväinenGeological Survey of FinlandEspooBedrock Construction andSite Assessment Unit

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RENGAS: overview of project goals andrelated activities at GTK

Riikka Kietäväinen

§ Geological,biological andengineeredenvironments§ Long term safety

of nuclear wastedisposal§ 14C release§ Cu corrosion§ Isolation of

deepgroundwaters

• Groundwater evolution andresidence time

• Mobility of dissolvedcomponents in the bedrock

• Energy sources for microbes• Method development

RENGASNutrients, energy

and gases inbedrock

biosphere

• Limits of microbiologicalsulphide formation (WP2)

• Importance of microbialreactions for the safety case(WP3)

MINDMicrobiology innuclear waste

disposal

KYT2018

Euratom/EU Horizon2020

• Formation/mobilization ofharmfulcompounds

• Time scales

• Importance ofmicrobiology

àRiskassessmentandmanagement

DataModels

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Evolution of deep bedrock groundwaters –model based on the study at Outokumpu

Riikka Kietäväinen

Kietäväinen, PhD Thesis 2017

South Africa

Canada

Fennoscandia1. Precipitation and infiltration at warmerclimate2. Isolation due to fracture minerals and crustaluplift3. Glaciations – no melt water infiltration4. Disturbance due to drilling and sampling(mixing and pressure release)

Time scales:

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Gases and nuclear waste disposal•CH4–rich groundwater atOlkiluoto• Gases may

• mobilise corrosive orradioactive compounds (e.g.sulphide, 14C)• increase microbial activity

• Gas migration may providetools to monitor changes inbedrock properties

• fracture density• deterioration/failure ofengineered barriers

Riikka Kietäväinen

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Riikka Kietäväinen

§ Energy densities (Er) inthe Outokumpu DeepDrill Hole for selectedreactions§ Sulphate reduction

combined withmethane oxidation(ANME)§ Autotrophic

methanogenesis§ Methanogenesis

from graphite andH2 up to 40 J/Lenergy available

Predicting CH4 formation pathways usingthermodynamics

Er = |ΔGr/vi|*[i]where i is thelimitingnutrient (notsolids)

9.12.2016

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Test, July 2016: Gas monitoring from the wellhead (results presented in our poster)

Riikka Kietäväinen

• Packer ~15 cm above the water table• Only slight pumping of gas• Correlation observed between Earth

tides and gas composition

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Sulphur in deep bedrock groundwaters

Riikka Kietäväinen

§ Mechanisms of sulphideformation traced by isotopegeochemical methods§ Good results from the

Pyhäsalmi mine§ Outokumpu Deep Drill Hole

samples more complex withpotential metastable S-compounds and matrixinterference from salts§ Method development still

needed

R-2247

R-2229

Sulphide Sulphate & Stot

Results from Pyhäsalmi:

Pyhäsalmidrill hole

Sulphate(mg/l)

Sulphide(mg/l)

TDS (g/l)

R-2229 145 0,1 43

R-2247 330 1,2 79

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Conclusions

§ Several new methods and devices are developed / underdevelopment§ In-situ corrosion monitoring probes§ Methods for analyzing sulphide formation§ Gas monitoring methods from the well head

§Geochemistry is strongly linked to microbiology§ Microbiology Geochemistry§ Deep bedrock ground water, final disposal repositories, Gas

generation experiment§ New data on the influence of microbes on

§ Corrosion, gas generation, bentonite structure etc.

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Example of publications

Itävaara, M., Salavirta, H., Marjamaa, K., Ruskeeniemi, T. 2016.Geomicrobiology and metagenomics of terrestrial deep subsurface microbiomes.In: Sariaslani, S. Gadd, G.M. (eds.), Advances in applied microbiology. Elsevier.94:1-77. doi: 10.1016/bs.aambs.2015.12.001

Kietäväinen R., Ahonen L., Niinikoski P., Nykänen H. & Kukkonen I.T. Abioticand biotic controls on methane formation down to 2.5 km depth within thePrecambrian Fennoscandian Shield. Geochimica et Cosmochimica Acta (inpress)

Rajala, P., Bomberg, M.,Huttunen-Saarivirta, E., Carpén. L.. 2017. Corrosion ofstainless steels AISI 304 and AISI 316 induced by sulfate reducing bacteria inanoxic groundwater. NACE CORROSION2017, C2017-9359.

Rajala, P., Bomberg, M. Vepsäläinen, M., Carpén, L.. 2017. Microbial foulingand corrosion of carbon steel in alkaline deep groundwater, Biofouling, 33(2):195-209

Small, J., Nykyri. M., Vikman, M., Itävaara, M. Heikinheimo, L., Thebiogeochemistry of gas generation from low-level nuclear waste: modelling after18 years study under in situ conditions, Applied Geochemistry, submitted

Vikman, M., Marjamaa, K., Itävaara, M. 2016. Microbiological degradation ofLLW under repository, Proceedings of MIND Project Annual Meeting, Granada,Spain, 3-4.5.2016. Luettavissa http://www.mind15.eu

383805/04/2017

Acknowledgements

§ KYT Finnish Research Programon Nuclear Waste Management§ Posiva Oy§ Teollisuuden Voima Oyj§ Fortum Power and Heat Oy§ Horizon 2020 project Microbiology

In Nuclear waste Disposal (MIND)through funding from the Euratomresearch and training programme2014-2018 under GrantAgreement no. 661880

Thank you for your attention