Microbiology in nuclear waste managementkyt2018.vtt.fi/kyt2018_loppuseminaari/Vikman...

Microbiology in nuclear waste management

KYT2018 seminar 29.1.2019

Minna Vikman, Hanna Miettinen,

Leena Carpén, VTT

Riikka Kietäväinen, GTK

Projects

Microbial sulphur cycle in final nuclear waste repository

conditions - Geobiokierto (Miettinen, VTT)

Microbiology related to geological disposal of low- and

intermediate level waste - MAKERI (Vikman, VTT)

Microbially induced corrosion of low and intermediate level

radioactive waste - CORLINE (Carpén, Rajala, VTT)

Nutrients, energy and gases in bedrock biosphere - RENGAS

(Kietäväinen, GTK)

• Microbiology is also studied in Coordinated project Capsule

(BASUCA, MICOR)

Figure: Posiva

Figure:TVO

Microbes

Microbes can be found ‘everywhere’

• Deep subsurface environments

Have been around 3.8 billions years

(long before plants and animals)

Microbes

• Bacteria

• Archaea

• Fungi

Most microbes 0.5 – 2 µm

Live in communities

11/02/2019 VTT – beyond the obvious 3

Where microbes come from:• Indigenous microbes

• Groundwater• Bedrock

• Man-introduced microbes

Microbes in final repositories

Produce gas (methane, CO2)

Produce corrosive components (e.g. sulphide, acetate)

Enhance/participate corrosion process

Produce metabolites that form complexants with

radionuclides

Change geochemical environment in repository (e.g. pH)

Change redox state of the radionuclides

Can affect the performance of engineered barrier

materials

Can influence the solubility, the sorption and the

mobility of radionuclides

Photo:Posiva

What microbes need ?


What microbes need?• Space to grow• Water availability• Major nutrients: C,H,N,O,P,S• Minor nutrients: e.g Fe, Ca, Mg,

Mn…• Available microbial energy: results

of oxidation and reduction• Certain environmental conditions

(pH, temperature etc.)

What can we do?• Restrict space: porosity >0.2 µm• Lower water availability • Limit supply of major and minor

nutrients (diffusion)• Create extreme physico-

chemical conditions (high pH, temperature etc.)

• Control microbial respiration to limit unbeneficial microbes

Information about microbial communities needed


Microbes can adapt to extreme conditions:

TemperaturepHSalinityPressureRadiationWater contentNutrient concentrationToxic compounds

Some bacteria can form spores that survive extreme conditions for long time


MicrobiologyGroundwater

Bedrock

Bentonite

Copper

Steel

Waste

Geology

Chemistry

Structuralanalytics

Photo: Posiva

Photos: VTT and commons wikimedia

Geobiokierto

Microbial sulphur cycle in final nuclear waste repository conditions

Hanna MiettinenMinna VikmanMirva PyrhönenMichał MatusewiczMalin Bomberg

Geobiokierto project had two 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 physical structure of bentonite and

its performance


Literature review:

35S tracer method for analysing microbialsulphate reduction in deep groundwater

35S tracer method for microbial sulphate reduction measurement developed

• 300 x more sensitive than the chemical method

Sulphate reduction detected also in deep groundwaters with low

concentration of sulphate

Sulphate reduction rate varies

between groundwaters

Sulphate reduction rate is affected

by addition of depleted electron

donors and acceptors

Master thesis on the method


Long-term microbial experiment with bentonite

2015 started with “worst case scenario” conditions

• Wyoming –type bentonite, Olkiluoto water, slurry, low concentration of electron

donors for microbes, sterilised control samples

• Anaerobic and aerobic start

Yearly studies of

• Chemistry (liquid phase: aluminium, silicon,

sulphate, iron; bentonite: CEC)

• Gas phase composition (N2, O2, CO2, CH4, H2)

• Microbiology (ATP, sulphate reduction 35S)

Bentonite is challenging material to study

• Method to extract DNA from bentonite developed

• Suitable methods to analyse bentonite structure screened for

• Atomic force microscopy of bentonite (AFM) and HR-TEM


Main results from long-term experiment

Microbial activity

• Generally microbes not very active based on (energy molecule ATP levels)

• Sulphate reduction ongoing in the anaerobic samples after one year

• Microbial activity seen as changes in gas concentrations (O2, H2)

• Levels microbes mostly at the same level or slightly decreased

as a function of time

• Energy for microbes levelling out due to the small sample

volume (80 mL) – not happening in real conditions

No changes in bentonite structure after two years of microbial storage

- If changes had been detected - a big problem

• Experiment will be continued in KYT 2022 - energy additions


MAKERI

Microbiology related to geological disposal of low- and intermediate level waste

Minna VikmanMirva Pyrhönen

Aims of MAKERI project:

To study the influence of microbial activity on

microbiological degradation of low- and

intermediate level waste, gas generation and

performance engineered barrier systems

Research environments

• Gas Generation Experiment (GGE) in VLJ

repository, Olkiluoto

• Simulation experiments in the laboratory

• LLW

• Bitumen


• Gaseous radionuclides (e.g.14C) can be transported to the biosphere in the form of methane gas (14CH4)

• Development of overpressure in the repository• Disruption of the engineered

barrier system (EBS)• Increase groundwater flow

rates

• Produced gas in the geosphere can enhance the activity of microbial communities

Microbial activity and pH

Extreme alkaline pH (pH > 12.5)

limits microbial activity in repository

conditions.

Microbial activity can reduce pH –

especially in heterogenous chemical

conditions.

Heterogenous chemical conditions in the GGE

In the beginning of the experiment

chemical conditions were

heterogeneous in different

compartments of the experiment

• Bottom level /Tank lid level

• Tank water / Inside drums

pH and dissolved organic carbon (DOC)

optimal niches for microbial activity

gas generation started after 1 year More methanogens and SRBs weredetected in bottom level of the tankon the basis of quantitative PCR

pH 7-8

pH 11

Gas generation

• Generation of methane require pH

within certain limit values and a low

sulphate concentration.

• Groundwater flow to the LLW

repository can reduce the gas

generation (activation of sulphate

reducers)

Hydrogen formed as a result of

corrosion of metals and in

biodegradation was utilized in

microbial in situ processes, and

hydrogen was not detected in the gas

phase.

Simulation experiment in the laboratory

Biodegradation of bitumen in finalrepository conditions

• The most relevant organic components in

ILW in Finland are ion exchange resins and

bitumen used for encapsulation.

• Organic part of ILW can degrade

microbiologically, chemically, by radiation, or

by combination of them.

• Bitumen contains aliphatic, aromatic and

heterocyclic hydrocarbons, which are shown

to be biodegradable at least in certain

environmental conditions.

• No significant microbiological

degradation of bitumen was observed

• Microbial acitivity was slighly

increased in bitumen containing

samples

Results and achievements

New information about microbiological degradation of LLW and

about factors affecting the gas generation

• Microbiological monitoring of GGE

• Simulation experiments in the laboratory with LLW and bitumen

International co-operation with National Nuclear Laboratory in

modelling of GGE

• Two manuscripts

• Presentation in Goldschmidt 2017-conference


CORLINE

Microbially induced corrosion of low and intermediate level radioactivewaste

Leena Carpén, Pauliina Rajala, Elisa Isotahdon VTT

Aims of CORLINE project

1. Development of reliable online in-situ methods for measuring

corrosion and water chemistry in drill holes

2. Determination of microbial action and it’s effect on corrosion rate

and mechanism of decommissioning metal waste in final

repository conditions of Finland

3. Exploitation the more throughout analysis of formed corrosion

products and gases in order to improve the understanding of

microbially induced corrosion


Examples of corrosion studies

Long term electrochemical studies on carbon

steel and stainless steels with and without

bacteria (acetogens, methanogens, SRB)

• Instantaneous corrosion rates in different

environments (vs. average rates by weight loss)

• Corrosion mechanisms

Sample characterizations after the tests

• Biofilm

• Corrosion products


Gas analysis and microbiology

Gas detection (CH4, CO2) in co-operation with Helsinki University

Metagenomics of drill hole water

Sequencing of biofilm

qPCR


Microbes and corrosion product on surface of carbon steel exposed to groundwater with

SRB and acetogen amendment (left). The number of bacterial 16S rRNA and dsrB genes

and archaeal 16S rRNA genes per cm2 or mL estimated by qPCR (middle). The average

corrosion rates of carbon steel with different amendments (right).


Field studies in VLJ cave In-situ measurements in drill holes

Electrochemical measurements for online

measurements of steel sample behaviour

OsmoSampler collecting water samples

water chemistry connected to corrosion rates


Results and achievements

Corrosion evaluation for carbon steel and the most commonly used

stainless steel grades (304 and 316) in relevant microbial environments

were done

• Corrosive effect of different microbes discovered

Results may be used to estimate the behaviour of decommissioning

waste

VLJ cave sample metagenomics – relevant metabolism routes were

found

In-situ measurements in drill holes were successful

International co-operation with DCO in metagenomics, Jamstec:

Hybridized Chain Reaction Fluorescent in situ Hybridization (HCR-FISH),

Stirling University (Mössbauer analysis)


RENGAS – Nutrients, energy

and gases in bedrock

biosphereRiikka Kietäväinen

Geological Survey of Finland

Espoo

Bedrock Construction and

Site Assessment Unit

WP1: Method development

Métivier et al. EPSL 200929.1.2019Riikka Kietäväinen

Monitoring of spontaneous gas flux from the bedrock at Outokumpu

Correlation between gas composition and solid earth tides

Potential as a monitoring tool in the repository: changes in gas flux

can indicate changes in e.g. fracture density, deterioration of

engineered barriers

29.1.2019Riikka Kietäväinen

Model of deep groundwater evolution Compilation and new determinations of groundwater

residence times• Pyhäsalmi mine, 160 Ma

Better understanding of time scales of (bio)geochemical processes, long term safety

South Africa

Canada

Fennoscandia

Kie

tävä

inen

, 20

17

Ph

DTh

esis

WP2: Evolution and residence times of bedrock

groundwaters

WP3: Sulphur in deep bedrock groundwaters

Study of sulphide production using stable sulphur isotopes

Observed isotope fractionation consistent with microbial sulphate reduction

Differences between the sites could reflect differences in the SRB populations (complete vs. incomplete oxidation of organic carbon or use of H2 as an electron donor)

Importance for corrosion


”Sulphate” = bulk – sulphide

Ion exchange resins used to separatesulphur from the samples

Oxidation:

Carbonlimitation:

Mixing:

Dia

gram

afte

rEt

iop

e2

01

7

WP4: Carbon in deep bedrock groundwaters

Abiotic isotope signal in methane

Could be also due to carbon limitation and isolation

In the upper 1 km depth considerable potential for microbial methanogenesis

High and continous CH4flux observed (two drill holes, total flux 40.000 l/day)

14C mobilisation,

microbial activity


1Purkamo et al. 2016, methanogenesis rate: 2Colwell et al. 2008, 6,2*10-12 mmol/cell/a, 3Simkus et al. 2016, 1,5*10-11 mmol/cell/a

Depth Methanogens Residence time Microbial CH4 Microbial CH4

Thermodynamic viewpoint

Case study: Outokumpu Deep Drill

Hole

Direction (Gibbs energies, ΔGr)

Energy densities (Er)

take the limiting compounds

into account

Methanogenesis from graphite and

H2 the most energetically favourable

reaction

Sulphate reduction is limited by

sulphate availability

Energy densities are low

(1/1000 of those find at ocean

floor sediments)

WP5: Synthesis of biogeochemical reactions in bedrock

groundwater


Dep

th (

m)

Dissertations and thesis

Pauliina Rajala, PhD thesis: Microbially-induced corrosion of

carbon steel in a geological repository environment (2017), VTT

Science, 155 p.

Kietäväinen Riikka, 2017. Deep Groundwater Evolution at

Outokumpu, Eastern Finland: From Meteoric Water to Saline Gas-

Rich Fluid. PhD Thesis, University of Helsinki, Geological Survey of

Finland Special Publication 97, 150 p.

Anna Manninen, Biological sulphate reduction potential in waters

from the geological repository site for nuclear waste disposal in

Olkiluoto, Finland.

Heikkinen Nina, 2016. Kaasujen liukoisuus Outokummun

syväkairareiän suolaisessa pohjavedessä. MSc thesis, University of

Helsinki, Department of Geosciences and Geography, 77 p.

Publications

Bomberg, M., Raulio, M., Jylhä, S., Mueller, C.W., Höschen, C., Rajala, P., Purkamo, L., Kietäväinen, R., Ahonen, L.

Itävaara M., 2017. CO2 and carbonate as substrate for the activation of the microbial community in 180 m deep

bedrock fracture fluid of Outokumpu Deep Drill Hole, Finland. AIMS Microbiology 3, 846-871.

Carpén, L., Rajala, P., Bomberg, M. 2015. Microbially Induced Corrosion in Deep Bedrock. Advanced Materials

Research Vol. 1130 (2015) pp. 75-78.

Kietäväinen R., Ahonen L., Niinikoski P., Nykänen H., Kukkonen I.T., 2017. Abiotic and biotic controls on methane

formation down to 2.5 km depth within the Precambrian Fennoscandian Shield. Geochimica et Cosmochimica Acta

202, 124-145.

Miettinen, H., Kietäväinen, R., Sohlberg, E., Numminen, M., Ahonen, L., Itävaara, M. 2015. Microbiome composition

and geochemical characteristics of deep subsurface high-pressure environment, Pyhäsalmi mine Finland. Frontiers

in Microbiology. 6:1203. doi: 10.3389/fmicb.2015.01203

Miettinen, H. 2016. 35S-tracer method for analyzing microbial sulfur compound cycling in oligotrophic anoxic

groundwater habitat. VTT Technology 249.

Miettinen, H., Bomberg, M., Vikman, M. 2018. Acetate activates deep subsurface fracture fluid microbial

communities in Olkiluoto, Finland. Geosciences. 8(11), 399. doi.org/10.3390/geosciences8110399


Publications

Nuppunen-Puputti M., Purkamo L., Kietäväinen R., Nyyssönen M., Itävaara M., Ahonen L., Kukkonen I.,

Bomberg M., 2018. Rare biosphere archaea assimilate acetate in Precambrian terrestrial subsurface at 2.2 km

depth. Geosciences 8, 418, doi:10.3390/geosciences8110418

Purkamo L., Kietäväinen R., Miettinen H., Sohlberg E., Kukkonen I., Itävaara M., Bomberg M., 2018. Diversity

and functionality of archaeal, bacterial and fungal communities in deep Archaean bedrock groundwater. FEMS

Microbiology Ecology 94, doi: 10.1093/femsec/fiy116

Rajala, P., Bomberg, M. Vepsäläinen, M., Carpén, L., 2017. Microbial fouling and corrosion of carbon steel in

alkaline deep groundwater, Biofouling, 33(2): 195-209.

Small, J., Nykyri, M., Vikman, M., Itävaara, M., Heikinheimo, L., The biogeochemistry of gas generation from

low-level nuclear waste: Modelling after 18 years study under in situ conditions, Applied Geochemistry.

Elsevier. Vol. 84 (2017), 360-372.

Vikman, M., Itävaara, M., Carpén, L., Matala- ja keskiaktiivisen ydinjätteen loppusijoituksen mikrobiologiset

riskit Suomessa, 2016. VTT Technology 273, 41 s. ISBN 978-951-38-8457-4 http://www.vtt.fi/julkaisut

Vikman, M., Marjamaa, K., Itävaara, M., Nykyri, M., Small, J., Paaso, N., Microbial degradation of low-level

radioactive waste in repository conditions, presentation, Goldschmidt 2017, 13 - 18 August 2017, Paris, France

2017. European Association of Geochemistry.


Acknowledgements KYT Finnish Research Program on Nuclear Waste Management

Posiva Oy

Teollisuuden Voima Oyj

Horizon 2020 project Microbiology In Nuclear waste Disposal (MIND) through

funding from the Euratom research and training programme 2014-2018 under

Grant Agreement no. 661880

Istituto Nazionale di Geofisica e Vulcanologia (INGV) Rome/Giuseppe Etiope

Deep Carbon Observatory (DCO)

First Quantum Minerals/Pyhäsalmi Mine

Callio Lab

International Continental Scientific Drilling Program (ICDP)

GFZ Potsdam

KYT2018 THEBES project

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