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 ?
11/02/2019 VTT – beyond the obvious 5
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
11/02/2019 VTT – beyond the obvious 6
Microbes can adapt to extreme conditions:
TemperaturepHSalinityPressureRadiationWater contentNutrient concentrationToxic compounds
Some bacteria can form spores that survive extreme conditions for long time
11/02/2019 VTT – beyond the obvious 7
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
11/02/2019 VTT – beyond the obvious 9
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
11/02/2019 VTT – beyond the obvious 10
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
11/02/2019 VTT – beyond the obvious 11
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
11/02/2019 VTT – beyond the obvious 12
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
11/02/2019 VTT – beyond the obvious 14
• 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
11/02/2019 VTT – beyond the obvious 19
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
11/02/2019 VTT – beyond the obvious 21
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
11/02/2019 VTT – beyond the obvious 22
Gas analysis and microbiology
Gas detection (CH4, CO2) in co-operation with Helsinki University
Metagenomics of drill hole water
Sequencing of biofilm
qPCR
11/02/2019 VTT – beyond the obvious 23
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).
11/02/2019 VTT – beyond the obvious 24
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
11/02/2019 VTT – beyond the obvious 25
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)
11/02/2019 VTT – beyond the obvious 26
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
29.1.2019Riikka Kietäväinen
”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
29.1.2019Riikka Kietäväinen
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
29.1.2019Riikka Kietäväinen
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
11/02/2019 VTT – beyond the obvious 34
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.
11/02/2019 VTT – beyond the obvious 35
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