KYT seminar 18.3.2015
Microbiology research in KYT2014
Merja Itävaara
VTT Technical Research Centre of Finland
www.vtt.fi
Contents
Introduction
- Microbiology in repositories – why microorganisms and their functions
should be studied
Microbiology research in KYT2014 (2010-2014)
GEOMICRO
SALAMI
GEOBIOINFO
VTT
GTK
Aalto
(2011-2014)
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
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Conclusions
Merja Itävaara, VTT
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IGD-TP Implementing Geological Disposal of
Radioactive Waste Technology Platform
http://www.igdtp.eu/
Euratom/Mind project (2015-2019)
to support the implementation of planned geological disposal
projects for higher-level radioactive wastes across the EU.
“high urgency” and “high importance” topics identified in the
most recent IGDTP Strategic Research Agenda, focusing
specifically on the influence of microbial processes on waste
forms and their behavior, and the technical feasibility and longterm performance of repository components.
15 European groups working on the impact of microbial
processes on safety cases for geological repositories across the
EU, focusing on key questions posed by waste management
organisations
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Introduction
What microbes can do in the repositories?
Microbes can
- degrade organic C and can disintegrate construction
materials
- 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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Metagenomics to study uncultivable
Only 1-5% of microbes are cultivable
Isolate (pure culture)
Genomics
Courtesy of Susannah Green Tringe, DOE JGI
Microbial community
Metagenomics
5
Opening the genetic code - Sequencing
Reading the nucleotide structure (A, G, T, C) of the
genome or metagenome
Sequencing technologies
- Sanger sequencing
- Next generation sequencing (high throughput
sequencing):
454 pyrosequencing,
Illumina sequencing; HiSeq,
MiSeq. Next Seq
Ion Torrent PGM,
PacBio RS
Oxford Nanopore
(Life technologies)
6
Bioinformatics for highthroughput data analysis
Fast 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 more
automatic large data analysis
What are they doing?
Total sequencing of the whole community to
study metabolic pathways:
IMG/M, MG-RAST, Megan, Uniprot,
KEGG,etc
New pipeline developed recently in HUMAnN
microbiome project
(Abubucker, S et al. 2012, Plos Computational
biology 8, 6)
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Future research is focusing on
OMIC’s approach in Systems Biology of living
Accurate
organisms
Link with cognate
Identification of enzymes
involved
in metabolite transformation
annotation of
ORFs
metabolic pathways
Metabolome
Metaproteome
Metabolism
Metatranscriptome
Metagenome
Maria-eugenia Guazzaroni and Manuel Ferrer 2011. Handbook of Molecular Microbial Ecology, Vol. I
Metagenic approaches in systems biology, Chapter 54
Final cellular
output
8
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Competences developed in the microbiology projects
DIVERSITY
QUANTIFICATION
qPCR
Geomicrobiology
DGGE,functional
genes,
454 pyrosequencing
16S rRNA
VTT
Functional genes
FUNCTION
INTERACTION
Radiolabelled
substrates, SIP,
Total sequencing,
Metabolic pathways’
Geosphere/biosphere
Deep biosphere consortium
SALAMI, GEOMICRO, GEOBIOINFO 2011-2014
SALAMI GTK Ilmo Kukkonen, L
Ahonen
-Deep groundwater sampling
techniques
-Geochemistry, gases, isotopes
-Online gas analysis
GEOMICRO M.
Itävaara/VTT
-Microbial sampling
-Microbial diversity anjd function,
highthrouhput sequencingi
-Anaerobic methane oxidation
-Chemical small molecules in
groundwaters
Collaboration:
GTK, Prof. Ilmo Kukkonen (HY),
Lasse Ahonen, Riikka Kietäväinen
Aalto University Prof. Juho
Rousu,
Funding:
Finnish National Research Program for nuclear
waste disposal (KYT2010, KYT 2014)
Finnish Academy; Projects 1. Deep Life, 2. Deep
metapathway
Finnish Natural Science Foundation
Ph.D grant
Posiva Ltd.
TVO Ltd.
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Merja Itävaara, VTT
Scientists involved at VTT
Microbial diversity
Malin Bomberg
Mari Nyyssönen
Lotta Purkamo
Maija Nuppunen-Puputti,
Pauliina Rajala,
Mari Raulio
Leea Ojala
Hanna Miettinen
Minna Vikman
Elina Sohlberg
Metapathway analysis and
enzymes
Heikki Salavirta
Kaisa Marjamaa
Peter Blomberg
Antti Nyyssölä
Mikko Arvas
Merja Oja
Fahad Syed
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Deep biosphere and major processes
Bacteria, archaea, fungi, viruses, nematodes
Chemolithotrophic processes
Iron and sulphur oxidation and reduction
Methane production, methane oxidation
Hydrogen use and production
Nitrogen fixation, ammonia oxidation, denitrification
Heterotrophic, organic compound degradation
Carbon dioxide fixation
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Goals
To Characterize microbial
diversity of Fennoscandian
Shield by highthroughput
sequencing
Combine geochemistry
and geology to microbial
data
To estimate the major
metabolic functions based
on whole genome
sequencing and metabolic
network analysis
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Sampling sites (2006-2014)
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To provide knowledge about microbial diversity and
metabolism in Finnish bedrock aquifers
Major sites
Outokumpu deep borehole (2.5 km) Cu-Zn-Ni-Cosulphide ore province,
2.5 km, Geolaboratory, research borehole
Olkiluoto: Nuclear waste disposal site, several
drillholes 300-1000m, 15 boreholes
Outokumpu deep borehole studies
Metapeltic rocks (mica gneiss) representing
original marine clay deposition (sulphides
present), metamorphosed and associated
with intrusives (granite, tonalite)
Pyhäsalmi
cave, 2 km
Kuhmo
600m
Outokumpu
2.5km
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
Olkiluoto
300-1000m
Palmottu
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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
Sites:
Outokumpu deep borehole 2.5 km
Kuhmo, Romuvaara 600m
Pyhäsalmi cave ca 2 km
Tube sampling, samples from 50 tai 100m
Pumping water samples
Pressure samples
Biotraps
Filtration of large water samples from
Pyhäsalmi cave, decrease in pressure
systems
Outokumpu: On-line monitoring (pH, EC,
T, O2, Eh)
Measurement of gas evolution
from the head space of the borehole
30l/day, major gas methane
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Pyhäsalmi
SEM & EDX analysis
Very small number of cells observed
A lot of crystals which plugged the filter
High pressure problems in sampling
Kuvat Mari Raulio
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Saline fluids, gases and microbes in
crystalline bedrock -SALAMI
•
Sampling for representative gas and microbiological samples
Pressurised
cylinder
Pressure
gauge
Vacuum
pump
Sampling at the Outokumpu Deep Drill Hole (2.5 km
deep)
Ultrasonic
bath
Sampling in the Pyhäsalmi mine
(-1430 m)
KYT2014 18.3.2015
Riikka Kietäväinen
Gas separation in a vacuum line
SALAMI: Residence times of deep groundwaters
•
•
•
Concentration of crustal
noble gas nuclides
Concentration of U, Th
and K + physical
properties of the
bedrock
accumulation rate
Noble gas components
• 3He/4He 1.5 · 10-8
No mantle
component
Modified after Ballentine & Burnard (2002) and Ballentine et al. (2002)
Mantle
•In Outokumpu the average
residence time 30 million years
(Kietäväinen et al. 2014, GCA)
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
Terminal electron acceptors in anaerobic methane oxidation
a) Mangane
CH4 + 4MnO2 + 7H+
b) Iron
CH4 + 8 Fe(OH) 3 + 15H+
c) Nitrite:
3 CH4 + 8NO2- + 8H+
d) Nitrate:
5 CH4 + 8NO 3- + 8H+
e) Sulphate:
CH4 + SO42-
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HCO3- + 4Mn2 + 5 H2O
HCO3- + 8Fe2+ + 21 H2O
3CO2 + 4N2 + 10 H2O
5CO2 + 4N2 + 14 H2O
HS- + HCO3- + H2O
Merja Itävaara
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Outokumpu deep borehole research
What has been done
- the diversity of microbial communities each 100m depths
- pathways (3 depths)
Bacteria
- borehole water and fracture microbiology
DGGE and 454
pyrosequencing
0%
10 %
20 %
30 %
40 %
50 %
60 %
70 %
80 %
90 %
100 %
Flavobacteria
OUTOII 150-250 m
Gammaproteobacteria
Actinobacteria
OUTOII 550-650m m
Alphaproteobacteria
Mollicutes
OUTOII 950-1050 m
Clostridia
OUTOII 1050-1150 m
Betaproteobacteria
unclassified_Bacteria
OUTOII 1450-1550 m
Deltaproteobacteria
OUTOII 1850-1950 m
Erysipelotrichi
Anaerolineae
OUTOII 2250-2350 m
Whole genome
sequencing and
pathways (MG/Rast)
Sphingobacteria
Archaea
1
2
3
4
0%
10 %
20 %
30 %
40 %
50 %
60 %
70 %
80 %
90 %
OUTOII 150-250 m
*
*
100 %
Methanolobus
Methanomethylovorans
OUTOII 550-650 m
OUTOII 950-1050 m
*
*
OUTOII 1050-1150 m
OUTOII 1850-1950 m
Methanobacterium
Methanosarcina
unclassified_Methanobacteriaceae
unclassified_Euryarchaeota
unclassified_Archaea
unclassified_Methanosarcinaceae
unclassified_Methanomicrobia
OUTOII 2250-2350 m
unclassified_Methanobacteriales
*
*
*
Itävaara, M., Nyyssönen, M., Kapanen, A.,Nousiainen, A.,
Ahonen, L., Kukkonen, I. 2011. Characterization of bacterial
diversity down to a depth of 1500 m of the Outokumpu deep
borehole. FEMS Microbiology 2011, 1-15,.
DOI:10.1111/j.1574-6941.2011.01111.x
Deep metapathways:
- Energy metabolism based on
metagenomic data
Nyyssönen, M. Hultman, J., Ahonen, L., Kukkonen, I., Paulin, L., Laine, P., Itävaara, M. and
Auvinen, P. 2014.. Taxonomically and functionally diverse microbial communities in deep
crystalline rocks of the Fennoscandian shield" - ISME J, 8: 126–138;
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Merja Itävaara, VTT
doi:10.1038/ismej.2013.125.
- What are the major metabolic
pathways driving the community
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Statistical analysis of Sulphate reducers,
geochemistry and geophysics
Viivi Uurtio graduation
thesis
Supervised by Prof.
Juho Rousu
/Aalto Yliopisto
Sites compared:
Olkiluoto,
Outokumpu,
Nummi-Pusula
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KKK-koe
Biodegradation of low-level
radioactive waste in
geological disposal
Merja Itävaara, Minna Vikman, Kaisa Marjamaa
VTT Technical Research Centre of Finland Ltd
Operating waste
generated during operation and maintenance
Low level:
Paper, cardboard, cotton
Fire-protected fabrics
Plastic wrappings and protective
clothing
Machinery parts and pipes
59% cellulose-based material
Itävaara, VTT
Silo for low level radioactiveMerjawaste
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Photo: Posiva Oy
Picture: Small et al., 200824
Microbiological risks in geological disposal of
LLW
Methane evolution
Organic C cause
accelerated biocorrosion
and increased activity of
microbes
Microbial metabolites
Low-level waste:
Cellulose-based waste:
radiation
paper, cardboard, cotton,
fabrics etc.
Environmental
conditions (pH)
Others:
PVC, PE, rubber, metals etc..
Migration of
radionuclides
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microbes
25
Results
Anaerobic biodegradation of LLW waste is ongoing
Constant biogas formation
Methanogens detected
Microbial groups related to the hydrolysis of cellulosic materials
Biodegradability of LLW was below 5% in 2013
The heterogenity in environmental conditions in different parts of
the tank can also be seen in microbial activity and microbial
diversity
highest microbial activity in the drum containing mostly biodegradable
waste
Corrosion of steel plates was proceeding more rapidly in the capsules
containg biodegradable waste
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Mikrobiologisen korroosion
riskit Suomen loppusijoitusolosuhteissa (REMIC)
Leena Carpén, Pauliina Rajala, Malin
Bomberg
KYT2014 Loppuseminaari
18.3.2015
Tavoitteet
Hankkeen tavoitteena oli arvioida biofilmien muodostumista ja mikrobiologisen
korroosion riskiä metallisille materiaaleille (purkujätemetallit) Suomen loppusijoitusolosuhteissa sekä kehittää luotettava lopusijoitusolosuhteita simuloiva koejärjestely.
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Mikrobiologiset tulokset – betonin ja ravinteet
Biotic
Biotic
Methane A
Biotic
Glucose B
Biotic
Glucose A
Biotic
Concrete B
Biotic
Concrete A
Biotic
B
A
Abiotic Abiotic
Methane B
Biotic
• Ravinteiden lisäys muutti bakteeriyhteisön
koostumusta ja kiihdytti hiiliteräksen
korroosiota
• Betoni inhiboi biofilmin muodostumista ja
hidasti korroosiota, mutta aiheutti
voimakkaammin korroosion paikallistumista
Betaproteobacteria
Deltaproteobacteria
Bacilli
Mikrobit korroosiokuopassa
Alphaproteobacteria
Kuva: Mari Raulio
B
Bacilli
A
0%
10%
Alphaproteobacteria
Sphingobacteriia
Gammaproteobacteria
Zetaproteobacteria
Armatimonadetes_gp4
Caldilineae
Epsilonproteobacteria
Planctomycetia
Anaerolineae
Nitrospira
Anaerolineae
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Erysipelotrichia
20%
30%
40%
50%
Deltaproteobacteria
Ignavibacteria
Actinobacteria
Clostridia
SR1_genera_incertae_sedis
Flavobacteriia
Negativicutes
Subdivision3
Chlorobia
Spirochaetia
Bacilli
Mollicutes
60%
70%
80%
90%
100%
Betaproteobacteria
Cytophagia
Acidobacteria_Gp6
Verrucomicrobiae
Anaerolineae
Parcubacteria
Bacteroidia
Acidobacteria_Gp16
Holophagae
Aquificae
Bacteroidetes_incertae_sedis
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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 geologisissa
olosuhteissa, monet happoja tuottavia
Mikrobien aktiivisuus on erittäin hidas geologisessa ympäristössä.
kuitenkin muutokset elektroniakseptorien saannissa voivat aiheuttaa
nopeita aktiivisuuden muutoksia esim. metaani pulssi maan kuoresta
Ihmisen muokkaama geologinen ympäristö altistuu mikrobien lisääntyvälle
toiminnalle ja vaikutukset huonosti tunnettu
Orgaanisen hiilen esiintyminen edistää biokorroosiota
Mikrobit voi muuttaa ympäristönsä kemiaa, pH:ta ja lämpötilaa
Geologia, geokemia, paine, syvyys, lt, geokaasut
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