November 2013

Working Reports contain information on work in progress

or pending completion.

The conclusions and viewpoints presented in the report

are those of author(s) and do not necessarily

coincide with those of Posiva.

Karsten Pedersen, Johanna Arl inger,

Andreas Bengtsson, Johanna Edlund,

Lena Eriksson, Lotta Hal lbeck, Jessica Johansson,

Anna Pääjärvi & Lisa Rabe

Microbial Analyt ics Sweden AB

Working Report 2013-57

Sulphate Reduction Experiment: SURE-1

 

ABSTRACT

It was previously concluded that opposing gradients of sulphate and methane, observations of 16S rDNA sequences displaying great similarity to those of anaerobic methane-oxidizing Archaea, and a peak in sulphide concentration in groundwater from a depth of 250–350 m in Olkiluoto, Finland, indicated proper conditions for methane oxidation with sulphate.

In the present research (SURE-1), pressure-resistant, gas-tight circulating systems were constructed to enable the investigation of attached and unattached anaerobic microbial populations from a depth of 327 m in Olkiluoto under in situ pressure (2.4 MPa), diversity, dissolved gas, and hydrochemical conditions of groundwater station ONK-PVA6. Three parallel flow cell cabinets were configured to allow observation of the influence on microbial metabolic activity of 11 mM methane, 11 mM methane plus 10 mM H2, or 2.1 mM O2 plus 7.9 mM N2 (i.e., air). The concentrations of these gases and of organic acids and carbon, sulphur chemistry, pH and Eh, ATP, numbers of cultivable microorganisms, and total numbers of cells and bacteriophages were subsequently recorded under batch conditions for 105 d.

The system containing H2 and methane displayed microbial reduction of 0.7 mM sulphate to sulphide, while the system containing only methane produced 0.2 mM reduced sulphate. The system containing added air became inhibited and displayed no signs of microbial activity. Added H2 and methane induced increasing numbers of lysogenic bacteriophages per cell. It appears possible that a microbial anaerobic methane-oxidizing process coupled to acetate formation and sulphate reduction may be ongoing in aquifers at a depth of 250–350 m in Olkiluoto, but clear evidence of such an AOM process was not obtained.

Keywords: Sulphate reduction, microbial, anaerobic methane oxidation, groundwater, flow cell.

SULFAATIN MIKROBIOLOGINEN PELKISTYMINEN, SURE-1 KENTTÄKOE TIIVISTELMÄ Olkiluodossa on aiemmissa tutkimuksissa havaittu syvyydellä 250−350 m pohja-vesiolosuhteet, jotka saattavat olla suotuisat sulfaatin läsnä ollessa tapahtuvalle metaanin hapettumiselle. Tähän viittaavat sulfaatin ja metaanin käänteiset pitoisuus-gradientit, havainnot hyvin samankaltaisista 16S rDNA sekvensseistä tunnettujen anaerobisesti metaania hapettavien arkkieliöiden (Archaea) kanssa sekä kohonneet sulfidikonsentraatiot tällä syvyydellä. Tässä tutkimuksessa (SURE-1) valmistettiin paineenkestävä ja kaasutiivis veden kier-rätyslaitteisto koejärjestelyä varten, jossa pystyttiin tutkimaan sekä vapaita että kasvu-alustaan kiinnittyneitä anaerobisia mikrobeja. Koejärjestelyssä käytettiin Olkiluodon kalliosta 327 m:n syvyydeltä peräisin olevaa pohjavesiaseman ONK-PVA6 pohjavettä ja koe suoritettiin pohjavesiasemaa vastaavassa paineessa (2.4 MPa) sekä geo-mikrobiologisissa ja hydrogeokemiallisissa (sisältäen liuenneet kaasut) olosuhteissa. Laitteisto käsitti kolme rinnakkaista läpivirtauskennostoa, joilla pystyttiin seuraamaan kolmen eri kaasukoostumuksen vaikutusta mikrobien aineenvaihduntaan ja aktiivi-suuteen. Kaasukoostumukset olivat 11 mM metaania, 11 mM metaania ja 10 mM vetyä sekä 2.1 mM hapea ja 7.9 mM typeä (eli ilma). Mainittujen kaasujen, orgaanisten happojen, hiilen ja rikin muotojen pitoisuudet sekä pH, Eh, ATP, viljeltyjen mikro-organismien määrä, solujen kokonaismäärä ja bakteriofagit monitoroitiin läpivirtaus-kennoista 105 päivän ajan. Kennostossa, johon oli lisätty vetyä ja metaania, havaittiin 0.7 mM sulfaattia pelkis-tyneen sulfidiksi, kun taas kennostossa, johon oli lisätty pelkkää metaania, havaittiin pelkistyneen 0.2 mM sulfaattia. Kennostossa, johon oli lisätty ilmaa, ei havaittu lainkaan mikrobiologista aktiivisuutta. Lisätty vety ja metaani kasvattivat lysogeenisten bakteriofagien määrää. Kokeet viittaavat siihen, että mikrobiologiseen anaerobiseen metaanin hapettumiseen (AOM) liittyvä asetaatin muodostuminen ja sulfaatin pelkisty-minen voivat olla mahdollisia Olkiluodon pohjavedessä 250−350 m syvyydellä, mutta yksiselitteistä todistusta ei kuitenkaan saatu tämän kaltaisesta AOM-prosessista. Avainsanat: Sulfaatin pelkistyminen, mikrobiologinen, anaerobinen metaanin hapettuminen, pohjavesi, läpivirtauskenno.

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TABLE OF CONTENTS

ABSTRACT

TIIVISTELMÄ

KEY TO ABBREVIATIONS USED FREQUENTLY IN THE TEXT ................................. 3

1 INTRODUCTION .................................................................................................... 5

1.1 The requirement for sulphate reduction experiments ......................................... 5 1.2 Microbial sulphate reduction with H2 and methane ............................................. 5 1.3 Methane and sulphate in Olkiluoto groundwater ................................................. 7 1.4 H2 from anaerobic corrosion of metals ................................................................ 7 1.5 Outline of the SURE program ............................................................................. 8 1.6 Objectives of SURE ............................................................................................ 8 1.7 This work ............................................................................................................. 9

2 MATERIALS AND METHODS .............................................................................. 11

2.1 Source of groundwater ...................................................................................... 11 2.2 Groundwater characterization ........................................................................... 11 2.3 Flow cell systems for field work ........................................................................ 11 2.4 Configuration of growth experiments ................................................................ 15 2.5 Eh in FCCs ......................................................................................................... 16 2.6 Sampling procedures ........................................................................................ 16 2.7 Ph and Eh analysis using a portable meter ....................................................... 17 2.8 Analysis of sulphate and sulphur isotope composition in sulphate ................... 17 2.9 Acetate, lactate, organic carbon, ferrous iron, and sulphide analysis ............... 17 2.10 ATP analysis ................................................................................................. 17 2.11 Total number of cells ..................................................................................... 18 2.12 Total number of virus-like particles ............................................................... 18 2.13 Gas sampling and analysis ........................................................................... 18 2.14 Analysis of most probable numbers of culturable anaerobic microorganisms .. ...................................................................................................................... 18 2.15 DNA extraction and concentration ................................................................ 19 2.16 Analysis using the G3 PhyloChip assay ....................................................... 19 2.17 Statistical analyses ....................................................................................... 19

3 RESULTS ............................................................................................................. 21

3.1 Groundwater characterization ........................................................................... 21 3.2 pH, Eh, and gases ............................................................................................. 22 3.3 Numbers of cells, VLP, and ATP ...................................................................... 24 3.4 FCC chemistry .................................................................................................. 24 3.5 Cultivated microorganisms and organic acids and carbon ............................... 24 3.6 PhyloChip analysis ............................................................................................ 26

4 DISCUSSION........................................................................................................ 33

4.1 The experimental conditions ............................................................................. 33 4.2 The cultivation approach ................................................................................... 33 4.3 Cell density control by bacteriophages ............................................................. 34 4.4 H2-induced sulphate reduction .......................................................................... 34 4.5 Methane-induced acetogenesis and sulphate reduction ................................... 34 4.6 Sulphate reduction rates ................................................................................... 35 4.7 Conclusions ...................................................................................................... 37

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REFERENCES ............................................................................................................. 39

A. APPENDIX ............................................................................................................ 43 

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KEY TO ABBREVIATIONS USED FREQUENTLY IN THE TEXT Abbreviation Meaning Brief description

AA Autotrophic acetogens Microbes able to produce acetate from carbon dioxide and H2

AGW Analytical-grade water Purified distilled water

AM Autotrophic methanogens Microbes able to produce methane from carbon dioxide and H2

ANME Anaerobic methane oxidation Microbes oxidize methane to H2 and carbon dioxide, and use H2 to reduce sulphate to sulphide

AOM Anaerobic oxidation of methane

Oxidation of methane under oxygen-free conditions

AODC Acridine orange direct count Method based on nucleic acid staining for determining cell numbers

ATP Adenosine triphosphate Energy carrier in living organisms

CFU Colony-forming unit A cell that has divided repeatedly, e.g., on an agar plate, forming a dense colony of many identical cells

CHAB Cultivable heterotrophic aerobic bacteria

Microbes able to live on O2 and organic carbon and that grow in the laboratory

CoA Coenzyme A A key coenzyme in cell metabolism

DNA Deoxyribonucleic acid The genetic code, which builds the genome unique to each organism

FC Flow cell Pressure resistant, flow controlled unit for biofilm growth and activity

FCC Flow cell cabinet Pressure , temperature and flow controlled cabinet with 4 flow cells

HA Heterotrophic acetogens Microbes able to produce acetate from organic carbon

HM Heterotrophic methanogens Microorganisms able to produce methane from organic carbon

IRB Iron-reducing bacteria Microbes able to reduce iron(III) in their respiration

Km Enzyme kinetics constant The concentration of a substrate at which the reaction rate of the enzyme equals Vmax/2

MPN Most probable number Method for enumerating microbes

MRB Manganese-reducing bacteria Microbes able to reduce manganese(IV) in their respiration

NRB Nitrate-reducing bacteria Microbes able to reduce nitrate in their respiration

rDNA Ribosomal DNA DNA encoding for the ribosome

SMTZ sulphate–methane transition zone

Positions where opposing sulphate and methane gradients meet

SRB Sulphate-reducing bacteria Microbes able to reduce sulphate in their respiration

TNC Total number of cells The number of cells in a water sample or on a solid phase, usually determined by means of microscopy using the AODC method

Vmax Enzyme kinetics constant The maximum reaction rate of an enzyme

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5

1 INTRODUCTION

1.1 The requirement for sulphate reduction experiments

Sulphide produced by microbial sulphate reduction under anaerobic conditions may corrode the copper canisters used for final disposal of spent nuclear fuel (SNF). Understanding microbial sulphate reduction processes is therefore a key task for safety assessment. In the programme for characterizing repository host rock in the ONKALO tunnel (Aalto et al. 2009), three issues have been identified connected to the process of microbial sulphate reduction:

Issue I-11: Evolution of groundwater composition – Flow paths to/from the host rock, especially at the near-surface interface: Our understanding of the connection between the near-surface water and groundwater types in bedrock has advanced, due to the development of near-surface hydrological modelling, though some questions remain. The infiltration experiment, which is currently in process, will provide data relevant to this issue.

Issue I-12: Evolution of groundwater composition – Impact of surficial water intrusion (pH, redox, and buffering capacity): The process of sulphate reduction is well understood and at groundwater temperatures, it is only possible through microbial activity, but the quantification of the associated microbial activity, particularly the rate of sulphate reduction, is uncertain. The infiltration experiment (see issue I-11) will provide data relevant to this issue. Detailed characterization and monitoring below fracture zone denoted HZ20 is important to evaluate sulphate reduction due to groundwater mixing.

Issue I-13: Evolution of groundwater composition – Formation of gas phase/dissolved gases in groundwater: The origin of the detected dissolved methane is uncertain and the current production rate and source of methane production are unclear. It is essential to obtain more gas content, microbial, and isotopic data from deep saline groundwater.

The ONKALO tunnel is well suited for research into issues I-12 and I-13. This report describes tunnel experiments examining microbial sulphate reduction processes, i.e., issue I-12. The ONKALO tunnel enabled investigations of microbial activity under in situ conditions similar to investigations previously performed in the Äspö Hard Rock Laboratory (HRL) tunnel. There, the influence of acetate, H2, and O2 on microbial activity could be investigated using flow cell (FC) circulating systems operating under in situ conditions at a depth of 450 m (Pedersen 2012a,b). The installation of FC circulating systems in the ONKALO tunnel at a depth of 327 m enabled the investigation of microbial sulphate reduction activity under conditions relevant to the mixing between methane- and sulphate-rich deep and upper bedrock groundwater, respectively.

1.2 Microbial sulphate reduction with H2 and methane

Sulphate reduction at the temperatures and pressures prevalent in the deep groundwater environment is a microbiological process. The chemical reduction of sulphate to

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sulphide under these conditions is very slow, as revealed by the calculated half life of thermo-chemical sulphate reduction in the presence of acetate and elemental sulphur at 100C, i.e., 372,000 years (Cross et al. 2004). The sulphate-reducing bacteria (SRB) use the sulphur atom in the sulphate molecule as an electron-acceptor and the reduced product is sulphide. The energy and electron donor for SRB can be either organic compounds or H2. In the case of a general organic compound, the reaction with sulphate can be written:

2(CH2O) + SO42−

+ 2H+→ 2HCO3− + HS− + 3H+ Eq. 1

Organic compounds that can be used by various SRB are fermentation products such as short-chain organic acids, fatty acids, and higher-molecular-weight hydrocarbons. Many SRB, especially of the genus Desulfovibrio, can grow on lactate. The lactate molecule is incompletely oxidized to acetate and carbon dioxide, and the electrons are transported to electron transport enzymes in the cell membrane and then further to the sulphate reduction enzymes in the cytoplasm. The overall reaction is written:

2 lactate + SO42− + 2H+ → 2 acetate + 2CO2 + HS− + H+ + 2H2O Eq. 2

In this metabolism, lactate is used as an energy and electron source as well as a carbon source for biomass production. Other SRB use H2 as an electron donor and energy source. The reaction for the reduction of sulphate with H2 is written:

4H2 + SO42− + H+ → HS− + 4H2O Eq. 3

Note that no carbon is involved in this energy-transforming reaction and that protons are consumed (Eq. 3). The carbon sources for H2-utilizing SRB are either short-chain organic compounds, such as acetate, or carbon dioxide. The carbon is used for biomass production and the biosynthesis consumes chemical energy. Acetate and carbon dioxide are incorporated into the cell metabolism via the molecule coenzyme A (CoA) to produce pyruvate in the following way:

CH3COO−–CoA + CO2 + H2 CH3COCOO− + CoA + H2O Eq. 4

The pyruvate then enters the cell metabolism and is incorporated into new biomolecules.

Finally, some microbial consortia can use methane as a source of energy and produce sulphide:

CH4 + SO42– HCO3

– + HS– + H2O Eq. 5

For methane to act as an energy source, the SRB are assumed to have a symbiotic relationship with methanogenic Archaea, a situation hitherto found only in sea bed sediments (Lösekann et al. 2007). Other metabolic pathways have been suggested as well (Caldwell et al. 2008). Hydrogeochemical, isotopic, and microbiological evidence indicates, however, that the process may occur under deep bedrock conditions in Olkiluoto (Pedersen et al. 2008; Aalto et al. 2009).

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1.3 Methane and sulphate in Olkiluoto groundwater

Depth profiles of anaerobic aquatic sediments where sulphate and methane meet with the concomitant occurrence of sulphide have been interpreted as evidence of anaerobic methane oxidation (AOM), with sulphate as the final electron acceptor (Zehnder and Brock 1980; Thomsen et al. 2001; Knittel and Boetius 2009). A typical such sulphate–methane transition zone (SMTZ), albeit on a much broader scale than in aquatic sediments, was indicated by the analysis of methane, sulphate, and sulphide over depth in groundwater below the island of Olkiluoto, Finland (Pedersen et al. 2008). In the 250–350 m depth zone, the sulphate concentration decreased with depth from 5 mM to <0.1 mM, while methane increased with depth from 40 µM to 4 mM. In addition, the sulphide concentration in this zone reached 30–300 µM at several sampled sites, which was more than one order of magnitude higher than that found in shallower or deeper groundwater. Similarly, the total number of cells (TNC), ATP concentration, and numbers of culturable SRB were greater in groundwater samples from this zone than in shallower or deeper samples (Pedersen et al. 2008). It was concluded that the SMTZ under Olkiluoto likely offered proper conditions for a microbial AOM process involving sulphate as an electron acceptor.

1.4 H2 from anaerobic corrosion of metals

Underground repositories for SNF will include large amounts of iron, i.e., steel rock support material such as rock bolts and wire mesh, that will corrode with the concomitant production of H2 (Reardon 1995). The oxidation of metallic iron with sulphate by microorganisms is regarded as the principal reaction in the anaerobic microbial corrosion of iron (Figure 1-1). The suggested reaction mechanism of the corrosion is that the negative redox potential (i.e., Fe2+/Fe, E0’ = −0.44 V) of iron can liberate H2 (i.e., 2H+/H2, E0’ = −0.41 V) and may in this way indirectly act as an electron donor for SRB (Cord-Ruwisch 2000). Another possible mechanism in anaerobic corrosion has been proposed: the direct microbial utilization of the electrons liberated during the oxidation of iron (i.e., Fe → Fe2+ + 2e−) (Dihn et al. 2004). This mechanism is kinetically more favourable than is the consumption of electrochemically formed H2.

A recent H2 evolution scenario of concern is a process in which copper reacts with water under the production of H2 (Hultquist et al. 2011). This process is thought to be determined by the partial pressure of H2 and to stop when the concentration of dissolved H2 approaches one µM. If microbial processes consume the H2, this process may continue uninterrupted (Hultquist et al. 2011) and iron corrosion processes may be accelerated as indicated in Figure 1-1 (Little and Jason 2007). Consequently, how microbial processes utilize H2 with the concomitant production of sulphide must be understood for the safety evaluation process.

Sulphide is a compound that can mediate anaerobic copper corrosion. The principal mechanism of anaerobic copper corrosion is thought to be the same as for the anaerobic corrosion of metallic iron. The complete mechanism of anaerobic copper corrosion is not yet fully understood, but the theory is that electrons from copper reduce protons to H2 in the presence of sulphide produced by SRB (see Eq. 1). The produced H2 can be used by SRB that produce more sulphide, which may react with the copper and produce

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copper sulphides. It is therefore crucial to characterize the potential for sulphide production in groundwater in an SNF repository. One set of crucial parameters is the growth kinetics of SRB when grown on different energy sources, such as H2 and methane.

2Cu + HS− + H+→ Cu2S + H2 Eq. 6

Figure 1-1. The principle of anaerobic corrosion of metallic iron (Fe) mediated by SRB. 1. The H2 produced by the reduction of protons by electrons from the oxidation of iron is consumed by SRB. 2. The H2 sulphide (H2S) produced precipitates with the ferrous iron (Fe2+) and is deposited as 3. iron sulphide (FeS), on the steel surface. 4. During the precipitation, protons (H+) are liberated.

1.5 Outline of the SURE program

This investigation seeks a better understanding of the processes underlying sulphide production (i.e., sulphate reduction) and involves the chemical characterization and detailed microbiological investigation of groundwater from a depth of 327 m from the ONKALO tunnel. A plan for a sulphate reduction experiment (SURE) has been formulated as part of the ReRoC programme (Aalto et al. 2009).

1.6 Objectives of SURE

The objectives of the SURE are:

to demonstrate the microbial reduction of sulphate via AOM,

to determine case-specific reduction rates (i.e., variable concentrations), and

to determine the renewal rates of energy sources for sulphate reduction.

The experimental plan of the sulphate reduction investigation reported here was divided into three different phases:

Phase I: Drilling and baseline characterization of the investigation drillholes in fall 2009/winter 2009–2010.

Phase II: Identification, characterization, and selection of groundwater in 2010.

Phase III: Microbiological in situ tunnel-based research using SO4- and CH4-rich water types in 2010–2011.

SRB

Anode Cathode

Fe2+

2e−FeS

2H+

H2S

H2 1

2

34

SO42−

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1.7 This work

In the present work, three parallel flow cell cabinets (FCCs) were configured with four FCs each to permit observation of the effect on microbial metabolic activity of 11 mM methane, 10 mM H2 plus 11 mM methane, or 2.1 mM O2 plus 7.9 mM N2 (i.e., air). The additions of 10 mM methane and hydrogen (methane concentration in the studied groundwater is about 1 mM) was considered to be enough to activate microbial sulphate reduction. The FCs were first inoculated under an in situ pressure of 2.4 MPa in the ONKALO tunnel with circulating groundwater from a selected aquifer at a depth of 327 m for 110 d, after which they were installed in the laboratory in three independent FCCs under in situ conditions and supplied with their respective added gases. The concentrations of H2, methane, sulphate, sulphide, ferrous iron, organic acids, and organic carbon as well as pH and Eh were subsequently recorded under batch conditions for 105 d. The numbers of cultivable heterotrophic aerobic bacteria (CHAB), SRB, nitrate-reducing bacteria (NRB), iron-reducing bacteria (IRB), manganese-reducing bacteria (MRB), autotrophic acetogens (AA), autotrophic methanogens (AM), and virus-like particles (VLPs) as well as the TNC and amounts of unattached and attached biomass measured as ATP were also monitored over this period. The microbial diversities of cultures and of attached microorganisms were compared using microarrays. The effects of the different gas additions were evaluated.

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2 MATERIALS AND METHODS

2.1 Source of groundwater

Groundwater source

A 76-mm-diameter borehole denoted ONK-PVA06 was drilled in a niche of the ONKALO tunnel at a depth of 318.7 m (Toropainen 2009). The drilling was done at a 14.3° inclination on 3–4 November 2009 to a total length of 35.15 m. A metal-free packer system isolated an aquifer in the borehole located 32.7–32.9 m from the tunnel rock face at a depth of 327 m; the groundwater was directed by this packer system to the flow cells described below and then back to the aquifer via two parallel, 1/8inch polyetheretherketone (PEEK) thermoplastic tubes of high-pressure liquid chromatography quality (IDEX Health and Science, Oak Harbor, WA, USA). The packer system is described in detail elsewhere (Pedersen 2005) and an overview is presented in Figure 2-1. A modification was made with a 6 mm stainless steel tube that was used to shield the PEEK tubing in the part of the drillhole exposed to air.

2.2 Groundwater characterization

The microbiology of the ONK PVA06 groundwater was analysed in samples collected on 23 September 2010 as described elsewhere (Pedersen et al. 2008). Groundwater chemistry was analysed in samples collected on 8 November 2010 and transferred to the Teollisuuden Voima Oy (TVO) laboratory directly after sampling. The chemical analyses were performed by TVO according to their protocols, or were subcontracted to external laboratories as described in detail elsewhere (see Supplementary Table 3 in Pedersen et al. 2008).

2.3 Flow cell systems for field work

Three identical field systems comprising four FCs each, a micropump (Micropump GAH series V21 J with a PEEK impeller; Labinett, Göteborg, Sweden), two pressure meters (S-11, 40 Bar 4-20 G1 ⁄ 2; WIKA – AB Svenska Industri Instrument, Göteborg, Sweden), a flow meter (Promag 50; Endress+Hauser Flowtech AG, Sollentuna, Sweden), and a 4 L expansion vessel (Pedersen 2005) were installed in a container placed in a niche at a depth of 320 m in the ONKALO tunnel as shown in Figure 2-2 and connected to the packer system in ONK-PVA06. The flow cells had a stainless steel shell (length 300 mm, diameter 65 mm) and were lined with polyvinyldifluoride (PVDF) plastic. Each flow cell had a 120 mm long PVDF insert with a 22 × 32 mm opening that supported 110 g of crushed rock grains (2–4 mm in diameter, see Figure 2-3) offering a rock surface area of approximately 5200 cm2 per FCC for microbial adhesion and biofilm development, assuming spherical rock grains with an average diameter of 3 mm. The drill core was crushed by ALS Scandinavia, Luleå, Sweden, concomitant with the analysis of elemental composition (analysis G-0, Table 2-1). The rock grains, which were heat sterilized (160 °C for 5 h), were obtained from the drill core of the ONK-PVA06 drillhole at the approximate place of the intersected aquifer. Three flow stabilizers at each end of the insert ensured even distribution and a slow laminar flow of water through each flow cell (Pedersen 1982). The flow cells were installed on 24 November 2010 and circulated with groundwater under the in situ

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pressure of 2.4 MPa for 110 d at a flow rate to and from the aquifer of 17–18 mL min1. The total volumes of groundwater circulated were 2597, 2840, and 2758 dm3 in the three field systems, respectively, as registered by flow meters.

Figure 2-1. The packer system. The yellow sections are expandable polyurethane packers, while the green rings are Teflon-coated stainless steel casings. The grey components are made of PEEK, as are the 1/8 inch (outer diameter) sampling tubes. Groundwater sampling and circulation are mediated via two small holes opposite each other in the grey portion in the middle of the packer assembly (the dark spot in the top drawing). The depicted section is 100 mm long.

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Figure 2-2. Top image: three circulation set-ups. Bottom image: four flow cells with a micropump, flow meter, pressure meters, and expansion vessel installed in a container 320 m underground in the ONKALO research tunnel.

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Figure 2-3. Three flow cell cabinets installed in the laboratory, showing the flow cells from the circulation set-ups depicted in Figure 2-2. Micropumps, pressure meters, flow meters, and expansion vessels similar to those shown in Figure 2-2 were permanently installed in these cabinets. A flow cell insert with rock grains is shown in the lower left image. The microelectrodes were installed on top of the cabinets as shown in the top image.

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Table 2-1. Elemental composition of the crushed rock used to fill the flow cells in the flow cell cabinets.

ELEMENT mg kg1 ELEMENT mg kg1 ELEMENT %

Ba 453 Nb 0.861 SiO2 74.7

Rb 139 Er 0.814 Al2O3 14

Sr 103 Gd 0.633 K2O 4.39

Cr 24.3 Eu 0.583 Na2O 3.12

Zr 23.7 Th 0.546 Fe2O3 1.61

Ce 16.6 Hf 0.507 CaO 0.835

Ni 15.6 Sm 0.458 MgO 0.487

Ga 14.4 Lu 0.405 P2O5 0.2

Y 8.42 Ho 0.272 MnO 0.0255

V 7.77 Ta 0.209 TiO2 0.0175

La 5.38 Tm 0.145

W 4.55 Tb 0.14

Be 4.38 Co <6

U 2.6 Mo <2

Nd 2.06 Sc <1

Dy 1.05 Pr <1

Yb 1.03 Sum 99.4

2.4 Configuration of growth experiments

The 12 FCs exposed to ONK-PVA06 groundwater for 110 d were transported under pressure from the ONKALO tunnel to the laboratory in Mölnlycke and installed four by four in three FCCs. These FCCs have been described in detail elsewhere (Pedersen 2005; Hallbeck and Pedersen 2008) and are shown in Figure 2-3. The interior tubes and valves of the FCC were sterilized with 20 mg L1 chlorine dioxide, rinsed with sterile water, and filled with N2 before installation of the FCs. Each cabinet was temperature controlled (16–18 C) and a pressure of 2.4 MPa was maintained. A total of seven expansion vessels (4 L each) were filled with groundwater from ONK-PVA06, shipped pressurized with the FCs and used to fill the FCCs with a total of 5000 mL groundwater at the start of the experiments as described previously (Pedersen 2012a,b). Thereafter, gases were added as follows. Three Teflon-lined, 500 mL stainless steel cylinders (304L-HDF4-500-T; Swagelok, Göteborg, Sweden) were filled at room temperature (RT; 20 °C) with air to a pressure of 220 KPa, methane to a pressure of 220 KPa, and 50 % H2 and 50 % methane to a total pressure of 440 KPa, respectively. The cylinders were filled with groundwater under pressure (2.4 MPa), resulting in a total circulating volume of 5500 mL per FCC. The gas additions then corresponded to final theoretical

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concentrations of 7.9 mM N2 and 2.1 mM O2, 11 mM methane, and 11 mM methane and 10 mM H2, respectively. These treatments are hereafter denoted O2:N2, CH4, and H2:CH4, respectively. The start date was 15 March 2011 (day 0) and the end date was 28 June 2011, resulting in an experimental duration of 105 d. The flow rate was kept at 20 mL min1 corresponding to a flow of approximately 1 mm s1 over the rock grains.

2.5 Eh in FCCs

Four pressure-resistant micro-sensor Eh electrode couples equipped with one platinum micro-electrode with a tip diameter of 400–600 µm (RD500; Unisense A/S, Aarhus, Denmark) and one Ag/AgCl reference electrode with a tip diameter of 90–110 µm in gel-stabilized electrolyte (REF100; Unisense) were installed in line in each FCC circulation system. The electrodes represented an adaptation of the standard glass Unisense micro-sensors mounted in the stainless steel flow cells. The electrodes were connected to two eight-channel mV amplifiers that transformed the recorded voltages into digital signals, subsequently collected and stored in Microsoft Office Excel files every 600 s using SensorTrace Basic software (version 1.9; Unisense A/S). Each amplifier collected data from two electrode couples per FCC, thereby creating a back-up function if one of the amplifiers should fail.

2.6 Sampling procedures

Complete sampling was performed six times, i.e., on days 3, 21, 42, 63, 84, and 105, for analysis as described below. A pilot sampling was performed on day 0 before the gas additions to test the sampling procedures. On each sampling occasion, 20 mL of circulating water was drained and disposed of; 5 mL of water was then sampled and analysed for pH and 10 mL of water was collected in a sterile 15 mL polypropylene (PP) tube (Sarstedt, Landskrona, Sweden) for immediate analysis of ATP. Thereafter, 4 × 10 mL of water was collected, using syringes, in butyl rubber-stoppered anaerobic glass tubes (no. 2048-00150; Bellco Glass, Vineland, NJ, USA) and 5 mL was collected for the analysis of CHAB. Two 10 mL volumes of water were collected in PP tubes, preserved with 0.02 µm of filtered, neutralized formaldehyde to a final concentration of 2.5 %, and analysed for TNC and VLP, respectively. Thereafter, 8 mL of water was sampled for sulphate analysis, 9 mL was sampled for sulphide analysis, and two × 5 mL volumes were sampled using a 0.2 µm syringe filter (Minisart, Sartorius syringe filter, hydrophilic; Fisher Scientific, Göteborg, Sweden) and stored at 20 C until analysis of acetate and lactate analysis, respectively. Then, 25 mL of water was sampled using a 0.2 µm syringe filter (Minisart) for immediate analysis of ferrous iron; thereafter, 100 mL of water was sampled and filtered for DNA extraction and qPCR analysis. This analysis was unfortunately unsuccessful, probably due to the small biomass in 100 mL of groundwater combined with the low extraction efficiency of the employed extraction kit (DNeasy Blood and Tissue Kit; Qiagen, Hilden, Germany) as described elsewhere (Lloyd et al. 2010). Finally, 10 mL of groundwater was used for the analysis of Eh and 190 mL was collected for the analysis of gas on sampling occasions 1–3, while approximately 70 mL was sampled on the three remaining sampling occasions. In total, 452 mL of water was sampled on each of the first three sampling occasions and 332 mL was collected on the last three sampling occasions. After sampling the water, approximately 10 + 10 batches of rock grains were collected from two FCs in each FCC for subsequent analysis of amount of attached ATP and DNA.

17

2.7 Ph and Eh analysis using a portable meter

Eh was analysed using a HACH HQ40d portable multi-parameter meter (HACH Lange AB, Sköndal, Sweden) equipped with a MTC101-05 probe (HACH Lange AB) installed in a flow cell connected to each analysed FCC. This installation prevented contact with the air, which might have influenced Eh due to the degassing of H2, carbon dioxide, and sulphide from the samples. The pH of 5 mL subsamples was determined immediately following extraction from the FCCs, using a Schott CG84310 pH meter fitted with a BlueLine 13 pH electrode (VWR, Stockholm, Sweden) calibrated according to the manufacturer’s instructions.

2.8 Analysis of sulphate and sulphur isotope composition in sulphate

Samples for sulphate analysis were collected in sterile 15 mL PP tubes and frozen at 20 C until analysis using the SulfaVer 4 method (method #8051, programme 680, range 0.03–0.73 mM with 95 % confidence limits of distribution of ±10 %; HACH Lange). Samples for the analysis of the δ34S values of sulphate were collected in 125 mL PP bottles (Nalgene–Thermo Fisher Scientific, Göteborg Sweden) and sent for analysis with an elemental analyzer-isotopic ratio mass spectrometer by Iso-Analytical Limited, UK (www.iso-analytical.com).

2.9 Acetate, lactate, organic carbon, ferrous iron, and sulphide analysis

Acetate and lactate concentrations were determined using the enzymatic UV method (kit no. 10148261035 for acetate and kit no. 10139084035, for lactate; Boehringer Mannheim/R-Biopharm AG, Darmstadt, Germany) using a Genesys 10UV spectrophotometer (Thermo Fisher Scientific) for detection. Samples for the analysis of dissolved organic carbon (DOC) were diluted 1–100 times before analysis to obtain the optimal analytical concentration range. Samples of 14 mL were filtered through 0.2 µm hydrophilic syringe filters (Minisart) and deep frozen at 20 C until analysis at the Department of System Ecology, Stockholm University, according to Swedish Standard SS-EN 1484. The detection limit for DOC was 20 µM and the uncertainty was ±20 µM < 420 µM and 7 % > 420 µM. Sulphide was analysed using a colorimetric methylene blue method with an uncertainty of ±17 % (Swedish Standard Method SIS 028115). Ferrous iron concentrations were determined using the 1-10 phenanthroline method (method no. 8146, programme 255, range 0.4–54 mM with 95 % confidence limits of distribution of ±11 %; HACH Lange AB).

2.10 ATP analysis

The ATP Biomass Kit HS (no. 266-311; BioThema, Handen, Stockholm) was used to determine total ATP in cells living in groundwater. The ATP biomass method used here has been described, tested in detail, and evaluated for use with Fennoscandian Shield groundwater (Eydal and Pedersen 2007). The method was used for biomass attached to the rock grains, but with the following modification: Approximately 10 rock grains were sampled from each of two flow cells per FCC and placed in ATP extraction solution and analysed. Samples were diluted before analysis to obtain the optimal analytical concentration range.

18

2.11 Total number of cells

The TNC mL1 was determined in 10 mL samples using the acridine orange direct count (AODC) method as devised by Hobbie et al. (1977) and modified by Pedersen and Ekendahl (1990).

2.12 Total number of virus-like particles

The total number of virus-like particles was determined using a direct count method with SYBR Gold (Invitrogen, Eugene, OR, USA) according to Noble and Fuhrman (1998) and Chen et al. (2001).

2.13 Gas sampling and analysis

Water samples were collected using a pressure vessel (PVB) as described elsewhere (Hallbeck and Pedersen 2008). The sample was transferred to a vacuum container and any gas in the water was boiled off under vacuum (i.e., water vapour pressure) at RT; the transfer time was approximately 20–30 min. After extraction, the gas was compressed and transferred to a 10 mL syringe (SGE Analytical Science, Melbourne, Victoria, Australia) and the volumes of extracted gas and water were measured. The captured gas was subsequently transferred to a 6.6 mL glass vial stoppered with a butyl rubber stopper and sealed with an aluminium crimp seal. The vial had previously been evacuated and flushed twice with N2, in two cycles, and left under high vacuum (1 Pa). A dehydrant (cobalt chloride) was added to adsorb any traces of water remaining in the gas. Analysis was thereafter performed using gas chromatography.

Two different chromatographs were used and equipped as follows. H2 (<20 ppm) was analysed on a KAPPA-5/E-002 analytical gas chromatograph (AMETEK/Trace Analytical, formerly Trace Analytical, Menlo Park, CA, USA) equipped with a 156 1/16 inch stainless steel HAYESEP column (Varian Analytical Instruments, Varian AB, Bromma, Sweden) in line with a 31 1/8 inch stainless steel molecular sieve 5A column (Varian ABwhich was subsequently attached to a reductive gas detector with N2 used as the carrier gas. Helium and H2 (>20 ppm) were analysed on a Varian Star 3400CX gas chromatograph (Varian AB) using a thermal conductivity detector with oven, detector, and filament temperatures of 65, 120, and 250 °C, respectively. The gases were separated using a Porapak-Q column (2 m 1/8 inch diameter) followed by a molecular sieve 5A column (6 m 1/8 inch) with argon as the carrier gas. Methane was analysed on a Varian Star 3400CX gas chromatograph using a flame ionization detector (FID) with an oven temperature of 65 °C and a detector temperature of 200 °C. The gas was separated using a Porapak-Q column (2 m 1/8 inch diameter) and analysed on the FID with N2 as the carrier gas.

2.14 Analysis of most probable numbers of culturable anaerobic microorganisms

Media were prepared for CHAB and for the most probable number analysis of NRB, IRB, MRB, SRB, AA, and AM as described elsewhere (Hallbeck and Pedersen 2008). The cultivation time was about eight weeks to ensure that slow-growing microorganisms would be included in the results. Sampling and analysis were done on

19

days 0 and 105 for IRB and MRB while NRB, SRB, and AA were sampled and analysed on all full sampling occasions.

2.15 DNA extraction and concentration

DNA was extracted using the MO BIO Power Biofilm DNA isolation kit (catalogue no. 24000-50; Immuno Diagnostic Oy, Hameenlinna, Finland) from biomass attached to rock grains collected from the CH4 and the H2:CH4 FCs on day 118. Four extractions from 10 grains each were performed according to the manufacturer’s protocol and the extracted DNA was concentrated on a Microcon centrifugal filter (Ultracel, YM-100, 42424; Millipore Sweden AB, Solna, Sweden). The CH4 FCs were re-sampled on day 120 and a total of 40 extractions of 10 grains each were concentrated to one sample. DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent (catalogue no. P7589; Invitrogen AB, Lidingö, Sweden) and totals of 84, 450, and 87 ng DNA were obtained from the CH4×4, CH4×40, and H2:CH4×4 extractions, respectively. The DNA was sent deep frozen (on CO2 pellets) for microarray PhyloChip analysis.

2.16 Analysis using the G3 PhyloChip assay

This analysis was performed by Second Genome Inc. (San Bruno, CA, USA) as briefly outlined here; the methodology is completely described elsewhere (Hazen et al. 2010). The bacterial 16S rRNA genes were amplified using the degenerate forward primer 27F.1 5’-AGRGTTTGATCMTGGCTCAG-3’and the non-degenerate reverse primer 1492R.jgi 5’-GGTTACCTTGTTACGACTT-3’. For each sample, amplified products were concentrated using a solid-phase reversible immobilization method to purify the PCR products and quantified by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). PhyloChip Control Mix was added to each amplified product. Thirty-five cycles of bacterial 16S rRNA gene PCR amplification were performed. Labelled bacterial products were fragmented, biotin labelled, and hybridized to the PhyloChip Array, version G3. PhyloChip arrays were washed, stained, and scanned using a GeneArray scanner (Affymetrix, Santa Clara, CA, USA); each scan was captured using standard Affymetrix software (GeneChip Microarray Analysis Suite). Hybridization values, i.e., the fluorescence intensity, were calculated for each taxon as a trimmed average, with maximum and minimum values removed before averaging.

2.17 Statistical analyses

The PhyloChip data were evaluated using Second Genome’s PhyCA-Stats analysis software package. Data graphics design and statistical analyses were performed in Statistica 10 (Statsoft Inc., Tulsa, OK, USA).

20

21

3 RESULTS

3.1 Groundwater characterization

There were 3.7 × 104 cells mL1 and 2.8 × 104 amol ATP mL1 in the groundwater analysed on 23 September 2010. NRB dominated the MPN determination, and the MPNs of all culturable microorganisms except methanogens were above the detection limit (Table 3-1). The groundwater analysed on 8 November 2010 had a pH of 7.6 (analysed in the TVO laboratory) and a conductivity of 1520 mS m1. The major dissolved solids were 150 mM Cl, 91 mM Na+, 28 mM Ca2+, and 1.0 mM SO4

2. The following were also analysed: PO4

and NO3 at <0.01 µM, 1.7 µM NH4

+, 36 µM Fe2+, <0.01 µM HS, and 230 µM HCO3. There were 0.07 µM H2 and 4.5 mM CH4 dissolved in the groundwater as analysed on 23 September 2010, and carbon dioxide and N2 were also present in considerable concentrations (Table 3-2). The concentrations of dissolved gases in the transported groundwater were in the same range.

Table 3-1. The most probable numbers of culturable microorganisms and the total number of cells in groundwater from the drillhole ONK-PVA06 sampled on 23 September 2010. SD = standard deviation.

Analysis (cells mL1)

SD (n = 3)

Upper–lower 95 % confidence

interval (cells mL1)

Total number of cells (TNC) 37,000 6100 -

ATP (amol mL–1) 27,300 3170 -

Cultivable heterotrophic aerobic bacteria 16,300 3210 -

Nitrate-reducing bacteria 90,000 - 30,000–290,000

Iron-reducing bacteria 500 - 200–2000

Manganese-reducing bacteria 220 - 100–580

Sulphate-reducing bacteria 80 - 30–250

Autotrophic acetogens 17 - 7–48

Heterotrophic acetogens 110 - 40–300

Autotrophic methanogens <0.2 - -

Heterotrophic methanogens <0.2 - -

22

Table 3-2. The concentrations of dissolved gases in ONK-PVA06 groundwater sampled on 23 September 2010 and in the groundwater from ONK-PVA06 used to fill the flow cell cabinet circulations.

Gas 2010-09-23

µM 2011-03-14

µM

H2 0.078 <0.01

Helium 267 89.8

Argon 58.3 29.5

N2 3270 3200

Carbon monoxide 0.047 0.17

Carbon dioxide 2.73 3.67

Methane 4540 2780

3.2 pH, Eh, and gases

The pH increased from the starting value of approximately 7.4 to approximately 8.5 in the H2:CH4 FCC after 40 d, but did not increase in the other two FCCs (Figure 3-1 A). The Eh of the H2:CH4 FCC rapidly decreased to approximately –400 mV, as registered by the internal microelectrodes (Figure 3-1B). The Eh of the CH4 FCC decreased as well, reaching a steady level of –300 mV after 35 d, while the Eh of the O2:N2 FCC slowly decreased to 0 mV by the end of the experiment. A technical problem occurred with the electrodes in the O2:N2, which lost contact with the FCC between days 90 and 110. Sampling the rock grains for ATP and DNA analysis inevitably introduced small amounts of O2 when the FCs were opened, as indicated by the peaks in Eh after each sampling occasion (Figure 3-1B). This effect diminished within a few days, as expected from previous Äspö HRL results, indicating the effect of adding 0.1–0.2 mM O2 (Pedersen 2012b). The internal Eh electrodes reported these effects in continuous mode and were superior in sensitivity to the external Eh electrode (Figure 3-1C). The addition of gases resulted in expected concentrations of H2 (10 mM) and methane (11 mM) (Figure 3-1D, E). The O2 concentration was 2.2 mM in the O2:N2 system on day 3 and decreased linearly to 0.2 mM by day 105 (Appendix Table A-2).

The H2 concentration decreased exponentially in the H2:CH4 FCC from just below 10 mM at the start to 0.1 mM after 105 d as a result of microbial consumption and diffusion out of the system (Figure 3-1E). Similar decreasing trends of H2 in the FCCs have been observed and discussed previously (Pedersen 2012a). Both the CH4 and the H2:CH4 FCCs were amended with 8 mM methane, which supplemented the in situ concentration of 2.7 mM found in the O2:N2 system, resulting in a total of just below 11 mM methane (Figure 3-1D). The methane concentration decreased exponentially in the CH4 and the H2:CH4 FCCs from just below 11 mM at the start to 5.5 mM after 105 d, possibly due to microbial consumption. Diffusion of methane out of the system, or aerobic oxidation, did not occur in the inactivated N2:O2 FCC, suggesting that the drop in methane concentration observed in the amended FCCs was due to an AOM process.

23

A

C

E

B

D

F

Figure 3-1. (A) The pH and (B) Eh measured using internal electrode couples, average of four electrode signals, (C) Eh measured using an external multimeter (mV), (D) concentration of dissolved methane, (E) concentration of dissolved H2, and (F) concentration of dissolved carbon dioxide in FCC circulations containing added 2.2 mM O2 and 7.9 mM N2 (, blue symbol in B), 11 mM methane (, black symbol in B), and 10 mM H2 and 11 mM methane (, red symbol in B).

0 20 40 60 80 100

Time (days)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

pH

0 20 40 60 80 100

Time (days)

-400

-300

-200

-100

0

100

200

Eh (

mV

)

0 20 40 60 80 100

Time (days)

-2000

0

2000

4000

6000

8000

10000

12000

H2

(µM

)

0 20 40 60 80 100

Time (days)

0

2000

4000

6000

8000

10000

12000

CH

4 (µM

)

24

3.3 Numbers of cells, VLP, and ATP

The rock grains in the FCCs were colonized by microorganisms, as revealed by the ATP (Figure 3-2B) and DNA (Figure 3-5) analyses. Applying the previously observed average ATP content of approximately 0.4 amol per cell (Eydal and Pedersen, 2007; Pedersen, 2012a), the number of attached cells was found to average 1.1 × 109 cells per FCC, i.e., 106 amol ATP g–1 × 440 g FCC–1/0.4 amol ATP cell–1. That is, each FC contained 110 g rock grains and there were 4 FC in each FCC which gives 440 g rock in total in each FCC. The average amount of ATP per bacterium has been determined to 0.4 amol. The approximate number of cells per FCC could then be calculated from these numbers as the amount of ATP per g rock was quantified (Figure 3-2B). The corresponding average number of unattached cells was 0.5 × 109 cells per FCC (i.e., 1 × 105 cells mL–1 × 5000 mL). Consequently, approximately two thirds of the biomass was attached and one third was unattached in the FCCs.

The amount of ATP mL–1 and the TNC mL–1 did not differ significantly between the CH4 and the H2:CH4 FCCs, but was significantly lower in the O2:N2 FCC (Figure 3-2A, C). In contrast, the amounts of ATP g–1 rock grains did not differ over time between any of the FCCs (Figure 3-2B). The number of VLPs increased rapidly after 20 d in both the CH4 and the H2:CH4 systems, as indicated by the numbers of VLPs per cell in Figure 2D, while the VLP counts decreased to below detection (<100 VLPs mL–1) in the O2:N2 FCC after 20 d.

3.4 FCC chemistry

The sulphate concentration decreased by approximately 700 µM by day 105 relative to the starting concentration in the H2:CH4 FCC (Figure 3-3A). Sulphate decreased by 200 µM in the CH4 FCC and did not change significantly in the O2:N2 system. The sulphide concentration increased to approximately 250 µM in the H2:CH4 FCC and was below detection (0.01 µM) in the other two FCCs (Figure 3-3B). Ferrous iron increased to approximately 50 µM in the CH4 FCC and was below detection in the other FCCs, except on day 20 in the H2:CH4 FCC when the concentration was 20 µM (Figure 3-3C). An increase in pH was observed in the H2:CH4 FCC due to the protons consumed by the process of sulphate reduction with H2 (i.e. eq 3: 4 H2 + SO4

2– + H+ HS– + 4 H2O). The increase in pH in the H2:CH4 FCC also reduced the amount of dissolved carbon dioxide due to the formation of hydrogen carbonate and carbonate ions.

3.5 Cultivated microorganisms and organic acids and carbon

The MPN of SRB increased in the CH4 and the H2:CH4 FCCs, with the largest values observed in the CH4 FCC, while the MPN of SRB decreased to below detection (0.2 cells mL–1) in the O2:N2 FCC (Figure 3-3D). The MPN of SRB was five to ten times larger in the CH4 FCC than in the H2:CH4 FCC, but sulphate reduction, observed as a decrease in SO4

2– (Figure 3-3A), was only about 200 µM compared with the 700 µM decrease in the H2:CH4 FCC. While 700 µM sulphate was consumed, 250 µM sulphide was observed (Figure 3-3B); the remaining sulphide was likely precipitated as iron sulphide, which was observed as a black precipitate in the flow cells when sampling the rock grains. Ferrous iron was observed after 20 d (Figure 3-3C), while sulphide was below detection. Thereafter, any produced ferrous iron would have been precipitated

25

with sulphide. There were significant 1.5‰ and 2.5 ‰ increases in the δ34SV-CDT for sulphate in the CH4:H2 and the CH4 FCCs, respectively, compared with the inactive O2:N2 FCC, which indicates that microbiological sulphate reduction did occur (Table 3-3). This fractionation was larger per amount of reduced sulphate in the CH4 FCC (1.5/200 = 0.008) than in the H2:CH4 FCC (2.5/700 = 0.004), possibly due to the difference in SRB diversity between the FCCs that was revealed by the PhyloChip analysis (described next). This is because SRB species vary greatly in fractionation ability (Detmers et al. 2001).

In the CH4 FCC, the MPN of IRB and the concentration of ferrous iron were higher on day 105 than on day 0, while the MPN of MRB remained essentially the same (Table 3-3). In contrast, the MPNs of IRB and MRB decreased to values close to the detection limits in the H2:CH4 and the O2:N2 FCCs.

The MPNs of NRB were similar in the CH4 and the H2:CH4 FCCs throughout the experiment at 3.2 × 104 cells mL–1, compared with what was observed in the groundwater at the start (9 × 104 cells mL–1, Table 1), but decreased to <500 cells mL–1 in the O2:N2 FCC (Figure 3-4A). Likewise, the numbers of CHAB were similar throughout the experiment in all FCCs and averaged at approximately 4 × 103 cells mL–1 in the H2:CH4 and the O2:N2 FCCs and 2.5 × 104 cells mL–1 in the CH4 FCC (Figure 3-4B). The MPN of AA increased concomitantly in the CH4 and the H2:CH4 FCCs to at most 1 × 104 AA mL–1 after 60 d (Figure 3-4C) and the acetate concentration increased to approximately 300 µM acetate after 105 d in both these FCCs (Figure 3-4D). The concentration of DOC increased to 1.3 and 1.7 mM in the CH4 and the H2:CH4 FCCs, respectively, and it increased to less than 0.6 mM in the O2:N2 FCC (Figure 3-4E). The AA decreased to below detection and the acetate concentration did not change in the O2:N2 FCC. The MPNs of AM were below detection limits on all sampling occasions in all FCCs. Lactate was present at low concentrations, i.e., 10–16 µM, until day 40 when the concentration of this compound declined to below detection in all three FCCs.

Sampling the rock grains for ATP and DNA analysis inevitably introduced small amounts of oxygen when the FCs were opened, as indicated by the peaks in Eh after each sampling occasion (Figure 3-1B). This effect diminished within a few days, as was expected from previous Äspö HRL results, where the effect of adding 0.1–0.2 mM oxygen diminished within a few days (Pedersen 2012b). The internal Eh electrodes, which reported these effects in a continuous mode, were superior in sensitivity to the external electrode (Figure 3-1C). Introduction of 2.2 mM oxygen into the O2:N2 FCC was chosen as a method to create an inactivated control, and this approach was successful in that the inactivation effect was sustained for 105 d. The obligate anaerobic microorganisms, i.e., the MPN of SRB and AA and of the facultatively anaerobic NRB all decreased towards their detection limits in the O2:N2 FCC (Figure 3-3D, Figure 3-4A, C), while the aerobic CHAB results remained at approximately the day 3 values for most of the experiment. Anaerobic microbial processes were obviously halted by the oxygen addition, but some aerobic microbial activity may have been ongoing as suggested by the slowly decreasing Eh (Figure 3-1B) and oxygen concentration (Appendix Table A-2) in this FCC. There was no acetate production or acetogen growth in the O2:N2 FCC, while the acetate concentration and MPN of AA increased in the other two FCCs, which further attests to the inhibiting effect of the aeration. It also demonstrated that the acetate production in these systems and in the preceding

26

experiments (Pedersen 2012a,b) was microbiological and not an artefact of the degradation of FC plastic construction materials.

3.6 PhyloChip analysis

H2 addition narrowed the subfamily richness in the H2:CH4 FCC, as revealed by the PhyloChip analysis, versus in the CH4 FCC. Adding excess H2 to an ecosystem adapted to high concentrations of methane and only traces of H2 narrowed the subfamily richness and markedly changed the species abundance (Figure 3-5) compared with the in situ diversity reflected in the CH4 FCC. Bacterial subfamily richness was 199, 301, and 373 in the H2:CH4 × 4, CH4 × 4, and CH4 × 40 extraction samples, respectively, while archaeal subfamily richness was 3, 4, and 7, respectively (see section 2.15 for an explanation of the ×4 and ×40 extractions). Methanomicrobiales and Methanosarcinales were present in all extractions in low but significant and comparable amounts. A larger percentage of Pseudomonadaceae appeared to be present in the CH4 × 40 than the CH4 × 4 sample, but a larger percentage of Pseudomonadaceae was present in the CH4 × 4 than the H2:CH4 × 4 sample. A larger percentage of Xanthomonadaceae was present in the H2:CH4 × 4 sample than in the other two samples; the H2:CH4 × 4 sample had no Micrococcaceae, while this family accounted for 4 % of the bacterial subfamily richness in the CH4 samples. The number of operational taxonomic units (OTUs) in the Deltaproteobacteria class accounted for 3.9, 4.0, and 2.7 % of the OTUs in the H2:CH4 × 4, CH4 × 4, and CH4 × 40 samples, respectively. A circular tree showed that Firmicutes and Bacteroidetes OTUs varied in their abundance scores between the two treatments (Figure 3-5). In particular, some of the largest differences were found for a Clostridium sequence (OTU 32005) that was much more abundant in the H2:CH4 than the CH4 samples and for Bacteroidales sequences (OTUs 46869 and 47213) that were much more abundant in the CH4 than the H2:CH4 samples. Large differences were evident in the Proteobacteria, as suggested by the different percentages of Pseudomonadaceae and Xanthomonadaceae in the samples. The tree consequently showed that most of the OTU abundance scores diverged between the CH4 and the H2:CH4 samples, i.e., the different gas treatments resulted in different OTU abundances and diversities between the samples.

27

A

C

E

B

D

Figure 3-2. (A) ATP in circulation groundwater, (B) ATP on rock grains, (C) total number of cells (TNC) in circulating groundwater, (D) number of virus-like particles (VLP) per cell in circulating groundwater calculated as the quotient of VLP mL1 over TNC mL1, and (E) total number of virus-like particles (VLP) in circulating groundwater in the three flow cell cabinets (FCCs) containing added 2.2 mM O2 and 7.9 mM N2 (), 11 mM methane (), and 10 mM H2 and 10 mM methane (). Whiskers indicate ±1 standard deviation; n = 3 in (A) and (B) and n = 6 in (C).

0 20 40 60 80 100

Time (days)

3.5

4.0

4.5

5.0

5.5

6.0

10Lo

g(T

NC

) ce

lls m

L

0 20 40 60 80 100

Time (days)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

10Lo

g(V

LP)

viru

s m

L-1

0 20 40 60 80 100

Time (days)

0

2

4

6

8

10

VLP

cel

l-1

28

A

C

B

D

Figure 3-3. Concentrations of (A) sulphate, (B) sulphide, and (C) ferrous iron and (D) the most probable number (MPN) of sulphate-reducing bacteria (SRB) in the groundwater circulating through the three flow cell cabinets containing added 2.2 mM O2 and 7.9 mM N2 (), 11 mM methane (), and 10 mM H2 and 11 mM methane ().

29

A

C

E

B

D

F

Figure 3-4. (A) The numbers of nitrate-reducing bacteria (NRB), (B) cultivable heterotrophic aerobic bacteria (CHAB), and (C) autotrophic acetogens (AA) as well as the D) acetate, (E) lactate, and F) DOC concentrations in the groundwater circulating through the three flow cell cabinets containing added 2.2 mM O2 and 7.9 mM N2 (), 11 mM methane (), and 10 mM H2 and 11 mM methane (). Whiskers in B indicate ±1 standard deviation; n = 3.

30

Figure 3-5. A circular phylogenetic tree comparing the microarray hybridization results for DNA extracted from biomass attached to rock grains from flow cells treated with methane (CH4) and hydrogen and methane (H2:CH4). DNA was obtained from pooled CH4 × 4, CH4 × 40, and H2:CH4 × 4 extractions, respectively. In total, 4694 OTUs were present in at least one of the samples. There was an abundance score for each OTU in each of the three samples. OTUs for which the difference between the maximum and minimum abundance scores exceeded 300 were selected, bringing the total to 1193 OTUs, in which 182 families were represented. A representative 16S rRNA gene from each of the 182 families was aligned and used to infer a phylogenetic tree. The tree, taxonomy labels, and abundance data were rendered using Interactive Tree of Life Software (iTOL; http://itol.embl.de). The rings around the tree, from innermost to outermost, are CH4 × 40, CH4 × 4, and H2:CH4 × 4. The tree shows each sample’s abundance score for an OTU divided by the average abundance score for that OTU for all three samples. Blue indicates that the OTU was less abundant in that sample, and red indicates that the OTU was more abundant. The colour saturation indicates the degree of difference from the mean abundance score of the three samples, dark blue indicating a ratio of 0.90, white = 1.0, and dark red = 1.17.

Tab

le 3

-3.

The

mos

t pr

obab

le n

umbe

r of

iro

n- a

nd m

anga

nese

-red

ucin

g ba

cter

ia (

IRB

and

MR

B,

resp

ecti

vely

) an

d th

e δ34

S-ra

tio

for

sulp

hate

in

the

thre

e F

CC

s co

ntai

ning

add

ed 2

.2 m

M O

2 an

d 7.

9 m

M N

2 (O

2:N

2),

11 m

M m

etha

ne (

CH

4),

and

10 m

M H

2 an

d 11

mM

m

etha

ne (

H2:

CH

4). A

ll i

soto

pe r

atio

s ar

e re

port

ed i

n δ34

S no

tati

on (

per

mil

, ‰

) re

lati

ve t

o th

e V

ienn

a C

anyo

n D

iabl

o M

eteo

rite

Sta

ndar

d (V

-CD

T).

Sam

ple

d

ay

IRB

(ce

lls

mL1

)

(95%

con

fid

ence

lim

its,

cel

ls m

L1

)

MR

B (

cell

s m

L1

)

(95%

con

fid

ence

lim

its,

cel

ls m

L1

)

34S

V-C

DT

(‰)

±SD

, n =

3

O2:

N2

CH

4 H

2:C

H4

O

2:N

2 C

H4

H2:

CH

4

O2:

N2

CH

4 H

2:C

H4

0 14

0

(60–

360)

14

0 (6

0–36

0)

80

(36–

250)

500

(2

00–2

000)

30

0 (

100–

1300

) 90

0

(300

–290

0)

29

.57

(±0.

22)

29.9

2 (±

0.14

) 30

.58

(±0.

13)

3 -na

-

-

- -

-

30.2

7 (±

0.19

) 30

.04

(±0.

21)

29.8

5 (±

0.20

)

105

2.3

(0

.9–8

.6)

>16

00

(-)

13

(5–3

9

1.1

(0.

4.2.

9)

500

(20

0–20

00)

1.7

(0

.7–4

.6)

29

.14

(±0.

24)

31.6

9 (±

0.21

) 32

.64

(±0.

19)

SD

= S

tand

ard

devi

atio

n, n

= 3

, na

= n

ot a

naly

sed

31

32

33

4 DISCUSSION

4.1 The experimental conditions

Previous experiments under in situ pressure and groundwater chemistry conditions were performed from start to end in the underground Äspö HRL (Nielsen et al. 2006; Anderson et al. 2007; Pedersen 2012a,b). In the present work, the transfer of the pressurized flow cells containing biofilms and groundwater to the FCCs in the ground laboratory offered improved continuous surveillance of the systems and more efficient sampling and analysis conditions than did the previous underground installation of the FCCs. The circulation of water was halted during transport, which may have triggered attached microorganisms to detach. The numbers of microorganisms were generally higher in the day 0 than the day 3 sampling results (appendix Table A-1). The day 0 samples were obtained before adding gases and filling the expansion vessels in the FCCs with new ONK-PVA06 groundwater, which explains the difference between the day 0 and day 3 results. Fewer cells were present in the groundwater taken directly from ONK-PVA06 than in the FCCs after transportation. The starting values on day 3 correlate well with the numbers observed in ONK-PVA06 groundwater (Table 3-1).

4.2 The cultivation approach

It is often argued that most environmental microorganisms cannot be cultured (Sharma et al. 2005). In the present work, we successfully applied various culture procedures developed for the sampled environment (Vartoukian, et al. 2010). The results of the MPN cultivation with the electron acceptors Fe(III), Mn(IV), NO3

–, SO42–, and CO2 and

the electron donors H2, acetate, and lactate indicated the presence of microorganisms capable of iron, manganese, nitrate, and sulphate reduction. The detection limit for the MPN analysis was 0.2 cells mL–1 (Greenberg et al. 1992), which makes this analysis very sensitive to cultivable microorganisms. Here, the proportion of TNC detected using MPN analysis was more than 50 % for NRB and SRB on several sampling occasions in the CH4 FCC and 10–50 % on most other sampling occasions in the CH4 and the H2:CH4 FCCs (see Appendix for details). Consequently, the varied cultivation approach was successful, revealing that the microbial populations were dominated by cultivable microorganisms capable of nitrate and sulphate reduction and of acetate formation in the CH4 and the H2:CH4 FCCs, while IRB and MRB were found in significant numbers in the CH4 FCC (Table 3-3). However, as many microorganisms are facultative with respect to electron donors and acceptors, one cannot conclude the presence or absence of a metabolic process based solely on MPN results. The observed production of ferrous iron (Figure 3-3C), sulphide (Figure 3-3B), and acetate (Figure 3-4D) indicates that the cultivated microorganisms were active and expressed the metabolic activities that correlated with the MPN results. The MPN results were also in line with those for the original ONK-PVA06 groundwater, in which NRB was the dominant cultivable group of microorganisms, followed by IRB and MRB (Table 3-1). It can be concluded that the CH4 FCC reproduced the growth conditions in the studied aquifer well, based on the similarity in the TNC, ATP, CHAB, and MPN numbers. The increase in the MPN and metabolic activity of SRB in the CH4 and the H2:CH4 FCCs is discussed below.

34

4.3 Cell density control by bacteriophages

The presence of a viral mechanism of population control, similar in function to a viral shunt (Suttle 2005), in deep groundwater was indicated in previous FCC experiments (Pedersen 2012a, b). It was hypothesized that bacteriophages exerted a significant mitigating effect on the numbers of microbial cells and on the observed production rate of sulphide, but VLP data were not obtained. In this investigation, the VLP/TNC data indicated that bacteriophages increased from 0.2 to 5 and 9 bacteriophages cell–1 after day 20 in the CH4 and the H2:CH4 FCCs, respectively (Figure 3-2D), and that the TNC did not exceed 2.6 × 105 cells mL–1 in any of the FCCs. In contrast, VLPs were below detection in the O2:N2 FCC, which indicates that the metabolic activity of microorganisms in this system was likely inactivated by the O2 addition to a level at which lytic phage activity was arrested. Taken together, it seems reasonable to conclude that bacteriophages controlled the number of cells in this experiment, keeping it at a cell density within the range observed in deep Fennoscandian groundwater, i.e., between 5 × 103 and 1 × 106 cells mL1 (Hallbeck and Pedersen 2012; Pedersen et al. 2008).

4.4 H2-induced sulphate reduction

The marked sulphate reduction induced by H2 was expected because many SRB have low half-saturation constants (Km) and maximum uptake rates (Vmax) for sulphate and hydrogen. For example, Sonne-Hansen et al. (1999) determined Km to be approximately 3 µM for sulphate and approximately 2 µM for H2 for two thermophilic SRB. That was in line with the constants determined for mesophilic SRB (Ks = 1 µM) (Kristjansson et al. 1982; Robinson and Tiedje, 1984). These numbers suggest that the SRB metabolized at the maximum sulphate-reduction rate, Vmax, in the H2:CH4 FCC, because the sulphate and hydrogen concentrations were much higher than the respective Km values. The stable isotope fractionation data confirmed that biological sulphate reduction was present. Although there was an excess of H2 in this FCC, which would suggest a rapid increase in the numbers of SRB, these numbers did not increase above 5000 cells mL–1, likely because the cell density was controlled by bacteriophages (Eydal et al. 2009).

4.5 Methane-induced acetogenesis and sulphate reduction

AOM is thought to proceed via several pathways with a range of final electron acceptors (Caldwell et al. 2008). Some of these pathways include the formation of acetate from methane (Valentine and Reeburgh, 2000). Both the CH4 and the H2:CH4 populations produced much more acetate and DOC than did the inactivated control (Figure 4-4D, F), and the produced acetate constituted approximately 1/3 of the DOC in both systems. The only available electron donor and source of reduced carbon that can explain the formation of organic carbon in these systems was methane. Various processes reduced the methane concentration by approximately 5 mM in the methane-amended systems, which more than balanced the amounts of DOC formed. If an AOM-driven metabolism were present, biomass should be formed as well; however, because phages were active (Figure 3-2D), a net increase in TNC or ATP was not observed (Figure 3-2A–C). Possibly, growing cells were lysed by phages and the released cytoplasmic material contributed to the increase in DOC concentration over time. Some of the difference between consumed methane (approximately 5 mM) and the produced DOC (approximately 1 mM) could be present as biomass in biofilms and as extracellular

35

polymers in the biofilms. Scanning electron microscopy has previously indicated that the biofilms in FCCs do produce such extracellular material, (Anderson et al. 2006). Total organic carbon in biofilms was not analysed this time; it can be added in next experiment. Anaerobic oxidation of methane will result in expiration of carbon dioxide from cell metabolism and some more of the discrepancy between consumed methane and produced DOC can be allocated to this process according to Eq 5 (See section 1.2). However, the amount of consumed sulphate did not balance the decrease in methane. This could partly be due to some re-oxidation of sulphide to sulphate but there may also be other, unknown, causes behind this discrepancy, including uncertainties in how well methane was kept from diffusing out from the FCCs added with methane and microorganisms that oxidise methane with other electron acceptors than sulphate.

The PhyloChip indicated a limited presence of the typical AOM orders Methanomicrobiales and Methanosarcinales (Knittel and Boetius 2009) in the rock grain biofilms; various groups of SRB were also indicated, but methanogens could not be cultured. AOM is assumed to involve methanotrophic Archaea and syntrophic partners, and the absence of a large representation of Archaea in the populations studied here could imply that the generally accepted AOM process (Knittel and Boetius, 2009) may not be relevant to the studied deep groundwater. An alternative possibility that merits more research could be unknown AOM consortia that include methanotrophic bacteria such as Methylomirabilis oxyfera (Wu et al. 2011), that utilize other electron acceptors than NO2

which is used by M. oxyfera, for example, Fe(III) or Mn(IV).

4.6 Sulphate reduction rates

When the FCCs were moved to the laboratory after 110 d in the ONKALO tunnel, microorganisms had attached themselves to the crushed rock grains in the flow cells and were present in the circulating groundwater. The amount of ATP per cell in deep groundwater is approximately 0.4 amol (Eydal and Pedersen 2007). The average numbers of attached and unattached microorganisms, considering the amount of ATP times 2, were similar in all FCCs, and together their numbers totalled from 5.4 × 109 to 12× 109 cells (Table 4-1). Sulphate reduction was ongoing in the H2:CH4 FCC from day 21 to day 63 as judged from the decrease in sulphate (Figure 3-3A), while the sulphate reduction appeared later, between days 63 and 105, in the CH4 FCC. This time interval, 42 d for both the H2:CH4 and the CH4 FCCs, was used in calculating sulphate reduction rates (Table A-5) and the results are shown in Table 4-1. Because 475 mL of water was removed on the three first sampling occasions, the number of unattached microorganisms and the amount of sulphide and sulphate decreased correspondingly; this decrease was considered by using the remaining volume of water on day 64 as an average for the amount of water in the FCCs.

Sulphate reduction was observed in the CH4 and the H2:CH4 FCCs but not in the O2:N2 FCC, where the change in sulphate concentration was below the 95 % confidence limits of distribution. Two cases were calculated for sulphate reduction, one in which all microorganisms were assumed to be sulphate-reducing and another in which it was assumed that 25 % of the populations were sulphate-reducing (Table 4-1). The exact numbers of SRB were impossible to establish. However, the MPN indicated that SRB constituted a few percent of the TNC in the H2:CH4 system and approximately 10 % in the CH4 system. The proportion of SRB among the attached cells could not be analysed,

36

but the PhyloChip indicated that SRB species were present with high hybridization scores. If SRB constituted 25 % of the total number of cells in the CH4 and the H2:CH4 FCCs, the sulphide production rates of SRB were then 0.25 × 1015 mol h1 cell1 in the CH4 FCC and five times higher, i.e., 1.25 × 1015 mol h1 cell1, in the H2:CH4 FCC (Table 4-1). This is within the range found in pure cultures under good growth conditions (i.e., 0.01–2 × 1015 mol h1 cell1) (Jörgensen 1978) and also similar to what has been found in Äspö HRL groundwater (Pedersen 2012a). Obviously, the SRB must have had good growth conditions in these FCCs. Still, the amounts of sulphide produced after 105 d, i.e., 0.75 and 3.2 mM, respectively, were very modest compared with what is often obtained in pure cultures, in which 15 mM can be produced in a few days (Reis et al. 1992). This could be explained by the observed phage control of the studied populations (Figure 3-2D, E). If phages grazed the SRB, they would have kept the numbers of SRB at the steady states observed (Figure 3-3D) which then resulted in a sustainable situation with respect to available electron donors and acceptors in which the SRB that survived phage attacks could be metabolically very active. The observed steady state in MPN of SRB could then represent the sum of cell growth and phage-induced cell lysis. Individual SRB cells may consequently have been growing and metabolizing at much higher rates than those observed for the total population, something clearly indicated by the high sulphide production rate per cell (Table 4-1).

Table 4-1. Average total populations of microorganisms in the three flow cell cabinets as analysed from days 3 to 105 and calculated sulphide production rates; see Table A-5 for details. These data are compared with data from previous experiments in the Äspö Hard Rock Laboratory (HRL) tunnel (Pedersen 2012a).

Treatment Population

size (cells FCC1)

Sulphide* (mol h1)

Sulphide* (mol cell1

h1)

Sulphide* (mol SRB1

h1)

ONK-PVA06 – ONKALO

2.1 mM O2 + 7.9 mM N2 5.40 × 109 <0.02 <0.01 × 1015 <0.05 × 1015

10 mM CH4 12.0 × 109 0.179 0.062 × 1015 0.25 × 1015

10 mM H2 + 10 mM CH4 9.90 × 109 0.753 0.32 × 1015 1.25 × 1015

KJ0052F01 – Äspö (HRL)

Control – no additions 1.91 × 109 0.090 0.22 × 1015 0.88 × 1015

2.4 mM Acetate 2.39 × 109 0.068 0.12 × 1015 0.48 × 1015

3 mM H2 + 0.75 mM CO2 3.52 × 109 0.208 0.27 × 1015 1.09 × 1015

*Sulphide was calculated from the amount of consumed sulphate as shown in Table A-5

37

4.7 Conclusions

The SURE programme aims to quantify microbial activity, particularly the rate of sulphate reduction, in Olkiluoto groundwater. This aim was met by using circulating systems with flow cells that mimicked in situ pressure, chemistry, and biology conditions. The first in a series of SURE investigations has now been completed and the following observations and conclusions were made:

The experimental conditions reproduced previously observed numbers and diversity of microorganisms in Olkiluoto groundwater.

Attached microorganisms constituted approximately 2/3 of the total biomass. The proportion of groundwater per surface area will be smaller in aquifers than was the case in this experiment. This observation emphasizes the importance of including analysis of attached biomass in ongoing site investigations.

The microbial populations were under population number control by bacteriophages. Despite a significant load of energy in the form of H2 and/or CH4, the numbers of cells were in a steady state. This important observation suggests that the overall rate of sulphate reduction will be limited by bacteriophages, even under conditions that should allow unlimited growth. Individual sulphate-reducing cells may be very active, but as the population size is controlled by bacteriophages, a steady state will be present, which may explain why the total number of cells never exceeded 106 cells mL1 in deep Fennoscandian groundwater.

H2 may be produced locally, for example from the anaerobic corrosion of iron in the repository, so the effect of H2 was investigated. Adding H2 changed the diversity of the population, narrowed the sub-family richness, and increased the sulphate reduction rate.

A preliminary estimate of sulphate reduction rates was obtained. The rate was 0.18 mol h1 under methanic conditions, increasing fivefold with the addition of H2 to 0.75 mol h1.

A growing number of indications attest to the presence of a microbiological anaerobic methane oxidation process with sulphate, but conclusive data are still lacking. These indications are: the depth profiles of methane, sulphate, and sulphide; obtained 16S rDNA data; and the results of this SURE investigation, i.e., the CH4 FCC results. Continued SURE investigations, combined with radioisotope experiments (14CH4) and stable isotope (13CH4) analysis of ONKALO cultures in the laboratory, may eventually settle this issue.

The growth-stimulating effect of hydrogen on the investigated communities was expected because this gas is readily used by many different phylogenetic traits of anaerobic microorganisms, such as SRB (Barton and Fauque 2009), acetogens (Drake et al. 2002), and methanogens (Ferry 1992). Regarding the anaerobic metabolism of methane, the situation is less clear. Although AOM with sulphate as the final electron acceptor is a well-documented process, detailed elucidation of the metabolic pathways of AOM awaits successful cultures (Knittel and Boetius 2009). The combined use of cultivation with microarrays and chemical analysis in the present work resulted in a large dataset. As discussed, the data indicated that methane was a source of electrons and energy for metabolic activity, possibly in combination with acetate formation and

38

sulphate reduction, but clear evidence of such an AOM process was not obtained. However, the present data form a solid basis for designing future experiments examining AOM with sulphate in deep Olkiluoto groundwater.

In the event that sulphate-rich groundwater penetrates to the SNF repository in Olkiluoto and mixes with deep, methane-rich groundwater, this work has demonstrated an apparent potential for microbial sulphate reduction to sulphide via two metabolic processes. First, in addition to the naturally occurring hydrogen in Olkiluoto groundwater, iron in water-filled deep underground SNF constructions is bound to corrode anaerobically with the concomitant production of H2 (Reardon 1995), which may induce SRB growth and sulphide production. Second, the in-mixing of sulphate-rich groundwater with the deep methane-rich groundwater at repository depth will induce SRB growth and activity. The electron donor may be methane, but more research is required before conclusions can be drawn regarding the detailed nature and extent of an AOM process in Olkiluoto.

39

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A.

AP

PE

ND

IX

Tab

le A

-1. p

H, E

h, a

nd c

hem

istr

y da

ta.

FC

C

Sam

ple

d

(Y-M

-D)

Tim

e (d

ay)

pH

R

edox

m

V

Fe2+

M

Su

lph

ide

M

Su

lph

ate

mM

A

ceta

te

M

Lac

tate

M

SU

RE

O2:

N2

20

11-0

3-14

0

7.45

80

.7

1.08

0.

00

1.27

9.

7 10

.7

SU

RE

CH

4 20

11-0

3-14

0

7.48

79

.3

0.72

0.

00

1.23

19

.3

16.0

SU

RE

H2:

CH

4 20

11-0

3-14

0

7.64

76

.3

0.90

0.

00

1.19

19

.4

21.4

SU

RE

O2:

N2

20

11-0

3-18

3

7.37

14

0 0.

18

0.00

1.

25

38.4

10

.7

SU

RE

CH

4 20

11-0

3-18

3

7.23

14

6 0.

54

0.00

1.

19

28.8

10

.7

SU

RE

H2:

CH

4 20

11-0

3-18

3

7.45

–1

55

0.36

0.

00

1.17

19

.2

10.7

SU

RE

O2:

N2

20

11-0

4-05

21

7.

10

200

0.54

0.

00

1.25

48

.2

10.7

SU

RE

CH

4 20

11-0

4-05

21

7.

24

–89.

9 20

.4

0.00

1.

25

57.8

16

.0

SU

RE

H2:

CH

4 20

11-0

4-05

21

7.

68

–309

20

.6

3.81

1.

19

38.7

16

.0

SU

RE

O2:

N2

20

11-0

4-26

42

6.

88

181

1.08

0.

00

1.23

67

.3

16.0

SU

RE

CH

4 20

11-0

4-26

42

7.

36

–134

38

.5

0.00

-

151

0.0

SU

RE

H2:

CH

4 20

11-0

4-26

42

8.

45

–342

0.

36

80.6

0.

90

114

10.7

SU

RE

O2:

N2

20

11-0

5-17

63

6.

98

200

1.08

0.

00

- 48

.4

0.0

SU

RE

CH

4 20

11-0

5-17

63

7.

51

–133

19

.7

0.00

1.

22

188

0.0

SU

RE

H2:

CH

4 20

11-0

5-17

63

8.

73

–342

0.

72

208

0.47

18

8 0.

0

SU

RE

O2:

N2

20

11-0

6-07

84

6.

86

171

0.36

0.

00

1.21

95

.7

0.0

SU

RE

CH

4 20

11-0

6-07

84

7.

21

–168

53

.8

1.44

1.

10

312

0.0

SU

RE

H2:

CH

4 20

11-0

6-07

84

8.

47

–338

0.

90

259

0.45

23

3 0.

0

SU

RE

O2:

N2

20

11-0

6-28

10

5 7.

20

125

0.36

0.

00

1.21

67

.5

0.0

SU

RE

CH

4 20

11-0

6-28

10

5 7.

26

–161

48

.2

0.00

1.

04

320

0.0

SU

RE

H2:

CH

4 20

11-0

6-28

10

5 8.

41

–333

0.

72

261

0.44

28

6 0.

0

43

Tab

le A

-2. G

as d

ata.

FC

C

Sam

ple

d

(Y-M

-D)

Tim

e (d

ay)

H2

M

CH

4 m

M

CO

2 M

H

e M

O

2 M

SU

RE

O2:

N2

20

11-0

3-14

0

<0.

0127

205.

33

88.8

<10

SU

RE

CH

4 20

11-0

3-14

0

<0.

0127

723.

67

89.8

<10

SU

RE

H2:

CH

4 20

11-0

3-14

0

<0.

0126

802.

41

88.8

<10

SU

RE

O2:

N2

20

11-0

3-18

3

<0.

0121

705.

28

45.5

2221

.94

SU

RE

CH

4 20

11-0

3-18

3

<0.

0199

008.

35

72.5

<10

SU

RE

H2:

CH

4 20

11-0

3-18

3

9830

1079

07.

65

81.4

<10

SU

RE

O2:

N2

20

11-0

4-05

21

<

0.01

2370

12.2

40

.818

81.3

2

SU

RE

CH

20

11-0

4-05

21

<

0.01

9280

14.1

36

.1<

10

SU

RE

H2:

CH

4 20

11-0

4-05

21

62

4091

708.

8 29

.8<

10

SU

RE

O2:

N2

20

11-0

4-26

42

<

0.01

2424

17.3

36

.213

55.3

3

SU

RE

CH

4 20

11-0

4-26

42

<

0.01

8190

- 22

.5<

10

SU

RE

H2:

CH

4 20

11-0

4-26

42

34

1083

101.

33

17.3

<10

SU

RE

O2:

N2

20

11-0

5-17

63

<

0.01

2160

27

27.3

752.

39

SU

RE

CH

4 20

11-0

5-17

63

<

0.01

6560

17.3

10

.4<

10

SU

RE

H2:

CH

4 20

11-0

5-17

63

16

8066

401.

08

14.6

<10

SU

RE

O2:

N2

20

11-0

6-07

84

<

0.01

2430

29.0

26

.241

0.64

SU

RE

CH

4 20

11-0

6-07

84

<

0.01

6890

28.0

9.

1<

10

SU

RE

H2:

CH

4 20

11-0

6-07

84

13

2072

400.

0 6.

9<

10

SU

RE

O2:

N2

20

11-0

6-28

10

5 <

0.01

2200

35.7

21

.621

6.17

SU

RE

CH

4 20

11-0

6-28

10

5 <

0.01

5950

32.3

11

.9<

10

SU

RE

H2:

CH

4 20

11-0

6-28

10

5 53

855

300.

00

8.0

<10

44

Tab

le A

-3. M

icro

biol

ogy

data

FC

C

Sam

ple

d

(Y-M

-D)

Tim

e (d

ay)

TN

C

(cel

ls

mL1

) S

D

n

VL

P

(vir

us

mL1

) S

Dn

A

TP

(a

mol

m

L1

) S

D

n

AT

P

(am

ol g

1)

SD

n

C

HA

B(c

ells

m

L1

) S

D

n

SU

RE

O2:

N2

20

11-0

3-14

0

1800

0022

000

3 37

800

- 1

1370

000

8020

03

1.25

E+

061.

26E

+05

6 69

000

1850

0 3

SU

RE

CH

4 20

11-0

3-14

0

1700

0049

000

3 40

000

- 1

2980

000

9380

03

1.16

E+

065.

90E

+04

6 10

8000

1770

0 3

SU

RE

H2:

CH

4 20

11-0

3-14

0

1600

0014

000

3 10

0-

173

1000

20

400

3 1.

24E

+06

2.48

E+

046

6070

015

500

3

SU

RE

O2:

N2

20

11-0

3-18

3

9300

019

000

3 81

4-

132

900

1710

3 9.

30E

+05

1.58

E+

056

7830

651

3

SU

RE

CH

4 20

11-0

3-18

3

8800

063

003

100

- 1

3000

0 25

803

1.55

E+

066.

33E

+04

6 40

7035

1 3

SU

RE

H2:

CH

4 20

11-0

3-18

3

6800

013

000

3 10

0-

124

600

1560

3 1.

72E

+06

1.48

E+

056

2600

1280

3

SU

RE

O2:

N2

20

11-0

4-05

21

98

0089

03

100

- 1

7410

41

53

1.15

E+

057.

27E

+04

6 11

0012

0 3

SU

RE

CH

4 20

11-0

4-05

21

97

000

4100

03

6840

- 1

1080

00

4010

3 8.

65E

+05

2.47

E+

056

1600

025

00

3

SU

RE

H2:

CH

4 20

11-0

4-05

21

11

0000

7600

3 18

700

- 1

4430

0 38

203

9.84

E+

051.

85E

+05

6 14

000

800

3

SU

RE

O2:

N2

20

11-0

4-26

42

11

0000

2400

03

4800

00-

114

300

407

3 4.

26E

+05

2.37

E+

056

7500

1100

3

SU

RE

CH

4 20

11-0

4-26

42

20

0000

3600

03

9560

00-

183

400

6890

3 8.

34E

+05

2.63

E+

056

2800

015

00

3

SU

RE

H2:

CH

4 20

11-0

4-26

42

17

0000

1100

03

3640

00-

135

400

3860

3 3.

71E

+05

8.65

E+

036

6900

250

3

SU

RE

O2:

N2

20

11-0

5-17

63

62

000

4800

3 30

4000

- 1

5510

25

23

1.38

E+

065.

79E

+04

6 36

0020

0 3

SU

RE

CH

4 20

11-0

5-17

63

15

0000

5200

03

2210

00-

142

600

1850

3 1.

21E

+06

9.20

E+

046

1600

080

0 3

SU

RE

H2:

CH

4 20

11-0

5-17

63

20

0000

3900

03

7940

0-

128

600

1060

3 1.

57E

+06

1.19

E+

056

3400

200

3

SU

RE

O2:

N2

20

11-0

6-07

84

31

000

9800

3 11

700

- 1

7660

79

93

2.28

E+

054.

28E

+04

6 58

0052

9 3

SU

RE

CH

4 20

11-0

6-07

84

26

0000

9800

03

9800

00

165

300

2490

3 7.

90E

+05

1.42

E+

056

6330

014

600

3

SU

RE

H2:

CH

4 20

11-0

6-07

84

16

0000

2300

03

6430

0

136

800

4060

3 1.

21E

+05

2.07

E+

046

3400

265

3

SU

RE

O2:

N2

20

11-0

6-28

10

5 51

000

6300

3 15

5000

1

1010

0 11

603

2.61

E+

056.

76E

+04

6 50

3080

8 3

SU

RE

CH

4 20

11-0

6-28

10

5 16

0000

4000

03

1780

00

147

000

3170

3 2.

35E

+05

9.86

E+

036

1600

019

90

3

SU

RE

H2:

CH

4 20

11-0

6-28

10

5 15

0000

2000

03

9490

0

120

600

1080

3 7.

01E

+04

6.94

E+

036

3970

1100

3

45

Tab

le A

-4. M

ost p

roba

ble

num

ber

data

; L

and

U li

mit

s ar

e th

e 95

% c

onfi

denc

e va

lues

.

FC

C

Sam

ple

d

(Y-M

-D)

Tim

e (d

ay)

NR

B

(cel

ls

mL1

)

L

lim

it

U

lim

it

SR

B

(cel

ls m

L1

) L

lim

it

U li

mit

A

A

(cel

ls

mL1

)

L

lim

it

U

lim

it

SU

RE

O2:

N2

20

11-0

3-14

0

<16

0000

--

220

100

580

220

100

580

SU

RE

CH

4 20

11-0

3-14

0

<16

0000

--

170

70

480

170

7040

0

SU

RE

H2:

CH

4 20

11-0

3-14

0

<16

0000

--

3000

10

00

1100

0 28

1269

SU

RE

O2:

N2

20

11-0

3-18

3

1700

070

0048

000

5 2

17

10

3

SU

RE

CH

4 20

11-0

3-18

3

1700

700

4800

8 3

25

0.4

0.1

1.5

SU

RE

H2:

CH

4 20

11-0

3-18

3

9000

4000

2500

030

10

12

0 0.

20.

11

SU

RE

O2:

N2

20

11-0

4-05

21

17

0080

041

000

0 1

0.4

0.1

1.5

SU

RE

CH

4 20

11-0

4-05

21

30

000

1000

013

0000

7000

30

00

2100

0 7

321

SU

RE

H2:

CH

4 20

11-0

4-05

21

30

000

1000

013

0000

3300

15

00

7700

14

636

SU

RE

O2:

N2

20

11-0

4-26

42

50

0020

0017

000

0.8

0.3

2.4

0.2

0.1

1.1

SU

RE

CH

4 20

11-0

4-26

42

50

000

2000

017

0000

1300

0 50

00

3900

0 50

2017

0

SU

RE

H2:

CH

4 20

11-0

4-26

42

30

000

1000

012

0000

3000

10

00

1200

0 3

112

SU

RE

O2:

N2

20

11-0

5-17

63

17

080

410

0.2

- -

0.2

--

SU

RE

CH

4 20

11-0

5-17

63

80

000

3000

025

0000

2400

0 10

000

9400

0 14

000

6000

3600

0

SU

RE

H2:

CH

4 20

11-0

5-17

63

17

000

7000

4800

050

00

2000

17

000

7000

3000

2100

0

SU

RE

O2:

N2

20

11-0

6-07

84

80

030

024

000.

2 -

- 0.

2-

-

SU

RE

CH

4 20

11-0

6-07

84

80

000

3000

025

0000

2200

0 10

000

5800

0 13

050

390

SU

RE

H2:

CH

4 20

11-0

6-07

84

11

000

4000

3000

017

00

700

4800

7

321

SU

RE

O2:

N2

20

11-0

6-28

10

5 35

016

082

00.

2 0.

1 1.

1 0.

2-

-

SU

RE

CH

4 20

11-0

6-28

10

5 13

000

5000

3900

024

000

1000

0 94

000

31

12

SU

RE

H2:

CH

4 20

11-0

6-28

10

5 70

000

3000

021

0000

3000

10

00

1200

0 17

748

46

47

Table A-5. Calculation of sulphate reduction rates shown in Table 4-1.

O2:N2 FCC

Sulphate, day 3: 1250 M

Sulphate, day 105: 1210 M

Reduced sulphate = formed sulphide: 40 M

Rate of sulphide formation, days 3–105: 0.016 M h1

Volume of water, day 64: 4150 mL

Total amount of formed sulphide, day 105: 166 mole

Average number of unattached cells (Table A-3) TNC = 1.5 × 109 cells

Average number of attached cells (Table A-3) ATP × 2 = 3.9 × 109 cells

Average total number of cells 5.4 × 109 cells FCC1

Sulphide production rate by all cells 166 × 106/(5.4 × 109 ×24 × 102) = 0.01 ×

1015 mole cell1 h1

Assuming 25% of total were SRB 1.3 × 109 SRB FCC1

Sulphide production rate by SRB 166 × 106 /(1.3 × 109 ×24 × 102) = 0.05×

1015 mole cell1 h1

CH4 FCC

Sulphate, day 63: 1220 M

Sulphate, day 105: 1040 M

Reduced sulphate = formed sulphide: 180 M

Rate of sulphide formation, days 3–105: 0.179 M h1

Volume of water, day 64: 4150 mL

Total amount of formed sulphide, day 105: 747 mole

Average number of unattached cells (Table A-3) TNC = 4.0 × 109 cells

Average number of attached cells (Table A-3) ATP × 2 = 8.0 × 109 cells

Average total number of cells 12 × 109 cells FCC1

Sulphide production rate for all cells 747 × 106/(12 × 109 ×24 × 42) = 0.062 × 1015

mole cell1 h1

Assuming 25% of total were SRB 1.3 × 109 SRB FCC1

Sulphide production rate by SRB 747 × 106 /(13 × 109 ×24 × 42) = 0.25×

1015 mole cell1 h1

48

H2:CH4 FCC

Sulphate, day 21: 1200 M

Sulphate, day 63: 440 M

Reduced sulphate = formed sulphide: 760 M

Rate of sulphide formation, days 3–105: 0.753 M h1

Volume of water, day 64: 4150 mL

Total amount of formed sulphide, day 105: 3154 mole

Average number of unattached cells (Table A-3) TNC = 3.6 × 109 cells

Average number of attached cells (Table A-3) ATP × 2 = 6.3 × 109 cells

Average total number of cells 9.9 × 109 cells FCC1

Sulphide production rate for all cells 3154 × 106/(9.9 × 109 ×24 × 42) = 0.32 ×

1015 mole cell1 h1

Assuming 25% of total were SRB 1.3 × 109 SRB FCC1

Sulphide production rate by SRB 3154 × 106 /(2.5 × 109 ×24 × 42) = 1.26×

1015 mole cell1 h1