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Energy and the Environment
The year-long development of microorganisms in uncompacted Bavarian bentonite slurries at 30 °C and 60 °C Nicole Matschiavelli, Sindy Kluge, Carolin Podlech, Daniel Standhaft, Georg Grathoff, Atsushi Ikeda-Ohno, Laurence Noel Warr, Alexandra Chukharkina, Thuro Arnold, and Andrea Cherkouk Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02670 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019
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Environmental Science & Technology
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The year-long development of microorganisms in uncompacted
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Bavarian bentonite slurries at 30 °C and 60 °C
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Nicole Matschiavelli,*,1 Sindy Kluge,1 Carolin Podlech,2 Daniel Standhaft,2 Georg
4
Grathoff,2 Atsushi Ikeda-Ohno,1 Laurence N. Warr,2 Alexandra Chukharkina,3
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Thuro Arnold,1 Andrea Cherkouk*,1
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1Helmholtz-Zentrum
7
Landstraße 400, 01328 Dresden, Germany
8
2University
9
Straße 17a, 17487 Greifswald, Germany
Dresden-Rossendorf, Institute of Resource Ecology, Bautzner
of Greifswald, Institute of Geography and Geology, Friedrich-Ludwig-Jahn-
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3Microbial
Analytics Sweden AB, Mölnlycke Fabriker 9, 43535 Mölnlycke, Sweden
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KEYWORDS high-level radioactive waste repository, sulfate-reduction, spores,
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thermophiles
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ABSTRACT In the multi-barrier concept for the deep geological disposal of high-level
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radioactive waste (HLW), bentonite is proposed as a potential barrier and buffer material
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for sealing the space between the steel-canister containing the HLW and the surrounding
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host rock. In order to broaden the spectra of appropriate bentonites, we investigated the
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metabolic activity and diversity of naturally occurring microorganisms as well as their time-
19
dependent evolution within the industrial B25 Bavarian bentonite under repository-
20
relevant conditions. We conducted anaerobic microcosm-experiments containing the B25
21
bentonite and a synthetic Opalinus Clay pore water solution, which were incubated for
22
one year at 30 °C and 60 °C. Metabolic activity was only stimulated by the addition of
23
lactate, acetate or H2. The majority of lactate- and H2-containing microcosms at 30 °C
24
were
dominated
by
strictly
anaerobic,
sulfate-reducing
and
spore-forming
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microorganisms. The subsequent generation of hydrogen sulfide led to the formation of
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iron-sulfur precipitations. Independent from the availability of substrates, thermophilic
27
bacteria dominated microcosms that were incubated at 60 °C. However, in the respective
28
microcosms, no significant metabolic activity occurred and there was no change in the
29
analyzed
30
microorganisms of B25 bentonite evolve in a temperature- and substrate-dependent
31
manner.
bio-geochemical
parameters.
Our
findings
show
that
indigenous
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INTRODUCTION
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The long-term disposal of high-level radioactive waste (HLW) is currently an issue of
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global importance. One solution for the disposal of HLW is the construction of deep
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underground repositories with a system of multiple containment barriers engineered to
36
isolate the waste from the biosphere. Such repositories need to remain intact for a long
37
period of time (one million years) so that the concentration of any residual radioactive
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substances reaching the groundwater surrounding the repository remains negligible.1 The
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proposed multi-barrier concept consists of the natural, geological barrier (host rock), a
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buffering and sealant material and the metal canister containing the HLW.2,3 Bentonite is
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a favored buffer material for HLW disposal and primarily consists of the smectite mineral
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montmorillonite, which is a 2:1 clay mineral characterized by interlayers with a weak
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negative layer charge. These interlayers are occupied by cations, which draw in water
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during hydration, resulting in a high swelling capacity and a very low hydraulic
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conductivity.4,5 In addition, these interlayer sites have the ability to adsorb and immobilize
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selective metals and radionuclides.2,6 A number of different aspects need to be
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considered when selecting a suitable, repository-relevant bentonite. Such specifications
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required for optimal performance include hydraulic conductivity, gas permeability, stability
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against iron corrosion and swelling capacity.7
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Since the bentonite will be in direct contact with the metal canister containing the HLW
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and, in the long-term, not all areas may remain compact,8 the potential for microbial
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metabolic activity within any selected sealant material should be considered. It is known
53
that microbial activity can affect the properties of smectitic clay minerals9,10,11,12 as well
54
as the adsorption of metals and actinides via a number of processes including the
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mobilization and immobilization of toxic elements and radionuclides.13,14,15 The microbial
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catalyzed reduction of sulfate leads to the formation of hydrogen sulfide, which is known
57
to promote the corrosion of the metal canisters.16,17,18 Therefore, microbial influenced
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corrosion (MIC) may lead to radionuclide release from the canisters.
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Although the influence of microorganisms in compacted bentonite is thought to be
60
limited by the high swelling pressure and the low water activity of the seal as well as by
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elevated temperatures,19,20,21,22,23 several studies demonstrated that microorganisms can
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survive under repository-relevant conditions. For instance, Wyoming MX-80 bentonite
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was used as an inoculum for enrichment cultures in order to demonstrate the viability of
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indigenous sulfate-reducing bacteria (SRB), even after a 20 h treatment of the bentonite
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in 100 °C dry heat prior to inoculation.24 Furthermore, mixed SRB suspensions (including
66
bacteria, which were isolated from deep Äspö Hard Rock Laboratory, Sweden) were used
67
as inoculum for compacted bentonite blocks that were exposed for 15 months to 20 °C -
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30 °C and 50 °C - 70 °C, respectively.25 By testing the cultivability of the introduced
69
microorganisms, it was shown that only the introduced spore-forming bacteria
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Desulfotomaculum nigrificans and Bacillus subtilis survived the high pressure and
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temperature.25 Similar results were obtained by incubating with swelling pressure
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oedometers at elevated temperatures (55 °C, 65 °C and 80 °C), showing that spore-
73
forming bacteria, which were added before, were the only cultivable cells.26
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Detailed biogeochemical processes caused by microbial activity in pore water and clay
75
medium were also studied experimentally. For instance, the pore water of the Opalinus
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Clay formation was analyzed in terms of its microbial activity.27 Although the origin of
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microorganisms in this study was not clear, active microbial populations were identified
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that likely played a role in the observed reduction of the redox conditions of the pore
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water. Additionally, sulfate-reducing bacteria (SRB) from the genus Desulfosporosinus
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were isolated from the pore water.27 In other experiments, microcosms were set up
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containing the commercially available MX-80 bentonite and an enriched medium.28 The
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microcosms were incubated for 120 days at 15 °C and 37 °C and continuously sampled.
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After 90 days incubation, spore-forming and/or sulfate-reducing bacteria were detected
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and their growth linked with the observed decrease of lactate and sulfate in the respective
85
supplemented set ups.28
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Since bentonites are not sterile, microorganisms can be expected to enter the repository
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and a number of experiments regarding the HLW repositories have focused on SRB
88
and/or spore-formers, using rich SRB-media, SRB inoculations or respective molecular
89
biological tools for detecting them.29 In this investigation, we conducted controlled,
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anaerobic long-term experiments relevant to HLW-repository conditions by using the
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industrial Bavarian B25 bentonite30 as inoculum for the enrichment of indigenous
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microorganisms in artificial pore water at temperatures that are expected to be present in
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a repository. Our main objectives of this study were: 1) to investigate which
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microorganisms occur naturally within the B25 bentonite and how the microbial
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communities change during one year of incubation at 30 °C and 60 °C in microcosms
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containing uncompacted bentonite; and 2) to elucidate to what extent they are
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metabolically active under relevant conditions and whether or not they are important to
98
the safety assessment when constructing Deep Geological Repositories (DGR).
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Microcosms were set up in a sterile, strictly anaerobic manner. To not disturb the systems,
100
we used a high number of serum glass vials with identical conditions and analyzed them
101
sequentially and separately at different time points. Following this procedure, the risk of
102
contamination was very low, the system was not disturbed during the sampling process
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and the evolution of indigenous microorganisms and geochemical parameters could be
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analyzed in detail under different conditions. Furthermore, we decided to use a synthetic
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Opalinus Clay pore water solution and avoided the addition of supplements such as yeast
106
extract, trace elements, vitamins and redox indicators. Since all microorganisms originate
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from the bentonite used, we could determine what influence the microbial activity had on
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the chemistry of the clay barrier and its pore water.
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Our new findings emphasize the importance of SRB in bentonites that may develop
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from spores in the presence of H2 gas and low nutrient-conditions during the development
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of the repository at low temperatures (30 °C) and the possibility of thermophilic bacterial
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activity under enhanced temperature conditions (60 °C); although the latter resulted in no
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detrimental effects on the properties of the bentonite over the one-year period of
114
experimentation.
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MATERIALS AND METHODS
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Bentonite. The commercial B25 Bavarian bentonite (mined in Hallertau, Bavaria,
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Germany) used in this study was dried and milled at IMERYS (Landshut, Bavaria,
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Germany). Temperatures during the drying process did not exceed 100 °C. The
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mineralogy, chemistry and cationic exchange capacity (CEC) of B25 bentonite are listed
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in Table S1. The respective methods are described in the SI part of this manuscript
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(Methods S1, S2, S3, S4 and S5).
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Microcosm Experiments and Sampling. Microcosm-experiments were set up by placing
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20 g of the respective bentonite powder into sterile glass bottles and subsequent addition
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of 40 ml sterile, anaerobic synthetic Opalinus Clay pore water solution (212 mM NaCl, 26
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mM CaCl2, 14 mM Na2SO4, 1.6 mM KCl, 17 mM MgCl2, 0.51 mM SrCl2 and 0.47 mM
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NaHCO3, degassed with a N2/CO2 gas mixture (80/20) while stirring).31 The final pH of
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the pore water was 7.3. The Opalinus clay pore water was chosen because it has been
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used in many chemical, physical and mineralogical experiments concerning the long-term
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safety and construction/planning of DGRs in general.32 Since we needed quite a large
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volume of Opalinus Clay pore water for all the microcosms, we used the described
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synthetic mixture. Selected microcosms were supplemented with sterile and anoxic
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50 mM acetate, 50 mM lactate, 10 mM lactate or 50 kPa H2 (=0.33 %vol). Control
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microcosms included sterilized bentonite (autoclaving for 20 min at 121 °C and 1 bar) or
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only the pore water without the addition of bentonite. The bentonite was not compacted
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and there was no additional pressure applied. Microcosms were incubated for
136
approximately one year at 30 °C and 60 °C in the dark without shaking. During the
137
incubation period, samples were taken at six different time-points and analyzed for bio-
138
geochemical parameters and microbial diversity. For each time point and each condition,
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duplicates (30 °C microcosms) and triplicates (60 °C microcosms) were set up,
140
respectively. The sampling was carried out under strictly anaerobic conditions in a glove
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box (MB-200B modular glove box workstation; M. BRAUN; Garching, Germany)
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containing an N2-atmosphere. Microcosms were introduced into the glove box, well
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mixed, and a portion of the suspension (10 ml) transferred by pouring it into sterile 50 ml
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tubes for geochemical analyses. Since freezing and storage at -70 °C is widely
145
considered suitable for preserving microbial composition,33,34 we stored the remaining
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suspension in the resealed bottle at this temperature for no longer than two months after
147
sampling prior to DNA-extraction. For iron-determination, 300 µl samples taken from the
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well-mixed suspension were placed in 1.5 ml tubes and treated as described below.
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Sensory measurements of O2-concentration, redox potential and pH were made using
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calibrated sensors. The centrifuged and filtered (0.2 µm filter) supernatant of the
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suspension was used for determining the concentration of organic acids and sulfate.
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Determination of Ferric and Ferrous Iron, Sulfate and Organic Acids. Ferric and ferrous
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iron were determined using a modified protocol of Voillier et al.35 Approximately 300 µl of
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well-mixed microcosm suspension were added to 300 µl of 12 M HCl. The samples were
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incubated in the dark overnight at room temperature within an anaerobic glove box.
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Afterward, the HCl-suspension-mix was centrifuged and 200 µl of the clear supernatant
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(or appropriate dilutions in 212 mM NaCl solution) applied to the ferrozine-assay. The
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colorimetric test was conducted as described35 and relative values of ferric and ferrous
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iron calculated by comparison with standard solutions prepared as dilutions of 2 mM FeCl3
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in 10 mM HCl (starting from 5 µM up to 100 µM). Organic acids were identified via HPLC
161
analysis with an “Agilent 1200” (Degasser G1322A, diode array detector G1315B,
162
quaternary pump G1354A). For separation, a Nucleogel Ion 300 OA column was used.
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The releasing agent was 5 mM H2SO4 with a flow rate of 0.4 ml/min at 70 °C. The sulfate-
164
concentration was determined by ion chromatography using a Dionex Integrion HPIC
165
(Thermo Scientific). For calibration, a K2SO4-standard solution was used (0.05 mg - 10.0
166
mg SO42- ) and, like the samples, separated by utilizing an AS23-column (Thermo
167
Scientific). A defined Na2CO3-H2CO3 mixture served as eluent with a flow rate of 250
168
µl/min.
169
Extraction, Purification and Sequencing of DNA. For analyzing the microbial diversity
170
within the respective samples, DNA was extracted using a protocol from Selenska-
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Pobell36 with the following adjustments: step 1: centrifugation of samples for 15 min at
172
6,800 x g; step 2: for removal of SDS, centrifugation speed was adjusted to 11,000 x g;
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step 3: addition of PEG-6000 to a final concentration of 40 % with a centrifugation speed
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set at 11,500 x g. For purification and concentration of extracted genomic DNA,
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NucleoBond® AXG 100 columns and recommended buffers (G4, N2, N3 and N5) were
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used following the instruction manual. Genomic DNA was then precipitated by using 0.4
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volumes of isopropanol (molecular grade). After sedimentation of DNA at 12,000 x g, it
178
was resolved in a TE-buffer. The extracted DNA was utilized for amplification of the V4-
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region of the 16S rRNA gene by using PCR.37 The PCR reactions contained 25 µl PCR
180
water, 5 µl MgCl2 (2.5 mM final concentration), 10 µl 5x buffer (Promega), 2.0 µl each of
181
oligonucleotides
182
(GGACTACHVGGGTWTCTAAT)38 (0.4 µM final concentration), 0.5 µl dNTPs (125 µM
183
final concentration), 0.5 µl Taq-Polymerase (Promega, 0.05 U/µl final concentration) and
184
5 µl genomic DNA. Reactions were held at 95 °C for 2 min to denature the DNA, with
185
amplification proceeding for 30 cycles at 95 °C for 30 s, 50 °C for 60 s and 72 °C for 60
186
seconds. A final step of 10 min at 72 °C was added to the procedure to ensure complete
187
amplification. The successful amplification of DNA was checked via gel electrophoresis.
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Successfully amplified DNA was purified with MSB® Spin PCRapace (Stratec molecular)
189
according to the manufacturer`s protocol. Purified amplicons were quantified with a Qubit
190
4 (Thermo Scientific™) and quality control achieved by using a NanoDrop™ (Thermo
515f
(GTGCCAGCMGCCGCGGTAA)
and
806r
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Scientific™) following the manufacturer`s protocol. The samples were sequenced with
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MiSeq Illumina at RTL Genomics (Texas, USA). Data analysis was undertaken following
193
the
194
(www.rtlgenomics.com/docs/Data_Analysis_Methodology.pdf).39 Retrieved 16S rRNA
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gene sequences are available at NCBI database with the bioproject accession number
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PRJNA507942 (https://www.ncbi.nlm.nih.gov/; see also Table S3).
guidelines
of
RTL
Genomics
197 198
RESULTS AND DISCUSSION
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H2 drives microbial sulfate-reduction in B25 Bavarian bentonite – but not at elevated
200
temperatures. Our results showed that indigenous microorganisms fuel their metabolism
201
with the applied H2, which is used as an electron donor for the stepwise reduction of
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sulfate as indicated by the apparent decrease in the electron acceptor from 9 mM (initial
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sulfate concentration in microcosms) to 6 mM (Figure 1, B).16 The subsequent formation
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of hydrogen sulfide can promote processes such as corrosion of the metal canisters18,32
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and the reduction of smectitic ferric iron to ferrous iron, leading to a potential
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destabilization of the smectite and, therefore, to a loss of its swelling and sealing
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function.22,40 This sulfide-mediated iron reduction could also explain the observed
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increase of ferrous iron and simultaneous decrease of ferric iron as well as the observed
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formation of grey precipitates, probably indicative of iron-sulfur particles (Figure 1, B;
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Figure S2, D). Furthermore, the observed iron-reduction can be additionally catalyzed by
211
microorganisms, via direct or indirect electron transfer,41 although no commonly
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researched iron-reducing microorganisms were detected in H2-containing microcosms
213
(Figure 3).
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The described observations, which point to active sulfate-reduction in B25 bentonite,
215
were only detectable at 30 °C. H2-containing microcosms incubated at 60 °C did not show
216
equivalent changes after one-year incubation (Figure 2, B), even under the ideal
217
conditions of no compaction with the availability of pore water and H2 as an electron
218
donor. However, the accelerated decrease of redox potential observed in H2- containing
219
microcosms appears to be independent of the temperature (compare Figure 1, B and
220
Figure 2, B). Since H2 is a very potent reducing agent, one would expect changes in redox
221
potential within the respective microcosms, in addition to the activity of microorganisms
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that contribute by reductive hydrogen sulfide gas formation. A reducing environment is
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also a necessity to promote anaerobic metabolic activity.42 Furthermore, there was no
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significant change in the measured pH attributable to microbial activity (Figure 1, B; Figure
225
2, B).
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The analysis of 16S rRNA gene sequences showed that Desulfosporosinus species
227
dominated the conspicuous H2-containing microcosms incubated at 30 °C, whereas this
228
genus was not detected in the raw material or in control microcosms (Figure 3). A reason
229
for this could be the formation of spores, which are difficult to extract DNA from by the
230
applied extraction protocol.43 Species belonging to the genus of Desulfosporosinus are
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known to be strictly anaerobic, sulfate-reducing and spore-forming organisms.44
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Furthermore, they are metabolically very adaptable and can use the applied CO2 for
233
autotrophic growth.44,45 Although autotrophy is a typical feature of acetogenic growth, no
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significant amounts of acetate were detected in the respective microcosms (Figure 1,
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B).46 A reason could be the low energy that is provided by this reaction resulting in very
236
poor growth46 or the consumption of formed acetate by further microorganisms.47
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Desulfosporosinus species have already been detected in other bentonites, including MX-
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80 bentonite,27,48,49 and are therefore considered to be of importance with respect to
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repository design.
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Low concentrations of lactate support microbial growth in B25 Bavarian bentonite – but
241
not at elevated temperatures. Besides H2, lactate is an electron donor for microbial
242
sulfate-reduction and has been shown to be present in pore water, which may potentially
243
enter the repository, although in low concentrations (0.7 µM up to 3,5 µM).50 However, in
244
order to maximize the effects and to shorten the incubation time, we used higher
245
concentrations of 10 mM and 50 mM, respectively.
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As observed in H2-containing microcosms at 30 °C, lactate-containing experiments
247
showed the formation of grey precipitates, an accelerated decrease in redox potential and
248
indications of iron reduction (Figure 1, C). Additionally, the respective microcosms
249
showed the presence of horizontal fissures in the bentonite and gas bubbles, very likely
250
formed by gas release (Figure 1, C). The decrease in lactate- and sulfate-concentration
251
was accompanied by the formation of acetate in the respective microcosms. Again,
252
members of the genus Desulfosporosinus dominated the microbial population in most of
253
the microcosms containing 10 mM lactate (Figure 3). The supplied lactate was consumed
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via the reductive acetyl-CoA pathway. The incomplete oxidation of lactate and
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concomitant reduction of sulfate led to the formation of acetate and hydrogen sulfide
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(Figure 1, C), which reacted with iron to form the observed precipitates and to reduce
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ferric iron species.46,51,52 The generated fissures observed in our experiments are likely
258
to result from hydrogen sulfide gas and a low overburden pressure. Fissure-formation
259
due to the microbial formation of gases may be of importance only in areas of poor
260
bentonite compaction and little overburden, such as in voids or reaction rims formed along
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corroded contacts between the bentonite seal and other materials in the repository,
262
namely the concrete, the metal canister or host rock.53,54 Increased filtration of pore water
263
along such fissures in the buffer could provide additional space for further microorganisms
264
and renewed gas generation. Thus, the antimicrobial pressure of the bentonite could in
265
this way be compromised with time. Although this mechanism can be considered as a
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“worst-case scenario”, the formation of gas-filled cavities by microbial activity is not
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expected to occur in bentonite of high packing density and overburden pressure.26,55 In
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the case of compacted bentonite with high yield strength, the gas can be expected to
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generate fractures (channels) that migrate toward interfaces and reseal after the release
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of the gas.56
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The formed acetate represents a possible substrate or supplement for numerous other
272
microorganisms and can potentially affect radionuclide mobility.47,57,58 However, acetate-
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containing microcosms did not show any effect regarding the analyzed parameters
274
(Figure 1, D). The supplemented concentration of 50 mM acetate could be too high, which
275
is also true for microcosms containing 50 mM lactate (data not shown). Additionally,
276
microcosms containing 50 mM lactate, resulted in the formation of pyruvate (up to 0.8
277
mM in the filtrated supernatant), independent of temperature (data not shown). Microbial
278
excretion of pyruvate has been observed in a number of studies.59,60,61 Since pyruvate is
279
a very valuable metabolite for every organism, excretion is here very likely due to a stress
280
reaction caused by the high lactate concentration within the microcosms compared to
281
starved microorganisms within the raw bentonite. Similar observations regarding the
282
pyruvate formation have been made for starved E. coli cells, showing excretion of
283
pyruvate due to inactive pyruvate dehydrogenase.61 Thus, high concentrations of
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organics appear to inhibit and/or limit the metabolic activity of inherent microorganisms in
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the B25 bentonite. For the studied Bavarian bentonite, an organic carbon content of 0.1
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% (w/w) was reported62 and the determination of organic carbon (Method S1) revealed
287
the presence of a variety of organic compounds. Although only qualitative data are
288
available and the precise concentrations cannot be given, these organics represent
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potential carbon-sources for microorganisms and include hydrocarbons in the form of
290
alkanes, toluenes and various aromatic compounds (Table S2). A number of these
291
compounds can be degraded under the anaerobic conditions present in a repository.63
292
Many microorganisms couple the anaerobic degradation of hydrocarbons to nitrate,
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sulfate or iron reduction or to fermentative or syntrophic growth.63 Furthermore, it has
294
been shown that leachates from buffer materials, which were subjected to heat, radiation
295
or combinations of these conditions, have a stimulating effect on viable cell numbers.64
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However, regarding the overall mineralogy, no significant changes were observed
297
between the samples studied (Table S1).
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The described sulfate-reducing activity of lactate-containing microcosms was only
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observed at 30 °C with an applied concentration of 10 mM. No significant changes were
300
observed regarding the experimental parameters measured after one-year incubation at
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60 °C or when higher lactate concentrations (50 mM) were present (Figure 2, C). Thus,
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elevated temperatures and higher concentrations of organics seem to prevent or reduce
303
the metabolic activity of inherent SRB in the B25 Bavarian bentonite, even when optimal
304
environmental conditions were applied.
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Despite the lack of detectable microbial activity in the 60 °C experiments, we were still
306
able to successfully isolate, amplify and sequence the 16S rRNA gene of lactate- and
307
acetate-containing microcosms. This was also possible for setups with no additional
308
electron donor after almost one year of incubation at 60 °C (Figure 4). Independent of the
309
presence or absence of electron donors, all three microcosms showed the dominance of
310
two thermophilic organisms (Figure 4). The detected Caldinitratiruptor microaerophilus is
311
a thermophilic microorganism which grows at temperatures between 50 °C and 75 °C
312
with an optimum at 65 °C at neutral pH.65 This bacterium can use various organic acids,
313
including amino acids, as electron donors, but is restricted to oxygen or nitrate as an
314
electron acceptor.
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The second thermophilic organism that was detected is Thermaerobacter marianensis,
316
which can grow at temperatures between 50 °C and 80 °C (optimum: 74 °C-76 °C) and
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can use organic compounds as their sole source of energy.66 Whether these identified
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organisms were metabolically active in the respective microcosms, is not clear. So far,
319
we rule out a significant influence on the analyzed parameters under the applied
320
conditions. Since nitrate could play a role as the electron acceptor for the detected
321
thermophilic genera, nitrogen-containing compounds should be considered in further
322
studies as compounds potentially influenced by the microbial activity in further studies. In
323
general, nitrogen-containing compounds are crucial for metabolic activity and for life to
324
evolve (e.g. synthesis of amino acids and nucleotides)67,68 and should be taken into
325
account. Since microorganisms will encounter temperatures around 60 °C in the bentonite
326
buffer,69 which is different from the host rock claystone, more attention should be given
327
to the role of thermophiles, as emphasized in previous studies of elevated temperature
328
conditions.24,25,27
329
Microbial diversity in bentonites. Although bentonites are known for their low organic
330
content, the potential for metabolically active microorganisms is feasible, as
331
demonstrated in other low-biomass environments.70,71 However, the low biomass makes
332
it difficult to extract sufficient DNA for the successful amplification of the 16S rRNA gene.
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Additionally, the extraction of DNA is hindered through the adsorption of negatively
334
charged DNA on the cationic surface of smectite minerals that dominate the bentonite.72
335
Despite these problems, an existing DNA-extraction protocol was successfully adapted
336
by modulating centrifugation speed and by applying more careful handling of the
337
precipitated DNA in order to increase the DNA amount and to reconstruct the microbial
338
community within the respective samples.
339
Bentonites are known to contain diverse microbial communities.29 The 16S rRNA gene
340
is the most widely used marker for the performance of phylogenetic analysis, allowing the
341
classification of many taxa. It is, therefore, an excellent phylogenetic marker.73 Depending
342
on the oligonucleotides used, it is possible to map a part of the microbial population of a
343
given environment. It has been shown that the oligonucleotides utilized in this study are
344
very well suited for this purpose.38 Many factors influence the sequencing results74
345
including the DNA-extraction method itself. Since many microorganisms, especially in
346
soils, form spores, it is very likely that we missed these microorganisms when analyzing
347
the isolated DNA. This is especially the case for the bentonite raw material since suitable
348
conditions for the germination of spores are not likely to be present.
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However, the presence of a certain DNA alone cannot be used as an indication for
350
microbial metabolic activity. Therefore, a combination of culture-dependent and -
351
independent methods, similar to those applied in this study, are required. As soon as
352
appropriate bentonites are selected, metabolic potential can be elucidated by using a
353
combination of these techniques to identify microbial communities and to estimate their
354
metabolic potential.
355
It is important to mention that the distribution of nutrients, minerals, dissolved ions and
356
microorganisms in environmental habitats is not homogeneous.75 This is very likely also
357
an explanation for the observation that not every microcosm evolved in the same manner,
358
even when the experimental conditions were the same (Figure 1, Figure 2). It has already
359
been demonstrated that the bacterial diversity in soils is positively correlated with soil
360
heterogeneity,76 which originates from spatial and temporal variability in hydrological and
361
biogeochemical processes.77 These processes are likely to be equally valid during the
362
formation of bentonites, which may also be further modified during mining and industrial
363
preparation.
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It is therefore evident that microorganisms and especially spore-forming organisms will
365
be introduced into the HLW repository together with emplacement of the bentonite seal.
366
These will be in their vegetative state and only become metabolically active under the
367
suitable conditions at 30 °C. Furthermore, our results show that thermophilic
368
microorganisms evolve at 60 °C but the nature of this activity requires further
369
experimentation.
370
Microbial metabolic activity and the safety case. The conditions in a DGR are generally
371
considered to be rather hostile to life. Parameters like desiccation (low water activity),
372
elevated temperatures, small pore size in the compacted bentonite and high pore fluid
373
pressures generally affect microbial activity negatively and promote rapid entry into a
374
dormant cell state in the form of spores.23,25,78,79,80,81 In general, the formation of spores
375
enables the respective organisms to be resistant to heat, pressure, desiccation, UV and
376
γ-radiation treatments, even if the respective exposure lasts for many years.43,82,83 The
377
timing of events is likely to be a crucial parameter in the opportunistic development of
378
microbiological assemblages. The canister harboring the HLW should be stable over a
379
period of 500 years and the DGR itself even for one million years.84 Right after
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construction and closure of the DGR, a necessary environment for enabling microbial
381
activity is considered to be unlikely.
382
The presented data refer to microcosms that were incubated under ideal conditions:
383
namely no pressure or compaction of the bentonite as well as the presence of the pore
384
water solution. Additionally, the added electron donors were present in much higher
385
concentrations than expected in a DGR. It was shown that the groundwater-flow will be
386
the main regulator of nutrient transport.55 Since most DGR`s are located to minimize any
387
pore water or groundwater entering the site, the amount of nutrient-transport into the DGR
388
is considered to be absent or minimal following closure. However, a saturation with
389
ground- or pore water cannot be ruled out over the long-term.85–87
390
Nevertheless, in some designs of repository test-sites water-saturated bentonite is used
391
(e.g. FEBEX full emplacement experiment) in order to seal gaps occurring during
392
construction.32 Gradients of water-activity, temperature and pressure will be present
393
within the bentonite barrier that will be influenced by the released heat from the spent-
394
fuel. To what extent these gradients will homogenize and evolve over time cannot be
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addressed that easily.32,88 Thus, model experiments are important and illuminate the
396
complexities related to the different conditions.
397
By adding enriched bacterial suspensions to MX-80 bentonite in swelling pressure
398
oedometers, it was demonstrated that compacted bentonite under high pressure and
399
elevated temperatures will significantly limit microbial activity.25,26 Mainly spore-forming
400
and sulfate-reducing bacteria were shown to dominate under such conditions.
401
However, many of the experiments were inoculated with enriched cultures and/or
402
enriched media selected for the various groups of bacteria present. This is required when
403
addressing the effects of pressure or water activity on the respective, stress-tolerant
404
microbial groups, but the approach does not allow assessment of real microbial potential
405
of selected bentonite buffers. Therefore, in our study, we omitted any enriched media in
406
order to analyze the pure metabolic potential of the Bavarian bentonite.
407
In accordance with previous research, our findings clearly indicate that the B25
408
Bavarian bentonite contains intrinsic microorganisms, especially spore-forming sulfate-
409
reducers, which become metabolically active when the environmental conditions become
410
favorable at mesophilic temperatures. Spores should be considered as potentially active
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cells with the ability to dynamically modify their environment in a DGR setting.82 As a
412
result of various biotic and abiotic processes that could take place in a nuclear waste
413
repository, H2 will arise from a number of processes89,90,91 and is likely to act as the initial
414
electron donor for these microorganisms, as shown in this study and previous work.92,93
415
The build-up of hydrogen gas is considered to depend on the corrosion rate, host rock
416
permeability, and water consumption rate within the nearfield of the repository.94 Since
417
these respective parameters will differ in each repository, it is difficult to estimate the real
418
H2 concentration and pressure-build at a given storage location. Further potential sources
419
for electron donors in a DGR site are contained within the bentonite itself and/or pore
420
water that may enter with time.31 As bentonites generally have a very low carbon content
421
and as long as no organic compounds enter the DGR, organics will play a rather minor
422
role regarding the metabolic activity of intrinsic microorganisms.95
423
Even if the tested ideal conditions are present, it remains unclear to which extent such
424
microbial metabolic activity poses a problem to the safety case of a DGR. At elevated
425
temperatures of 60 °C, we did not observe any significant changes in the relevant
426
parameters (Eh, pH) measured (Figure 2). Although thermophilic microorganisms were
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detected to be present and appeared as the dominant microbial community within the
428
respective microcosms (Figure 4), no significant metabolic effects were observed,
429
indicating that, no detrimental metabolic activity will occur at elevated temperatures, even
430
when suitable electron donors are present. However, as soon as microorganisms evolve,
431
they are in constant, dynamic exchange with their environment.9 Thus, it is important to
432
determine whether their metabolisms might influence other bentonite properties such as
433
cation exchange capacity, hydraulic conductivity and swelling capacity as well as more
434
general changes in mineral composition not addressed in this study. The
435
formation/consumption of several gases involved in biochemical reactions within the
436
bentonite also deserves further attention. These results are of particular relevance when
437
considering the predicted time-dependent temperature distribution in the horizontal
438
direction of the emplacement tunnels where the HLW canisters are to be placed.69 In the
439
case of using bentonite with low thermal conductivity (low moisture content), the
440
temperature of the contacting bentonite (mid-bentonite) will be around 90 °C following
441
emplacement. This will then decrease to 60 °C after 200 years and 30 °C after 10,000
442
years.69 In the case of using bentonite with higher thermal conductivity (high moisture
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content), the initial temperature will be around 70 °C, reaching 60 °C after 100 years.69
444
However, under the conditions applied in this work, the metabolic activity of thermophilic
445
microorganisms produced no detectable effects. When considering the additional role of
446
compaction, pressure and water content, which were not addressed in our work, it
447
appears unlikely that this type of microbial activity will have any effect on the stability of
448
the bentonite, at least over the short time. The activity of thermophilic assemblages does,
449
however, warrant a more detailed study.
450
Our year-long experiments support the need for careful consideration and debate as to
451
the potentially negative influence that bentonite-related microorganisms may play for
452
securing the long-term stability of the repository and these aspects do require attention
453
when planning and constructing a DGR, which should remain intact for at least 200,000
454
years.96 Such microbial metabolic activity may become crucial with respect to the safety
455
case many years after the closure of the DGR. Potentially seeping pore water could be
456
an issue then.29,58 At this stage, the remaining radioactive compounds are still harmful
457
and should not enter the biosphere. Hence, the observed microbial formation of gases
458
and gas-related fissures in the bentonite, as well as the chemical effects of metabolic
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activity in general, such as changes in redox potential and pH, are dynamic changes in
460
the bentonite seal that may compromise its long-term effectiveness. As a result, microbial
461
activity should be given adequate consideration when assessing the long-term safety of
462
underground repositories.24,25,81
463
AUTHOR INFORMATION
464
Corresponding Authors
465
*Nicole Matschiavelli,
[email protected], +49 351 260 3375
466
*Andrea Cherkouk,
[email protected], +49 351 260 2989
467
Author Contributions
468
The manuscript was written through the contributions of all authors who have given their
469
approval to the final version of the manuscript.
470
Funding Sources
471
This work has received funding from the Euratom research and training programme
472
2014-2018 under grant agreement No. 661880.
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ACKNOWLEDGMENTS
474
We thank Falk Lehmann for HPLC-analyses and Carola Eckardt for IC-measurements
475
(HZDR) as well as Karsten Pedersen and colleagues (Microbial Analytics Sweden AB,
476
Sweden) for fruitful discussions. Furthermore, we thank Stephan Kaufhold (BGR,
477
Hannover, Germany) for providing the B25 Bavarian bentonite. For helpful proof reading,
478
we thank Johannes Raff (HZDR, Dresden, Germany).
479 480
ABBREVIATIONS
481
HLW, high-level radioactive waste; PCR, Polymerase chain reaction; DGR, Deep
482
Geological Repositories
483
ASSOCIATED CONTENT
484
Supporting Information.
485
The
486
publications website at ….This includes additional descriptions of materials and methods,
487
as well as SEM/EDX analysis of selected samples not shown in the main text.
supporting
information
is
available
free
of
charge
on
the
ACS
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FIGURES AND LEGENDS
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
-250 -500
0
70
140 210 tim e [d]
500
0
-250 -500
C
0
70
140
210
500
0
-250 -500
D
0
70
140 210 tim e [d]
500
0
-250 -500
774
0
70
140 210 tim e [d]
6
0
70
140 210 tim e [d]
280
350
0
70
140 210 tim e [d]
280
350
0
70
140 210 tim e [d]
280
350
lactate
0
70
140 210 tim e [d]
280
350
acetate
no additional electron dornor
60 30 12 9 6 3 0
H2
90 60 30 12 9 6 3 0
pH
Eh [m V]
250
9
90
pH
Eh [mV]
250
30 12
0
pH
E h [mV]
250
60
3
Metabolites [mM]
Eh [mV]
pH
0
Metabolites [mM]
280
250
B
90
12 11 10 9 8 7 6 5 4 3 2 1 0 350
500
Metabolites [mM]
A
90 60 Metabolites [mM]
773
Environmental Science & Technology
30 12 9 6 3 0
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775
Figure 1: Change of geochemical parameters and metabolites recorded in B25 bentonite
776
microcosms that were incubated at 30 °C for 305 days. Shown are the results from
777
microcosms containing no additional electron donor (A), 50 kPa H2 (B), 10 mM lactate (C)
778
or 50 mM acetate (D). The left panel shows the change in redox potential (Eh;□) and pH
779
(■). The right panel depicts the concentration of lactate (■), acetate (●), sulfate (▲), HCl-
780
extractable ferric iron (○) and HCl-extractable ferrous iron (●). Shown are mean values
781
with standard deviations measured on two independent microcosms. The photographic
782
images show representative microcosms after 181 days incubation (marked with a blue
783
bar in the panels). White arrows indicate the formation of black spots and grey
784
precipitates, yellow arrows indicate the formation of horizontal fissures and cavities due
785
to gas formation.
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280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
280
12 11 10 9 8 7 6 5 4 3 2 1 0 350
0
-250 -500
B
0
70
140 210 tim e [d]
500
0
-250 -500
C
0
70
140 210 tim e [d]
500
0
-250 -500 0
D
70
140 210 tim e [d]
500
0
-250 -500 0
786
70
140 210 tim e [d]
9 6
0
70
140 210 tim e [d]
280
350
0
70
140 210 tim e [d]
280
350
0
70
140 210 tim e [d]
280
350
0
70
140 210 tim e [d]
280
350
no additional electron donor
90 60 30 12 9 6 3 0
pH
Eh [m V]
250
30 12
0
pH
Eh [m V]
250
60
3
pH
Eh [m V]
250
Metabolites [mM]
pH
Eh [m V]
250
90
Metabolites [mM]
500
H2
90 Metabolites [mM]
A
60 30 12 9 6 3 0
lactate
90 Metabolites [mM]
Page 47 of 50
60 30 12 9 6 3 0
acetate
787
Figure 2: Changes in geochemical parameters and metabolites measured in B25
788
bentonite microcosms that were incubated at 60 °C for 323 days. Shown are results from
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789
microcosms containing no additional electron donor (A), 50 kPa H2 (B), 50 mM lactate (C)
790
or 50 mM acetate (D). The left panel shows the change in redox potential (Eh;□) and pH
791
(■). The right panel depicts the concentration of lactate (■), acetate (●), sulfate (▲), HCl-
792
extractable ferric iron (○) and HCl-extractable ferrous iron (●). Shown are mean values
793
with standard deviations taken from three measurements of three independent
794
microcosms.
795 796
Sample
Time of Incubation [d] 0
raw material sterile
Relative abundance [% ]
20
40
60
80
100
305
181 no additional electron donor 305
181 10 mM lactate 305
Actinobacteria
Ammoniphilus Bacillus Brevibacillus Desulfosporosinus Desulfotomaculum Effusibacillus Limnochorda
Firmicutes
Hydrogenophaga Limnobacter Massilia Polaromonas Ramlibacter Thiobacillus
181 H2
Arthrobacter Nocardia Nocardioides Rhodococcus Streptomyces Yonghaparkia
238 305
β-Proteobacteria
Acinetobacter Pseudomonas
γ-Proteobacteria
Candidatus Phytoplasma
Tenericutes
Archaea 50 mM acetate
797
181
no hit others (unclassiefied, unknown and/or below 1 % )
305
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798
Figure 3: Microbial diversity of selected B25 bentonite microcosms that were incubated
799
at 30° C. Shown is the relative abundance of detected genera and their dependence on
800
the added electron donor (sample) and incubation time. Two bars at one time-point reflect
801
the community of two distinct microcosm-flasks incubated under the same conditions.
802
The microbial community was analyzed by amplifying and sequencing the V4-region of
803
the 16S rRNA gene via MiSeq Illumina. The respective sequence data are available under
804
the bioproject accession PRJNA507942 (Table S3; https://www.ncbi.nlm.nih.gov/sra)
Sample
Time of Incubation [d] 0
no additional electron donor 323
Relative abundance [% ]
20
40
60
80
100
Caldinitratiruptor microaerophilus Thermaerobacter marianensis Methylobacterium Sphingomonas Curvibacter
50 mM lactate
323
50 mM acetate
323
Ralstonia
Firmicutes
α-Proteobacteria
β-Proteobacteria
Archaea
805
no hit others (unclassiefied, unknown and/or below 1 % )
806
Figure 4: Microbial diversity of selected B25 bentonite microcosms that were incubated
807
at 60° C for 323 days. Shown is the relative abundance of detected genera and their
808
dependence on the added electron donor. The microbial community was analyzed by
809
amplifying and sequencing the V4-region of the 16S rRNA gene via MiSeq Illumina. The
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810
respective sequence data are available under the bioproject accession PRJNA507942
811
(Table S3; https://www.ncbi.nlm.nih.gov/sra)
812
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