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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

2

Bavarian bentonite slurries at 30 °C and 60 °C

3

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

5

Thuro Arnold,1 Andrea Cherkouk*,1

6

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-

10

3Microbial

Analytics Sweden AB, Mölnlycke Fabriker 9, 43535 Mölnlycke, Sweden

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KEYWORDS high-level radioactive waste repository, sulfate-reduction, spores,

12

thermophiles

ACS Paragon Plus Environment

1


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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

16

for sealing the space between the steel-canister containing the HLW and the surrounding

17

host rock. In order to broaden the spectra of appropriate bentonites, we investigated the

18

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


2

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microorganisms. The subsequent generation of hydrogen sulfide led to the formation of

26

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


3


32

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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

35

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

38

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

42

montmorillonite, which is a 2:1 clay mineral characterized by interlayers with a weak

43

negative layer charge. These interlayers are occupied by cations, which draw in water

44

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

49

against iron corrosion and swelling capacity.7

50

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

55

mobilization and immobilization of toxic elements and radionuclides.13,14,15 The microbial

56

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.

59

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

61

elevated temperatures,19,20,21,22,23 several studies demonstrated that microorganisms can

62

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

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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-

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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

77

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

81

containing the commercially available MX-80 bentonite and an enriched medium.28 The

82

microcosms were incubated for 120 days at 15 °C and 37 °C and continuously sampled.

83

After 90 days incubation, spore-forming and/or sulfate-reducing bacteria were detected

84

and their growth linked with the observed decrease of lactate and sulfate in the respective

85

supplemented set ups.28

86

Since bentonites are not sterile, microorganisms can be expected to enter the repository

87

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

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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

92

microorganisms in artificial pore water at temperatures that are expected to be present in

93

a repository. Our main objectives of this study were: 1) to investigate which

94

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

96

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).

99

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

103

and the evolution of indigenous microorganisms and geochemical parameters could be

104

analyzed in detail under different conditions. Furthermore, we decided to use a synthetic

105

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

107

from the bentonite used, we could determine what influence the microbial activity had on

108

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

110

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

112

activity under enhanced temperature conditions (60 °C); although the latter resulted in no

113

detrimental effects on the properties of the bentonite over the one-year period of

114

experimentation.

115

MATERIALS AND METHODS

116

Bentonite. The commercial B25 Bavarian bentonite (mined in Hallertau, Bavaria,

117

Germany) used in this study was dried and milled at IMERYS (Landshut, Bavaria,

118

Germany). Temperatures during the drying process did not exceed 100 °C. The

119

mineralogy, chemistry and cationic exchange capacity (CEC) of B25 bentonite are listed

120

in Table S1. The respective methods are described in the SI part of this manuscript

121

(Methods S1, S2, S3, S4 and S5).

122

Microcosm Experiments and Sampling. Microcosm-experiments were set up by placing

123

20 g of the respective bentonite powder into sterile glass bottles and subsequent addition

124

of 40 ml sterile, anaerobic synthetic Opalinus Clay pore water solution (212 mM NaCl, 26

125

mM CaCl2, 14 mM Na2SO4, 1.6 mM KCl, 17 mM MgCl2, 0.51 mM SrCl2 and 0.47 mM

126

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

128

used in many chemical, physical and mineralogical experiments concerning the long-term

129

safety and construction/planning of DGRs in general.32 Since we needed quite a large

130

volume of Opalinus Clay pore water for all the microcosms, we used the described

131

synthetic mixture. Selected microcosms were supplemented with sterile and anoxic

132

50 mM acetate, 50 mM lactate, 10 mM lactate or 50 kPa H2 (=0.33 %vol). Control

133

microcosms included sterilized bentonite (autoclaving for 20 min at 121 °C and 1 bar) or

134

only the pore water without the addition of bentonite. The bentonite was not compacted

135

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,

139

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

141

box (MB-200B modular glove box workstation; M. BRAUN; Garching, Germany)

142

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

144

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

146

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

148

well-mixed suspension were placed in 1.5 ml tubes and treated as described below.

149

Sensory measurements of O2-concentration, redox potential and pH were made using

150

calibrated sensors. The centrifuged and filtered (0.2 µm filter) supernatant of the

151

suspension was used for determining the concentration of organic acids and sulfate.

152

Determination of Ferric and Ferrous Iron, Sulfate and Organic Acids. Ferric and ferrous

153

iron were determined using a modified protocol of Voillier et al.35 Approximately 300 µl of

154

well-mixed microcosm suspension were added to 300 µl of 12 M HCl. The samples were

155

incubated in the dark overnight at room temperature within an anaerobic glove box.

156

Afterward, the HCl-suspension-mix was centrifuged and 200 µl of the clear supernatant

157

(or appropriate dilutions in 212 mM NaCl solution) applied to the ferrozine-assay. The

158

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

160

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.

163

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-

171

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;

173

step 3: addition of PEG-6000 to a final concentration of 40 % with a centrifugation speed

174

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

176

used following the instruction manual. Genomic DNA was then precipitated by using 0.4

177

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-

179

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.

188

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


13


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191

Scientific™) following the manufacturer`s protocol. The samples were sequenced with

192

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

195

gene sequences are available at NCBI database with the bioproject accession number

196

PRJNA507942 (https://www.ncbi.nlm.nih.gov/; see also Table S3).

guidelines

of

RTL

Genomics

197 198

RESULTS AND DISCUSSION

199

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

202

sulfate as indicated by the apparent decrease in the electron acceptor from 9 mM (initial

203

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

205

and the reduction of smectitic ferric iron to ferrous iron, leading to a potential

206

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

208

increase of ferrous iron and simultaneous decrease of ferric iron as well as the observed

209

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

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microorganisms, via direct or indirect electron transfer,41 although no commonly

212

researched iron-reducing microorganisms were detected in H2-containing microcosms

213

(Figure 3).

214

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

222

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

224

significant change in the measured pH attributable to microbial activity (Figure 1, B; Figure

225

2, B).

226

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

231

known to be strictly anaerobic, sulfate-reducing and spore-forming organisms.44

232

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,

235

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

237

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

239

repository design.

240

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.

246

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

256

(Figure 1, C), which reacted with iron to form the observed precipitates and to reduce

257

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

266

“worst-case scenario”, the formation of gas-filled cavities by microbial activity is not

267

expected to occur in bentonite of high packing density and overburden pressure.26,55 In

268

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

270

of the gas.56

271

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-

273

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

284

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

286

% (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

289

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,

293

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

296

However, regarding the overall mineralogy, no significant changes were observed

297

between the samples studied (Table S1).

298

The described sulfate-reducing activity of lactate-containing microcosms was only

299

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,

302

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.

305

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.

315

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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317

can use organic compounds as their sole source of energy.66 Whether these identified

318

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.


22

Page 23 of 50


333

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.


23


Page 24 of 50

349

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.


24

Page 25 of 50


364

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


25


Page 26 of 50

380

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


26

Page 27 of 50


395

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


27


Page 28 of 50

411

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


28

Page 29 of 50


427

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


29


Page 30 of 50

443

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


30

Page 31 of 50


459

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.


31


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473

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


32

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488 489

490

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44

Page 45 of 50

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


30 12 9 6 3 0


45


Page 46 of 50

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.


46


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


47


Page 48 of 50

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


48

Page 49 of 50


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


49


Page 50 of 50

810

respective sequence data are available under the bioproject accession PRJNA507942

811

(Table S3; https://www.ncbi.nlm.nih.gov/sra)

812


50

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