Sulfide Corrosion by Sulphate-Reducing Bacteria in MX-80 Bentonites
Szu-Wei Lee*, Ching-Tu Chang, Chun-Cheng Lin, Tsong-Yang Wei
Institute of Nuclear Energy Research, Atomic Energy Council
1000 Wenhua Rd. Jiaan Village, Longtan District, Taoyuan City 32546, Taiwan

Key words; sulphate-reducing bacteria, safety assessment, diffusion coefficient

Abstract
In the safety assessment of the Spent Nuclear Fuel Final Disposal Program, the corrosion behavior of copper
canister caused by the sulfide is an important issue. Bentonite is one of the engineered barrier materials adjoiningthe canisters to prevent the harmful substances from the outside to be in contact with the canisters; however, thecorrosive sulfide could exist in bentonites, and corrosion of the copper canisters could happen. One of theimportant sources of sulfide comes from the sulphate-reducing bacteria (SRB) group, which live naturally inunderground water and clays. When active, the bacteria will reduce sulfate and produce sulfide; therefore, theknowledge of the SRB sulfide production and canister corrosion mechanisms, and relevant tests should bedeveloped.
One of the strong restrictions against the microbial activity is the swelling pressure of the bentonite, which isdependent on compacted density of it. The investigation of how well the SRB activity is inhibited by theincreasing density of MX-80 bentonite is developed, and related works are done in progress. In addition, thediffusion experiments of sulfate are set up to obtain diffusion coefficients in the saturated MX-80 compactedbentonite.
Test of SRB activity against the change of buffer
density is designed based on the previous work doneby Swedish Nuclear Fuel and Waste ManagementCompany (SKB) [3]. In the experiment, test cells aredesigned to hold the bentonite clay in a constantvolume, with isolated and anaerobic environments.
Different conditions of cells are set with different
saturation density, different treatments of bentonite,and experiment time. In addition, the anaerobic SRB
growth medium is used in saturation process. After
saturation, radioactive tracers, 35SO42–, is added at
one side of bentonite clay, and copper disc is installedat another side. It is expected that 35SO42– is diffusedthrough the bentonite clays and at the same time
consumed by actived SRB to produce 35S2–.when35 2–
S is diffused and reaches copper discs, reaction
occurs and Cux35S is formed on the disc. When thecells are disassembled, the amount of 35S2– can bedetected by radiation testing equipment. Using
computer simulation program, evaluation of how
microbial activity decreases as buffer density andswelling pressure increase will be obtained.
In addition, the diffusion experiments are set up toobtain apparent diffusion coefficients of sulfate in thesaturated MX-80 compacted bentonite with different
compacted density. The data, as a characteristic ofsulfate in MX-80 bentonite, can be used in the SRB

1. Introduction
Sulphate-reducing bacteria (SRB) can obtainenergy by oxidizing organic compounds or molecularhydrogen (H2) while reducing sulfate (SO42−) tohydrogen sulfide (H2S). Sulphide is a very corrosive
molecule that can corrode the copper canister; on theother hand, microbes can withstand harsh conditions
generally, and it is no exception to SRBs: they arevery common in deep soil and groundwater.
Therefore, sulphide production and possible effects
on the repository function must be considered in thesafety assessment.
Due to the fact that sulphide is usually precipitatedwith ferrous iron, high concentrations do not occur ingroundwater. In addition, its diffusive transportcapacity is very low in bentonite buffer. Thus,
sulphide formation must take place in the buffer, near
the canister and be of considerable extent forcorrosion to be a threat to canister integrity [1].
In the SR-Can safety assessment, the low limit ofclay saturation density for microbial sulphideproduction was set at 1,800 kg/m3, based on theexperiments of microbial survivability. Microbialactivity was assumed not to occur at densities above
this limit [2].
1

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japanactivity experiment as well as other relatedexperiments. In this experiment, the diffusion cells
are designed to hold the bentonite clay in a constantvolume, and through-diffusion test is done withconstant concentration in the both sides [4]. Based on
the diffusion theory, the setting initial conditions andthe boundary conditions, the apparent diffusion
coefficients can be evaluated.

In saturation process, the anaerobic SRB growth
medium without lactate and sulfur is used. The recipe
is shown as table 2-2. In consequence, clays in thecells are kept in the better surroundings for microbes,
and rich of SRB. After saturation, the sulfur
radiotracers 35SO42– and the copper disc will be added.
First of all, the top lid is unscrewed, and the piston isremoved from the cells. 35SO42– is added at thesurface of clays by pipette, with the concentrationand the points of addition recorded. Secondly, thebottom lid is unscrewed, filter and support disc areremoved, and the space is replaced with a cleaned
copper disc with the same size. Finally, the test cell isreassembled, and the screws are adjusted to keep theswelling pressure same as the recorded pressure when
the test cell is opened.
After several days, based on the settings, the test
cells are disassembled. The copper discs are removed
from the bottom lids and carefully washed. Using
radiation testing equipment, radioactivity counts andthe distribution at different points of the disc can begained with final amount of the sulfur tracers, whichis adjusted for the half-life of the 35S isotope (87.4days).
A computer program will be used to simulate thediffusion and the reactions of the sulfur tracers. In theprogram model, space of the clay will be divided into
serval parts, and concentrations of 35SO42– and 35S2– ateach small space. With the simulation of a short timeinterval, molecules will diffuse to adjacent space, andin each space, 35SO42– will be reduced into 35S2– bySRB. By iterative calculations, complete experimenttime can thus be simulated through accumulations ofeach computed short time interval. The diffusion
process is computed with Fick’s law [5], shown as
below; sulfate reduction rate is computed as equation
below.

2. Materials and Methods
2.1 SRB activity experiment
In this experiment, six test cells are designed with
the inside volume, a cylinder with 50 mm in diameterand 20 mm in height, and are set to seal bentonite
clays (Figure 2-1). These cells are made of stainlesssteel 316, which has high corrosion resistantproperties, for the purpose of limiting the interactionof sulfide and the cell inner wall. The test cell is
consisted of a top lid, a bottom lid, a cylinder, and apiston. A tunnel through the piston is designed to link
the cell with the air vacuum system, and the othertunnel through the bottom lid is designed to link withthe water saturation system. Swelling pressure ofbentonite can be monitored and recorded through theforce transducer, which is set between the top lid andthe piston, by a data logger. A 40 µm pore size
stainless steel filter accompanied by a support discand a copper disc with the same size are designed to
be installed at the bottom lid.
Different conditions of cells are set, shown as table2-1. Two different saturation densities of bentonite
are prepared, 1,750 kg/m3 and 2,000 kg/m3, to verify
how microbial activity decreases as bentonite density
increases. In one set of three cells with same
bentonite density, different experiment time, 47 days
and 77 days, is designed; one cell is installed withsterilized bentonite as negative controls; others are
pre-mixed with SRB.

Figure 2-1 Left: Exploded view of the test cell. Right: Assembled form of the test cell.
26th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan(a)Table 2-1 Setting conditions of each test cellsaturation
Bentonite clays treatment
densitysterilized pre-mixed with SRB(kg/m3)
1,750

Cell 1

Cell 2Cell 32,000

Cell 4Cell 5Cell 677

4777

Experiment
Time (day)(b)

Table 2-2 SRB growth medium for saturation
composition

amountAnalytical grade water

1,000 mL

NaCl

7.0 g/L

CaCl2·2H2O

1.0 g/L

KCl

0.67 g/LNH4Cl

1.0 g/L

KH2PO40.15 g/L

MgCl2·6H2O

0.5 g/L(c)

Figure 2-2 Illustrations of concept of the model.
(a) Initial concentration contribution. Red arrows
represent the transportation through diffusion; blue
arrow represents SO42− reduction. (b) Concentration
contribution after a time interval. (c) Concentration
contribution after another time interval.

m’ = ΔC × D × A/d (Fick’s law)(1)m’ = transport rate (mol/s)
ΔC = concentration difference (mol/m3)
D = diffusion coefficient (m2/s)
A = boundary area between two adjacent space (m2)d = distance between space centers (m)based on the chosen porous supports (Figure 2-3).
These cells are made of Polytetrafluoroethylene(PTFE), which has high corrosion resistant properties,
for the purpose of limiting the interaction of salt andthe cell inner wall. The diffusion cell is consisted of 2
opposite reservoirs; changeable porous supports withdifferent length; a central cylinder. Bentonite can becompacted directly in the central cylinder. Aftercompaction, a filter paper and a stainless steel filter isinstalled at each side of the bentonite clays, andporous supports with proper size is installed to fill therest of the space of the central cylinder.
Through-diffusion with constant concentration inboth sides is adopted as the experimental method [4].
Based on the diffusion theory, using Fick’s 2nd law,considering 1-D unsteady state diffusion, the initialcondition and the boundary conditions, theconcentration profile can be determined. Expressionof the cumulative mass of diffused solute verse
diffusion time is shown as figure 2-4. When thesystem reaches steady-state conditions, theexpression can be simplified, and the diffusion
coefﬁcient can be calculated from the slope of thestraight line ﬁtting in the steady-state region on thefigure. The intercept of ﬁtting line with the time axis
is denoted as time-lag (te) [6]:dSO4Red = RedRate × V × dT
(2)
dSO4Red = amount of reduced SO42− to S2− during atime interval (mol)RedRate = SO42− reduction rate (mol/m3 s)
V = volume of a small space (m3)dT = time interval (s)In consequence, with the simulation conditions
same as the experiment, when proper reduction rate ischosen, result amounts of the 35S2– at the bottom
space of the clay will match the results gained on thecopper disc. Illustrations of concept of the model are
shown as figure 2-2. Using computer programs tosimulate the diffusion of sulfate tracers and thereduction of 35SO42– to 35S2– done by SRB, and thencomparing the resulting amounts of 35S2– at thecopper side with amounts found in the experiment,
reduction rates can be calculated.
2.2 Diffusion experiment of sulfate
In this experiment, the diffusion cells are designedto hold the bentonite clay in a constant volume, acylinder with 50 mm in diameter and a fixed height3

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan

Porous supports

20cm5cm
Inreservoir

Filter papers and stainless steel filters

Outreservoir

Compacted clays

Figure 2-3 Left: Exploded view of the diffusion cell. Right: Assembled diagram of the diffusion cell.

Da = d2/6te
Da = apparent diffusion coefficientd = thickness of the clay
te = time-lag

out-reservoir, the solution is sampled and changed
periodically to maintain the concentration near tozero. Experiment time lasts no less than the expected
time to reach steady state for each cell.

(3)

3. Results and Discussion
The dimensionless time can be used for evaluationof experiment time [6]:td = Dat/d2
td = dimensionless time
Da = apparent diffusion coefficientd = thickness of the clayt = experiment time

3.1 Results of SRB activity experiment
Several instruments and standard operatingprocedures have been being established including thetesting of six test cells, compaction method of
bentonite clays, chemical cleaning process of copper
disc, and the detection method of tracers on thecopper discs using white image plate, etc.
Incubation of microbes is done by Nuclear Scienceand Technology Development Center at National
Tsing Hua University, Taiwan. Three different
species of SRB is chosen following the experiments
done by SKB[3], Desulfotomaculum nigrificans(DSM 574) Desulfosporosinus orientis (DSM 765)
and Desulfovibrio aespoeensis (DSM 10631), andare well cultivated following the instructions byGerman Collection of Microorganisms and Cell
Cultures (DSMZ). Method of pre-mixing bentonite
clays with SRB before compaction is established aswell.
The saturation process is carefully test to maintainthe system inside the cell in anaerobic conditions.
First of all, all of the cells and the tubes in theexperiment are sterilized before use. The compacted
bentonite clays, a cylinder with 50 mm in diameterand 20 mm in height with different setting, areinstalled in the cells. The cells are assembled with thefilters and support discs installed at bottom lids andlinked with air vacuum system and water saturation
system. Secondly, through the tunnels at the pistonside, the cells are flushed by nitrogen, and thenevacuated to < 10 Pa using a vacuum pump. This
process is repeated several times in order to remove
all O2 in the system, and then the cells are kept at
evacuated state. Finally, saturation medium (Table2-2) is injected through the tunnels at the bottom side.
A gas pressure of around 2 bars is added to apressure-converted container made of stainless steel,and through a piston in the container, medium is(4)When td reaches 0.45, the system can be seenreaching steady state at referring experiment time.
Distilled water is used to fill both reservoirs andcirculated to saturate the clay. Saturation process
takes about 30 days to ensure the full saturation ofclays, as well as for the dissolution of small amountsof resident sulfate in the clays to some extent. After
saturation process, the solution at in-reservoir isreplaced with sodium sulfate solution withconcentration of 0.1M. Then the in-reservoir is linked
with the container which has a large volume ofsolution (4L), and a pump is used to circulate thesolution to minimize the concentration change; in the0.3

0.2
Normalized
cumulative
0.1
mass

000.2

te0.4
0.6
Normalized
diffusion time

0.81

Fig 2-4 Normalized cumulative mass of diffusedsolute verse normalized solute diffusion time.
Time-lag can be gain by the ﬁtting line at thesteady-state region.
4

6th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japanpushed into the cells. Saturation process will lasts
until the monitored swelling pressure reaches theTheoretical value, which depends on the clay density.
In addition, for the purpose of testing theexperimental procedures, a blank test is done using
bentonite clays with no extra treating, and saturated
with distilled water. Experiment can start once all theprocedures are set up completely.

(a)
4000
y = 141.69x - 510.85
R² = 0.996
3000

ΣC 2000(ppm)
1000

3.2 Results of diffusion experiment
Two diffusion cells are installed with different
conditions of compacted MX-80 bentonite clays, inconsideration of the time each system takes to reachsteady state. Settings of bentonite clays are shown astable 3-1. After experiment, samples are analyzed
using ion chromatography system to detect theconcentration of sulfate. Results are shown as figure
3-1.
The calculation results are shown as table 3-2. The
resulting apparent diffusion coefficients are closed toother studies using similar situations [7].

005

10

15

20

2530time(day)

(b)
4000
y = 105.31x - 428.08
R² = 0.9954
3000

ΣC 2000(ppm)
1000

4. Conclusions
In this report, instruments and procedures of theSRB activity experiment are introduced. The test cellis designed to seal the saturated clays pre-mixed withthe well-incubated SRB in the anaerobic conditions;sulfur tracers 35SO42– are used as the reactant of
microbial reduction reaction; computer programs areused to simulate the experiment and calculate themicrobial activities. Related standard operatingprocedures have been being established.
Diffusion experiment of sulfate is done using two
settings with different saturation density andthickness of the bentonite clays. Method ofthrough-diffusion with constant concentration in bothsides, and related initial and boundary conditions, areused in the experiment, and apparent diffusion
coefficients are calculated using diffusion theories;
the results are closed to other studies using similar
situations. Further studies with this experiment will
include uncertainty analysis, sensitivity analysis andthe replication experiments.

000.5

1,750

102

0.25

2,000

15203040time(day)Figure 3-1 Cumulative concentrations of diffused
sulfate verse diffusion time. (a) Cell No.1. (b) Cell
No.2.
Table 3-2 Results of diffusion experiment ofsulfate.
Cell No.

1Clay saturation
1,745
density (kg/m3)
Time to reach steady
9.73
state (day)22,004
10.97

d (m)

0.00485

0.00215te (day)

3.60

4.07

Da(m2/s)
1.26×10-11Da(theoretical value)
1.2×10-11(m2/s)
Error
4.88%

Table 3-1 Settings of bentonite clays in diffusion
experiment of sulfate.
Clay
Clay
Estimated time
Cellsaturation
thicknessto reach steadyNo.
density(cm)state (day)(kg/m3)110

2.19×10-12
2×10-12
9.68%

5. References[1] Svensk Kärnbränslehantering AB, “Buffer,backfill and closure process report for the safetyassessment SR-Site,” SKB Technical Report
TR-10-47 (2010).
[2] Svensk Kärnbränslehantering AB, “Long-termsafety for the final repository for spent nuclear
fuel, Main report of the SR-Site project, VolumeI,” SKB Technical Report TR-11-01 (2011).
56th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan[3] Andreas Bengtsson, Johanna Edlund, Björn
Hallbeck, Carl Heed and Karsten Pedersen,“Microbial sulphide-producing activity in
MX-80 bentonite at 1750 and 2000 kg m–3 wetdensity,” SKB Report R-15-05 (2015).
[4] García-Gutiérrez M, Cormenzana J L, Missana T,Mingarro M, Molinero J, “Overview oflaboratory methods employed for obtaining
diffusion coefficients in FEBEX compactedbentonite,” Journal of Iberian Geology 32, 37–53(2006).
[5] Coulson J M, Richardson J F, “Chemicalengineering,” 4th ed. Vol. 1. Oxford: Pergamon(1990).
[6] J. CRANK, “The Mathematics of Diffusion,”
49-52 (1975).
[7] Pedersen K, “Analysis of copper corrosion incompacted bentonite clay as a function of clay
densityandgrowth
conditions
forsulphate-reducing bacteria,” Journal of Applied
Microbiology 108, 1094–1104 (2010).

66th East Asia Forum on Radwaste Management Conference
November 27-29, 2017, Osaka, Japan