Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 43-50, 2005

Accepted
Evaluation of Microbial Activity for Long-Term Performance Assessments of Deep
Geologic Nuclear Waste Repositories
Yifeng Wang*, a and Arokiasamy J. FrancisbaSandia National Laboratories, P. O. Box 5800, MS 0776, Albuquerque, New Mexico 87185, USA
Brookhaven National Laboratory, Upton, New York 11973, USAb

Received: November 15, 2004; In Final Form: November 15, 2004
Microorganisms are ubiquitous in subsurface environments and play a major role in the biogeochemical recyclingof various elements. In this paper, we have developed a general approach for a systematic evaluation of microbial
impact on the long-term performance of the repository. We have demonstrated that data on microbial population
alone are not sufficient for the evaluation of microbial impact on repository performance and a sensible approachfor such evaluation must be based on the consideration of environmental constraints on microbial reaction pathways. We have applied our approach to both the Yucca Mountain (YM) repository and the Waste Isolation Pilot
Plant (WIPP). We have demonstrated that the effect of microbial activity on the near-field chemistry in the Yucca
Mountain repository is negligible because of limited nutrient supply and harsh environmental conditions createdby waste emplacement. Whereas for the WIPP, we have shown that, due to the presence of a large quantity oforganic materials and nutrients in the wastes, a significant microbial activity can potentially be stimulated and its
impact on repository performance can be evaluated with carefully designed incubation experiments coupled withperformance assessment calculations. The impact of microbial gas generation on disposal room chemistry in the
WIPP can be mitigated by introducing MgO as a backfill material.

1. Introduction
Numerous studies have been conducted on microbialactivity related to nuclear waste disposal in deep geologic
repositories. Microorganisms have been detected in backfill
materials, natural analogue sites, plutonium-contaminatedsoils, low-level radioactive wastes, and waste-repository sites
selected for high-level radioactive wastes. 1−13 West et al.
surveyed microbial population distributions in water andsubsoil samples from deep mines designated for disposal ofhigh-level radioactive waste in Europe. 14, 15 They reported
finding a variety of organisms that include native organisms
and organisms introduced from mining operations. The presence of viable microbial populations in subsurface environments clearly suggests that, under certain conditions,significant microbial activity can be stimulated that can potentially impact the transformation and transport of radionuclide
in repository environments.
The impacts of microbial activity on repository performanceare several-fold:16 (1) Microbes may be directly involved inredox reactions of multivalent radionuclides such as uranium
and plutonium,17 whose solubilities highly depend on oxidation
states. (2) Microbial reactions may play an important role inregulating water chemistry (e.g. pH, Eh, and ΣCO2) as well as
secondary mineral distributions (i.e., radionuclide sorption
sites). (3) Microorganisms release low molecular weightorganic acids including acetate, lactate, and formate, which areable to complex with radionuclides. On the other hand,biodegradation may eliminate organic ligands and reduce themobility of radionuclides. (4) Microbial cells may act as
colloidal particles and thus facilitate radionuclide transport.
(5) Additionally, microbial reactions may impact metal corrosion and therefore the integrity of the engineered barriersystem. The combination of radionuclide mobility and EBS
integrity ultimately controls total radionuclide release from a*Corresponding author. E-mail: ywang@sandia.gor. : +81-47308-8866.

repository.
In this paper, we develop a general approach for a systematic evaluation of microbial impact on the long-term performance of the repository, based on the consideration ofenvironmental constraints on microbial processes. We thenapply this approach to both the Yucca Mountain (YM) repository and the Waste Isolation Pilot Plant (WIPP). We demonstrate that the effect of microbial activity on the near-fieldchemistry in the Yucca Mountain repository is negligible
because of limited nutrient supply and harsh environmental
conditions created by waste emplacement. Whereas for the
WIPP, we show that, due to the presence of a large quantity oforganic materials and a significant amount of nutrients in thewastes, a significant microbial activity can potentially be stimulated and its impact on repository performance can be evaluated with carefully designed incubation experiments.
2. Systematics of Repository Biogeochemistry
Population distribution vs. activity. Microbial populations surveyed at sites related to radioactive waste disposal are
listed in Table 1. It can be seen from the table that microbial
population varies greatly between locations within a specificsite and between different sites. Microbial populations at thesesites are generally lower than 10 7 cells per gram, with the
exception of populations found within some bentonite materials, in which the population can be higher than 1011 cells pergram. Bacteria dominate the whole microbial population, withfungi only accounting for ~ 1%. Note that, at the same site,
the cell density in water is much lower than that in the associated sediments, indicating the tendency for microbial cells to
attach solid surfaces. Generally, it is difficult to correlate thepresence of specific organisms with site conditions. The presence of a specific set of bacteria does not guarantee that they
will be active; some of them may be dormant if conditions are
not conducive to their active state. This implies that data on
microbial population alone are not able to provide sufficient
information on the geochemical impact of microbial activity.

© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

Wang

TABLE 1: Abundance of Microorganisms at Radioactive Waste Disposal Related Sites
Sample Source
Soils, Area 13, Nevada Test Site

Microorganisms Detected
Bacteria: 2.9 ± 0.3 to 8.0 ± 0.7 × 106/g
Fungi: 3.1 ± 0.7 to 24.6 ± 2 × 103/g

Reference
1

6

Los Alamos National Labs TRU waste shallow burial site
TA-54, Area C

Bacteria: aerobes: 2.3 × 10 /ganaerobes: 3.3 × 106/g
Fungi: 4.9 × 104/g

2

Sediment/water, Rocky Flat Plant Pond B1

Bacteria in sediments: aerobes: 3 – 300 × 106/mL
anaerobes: 0.2 – 2.0 × 106/mL
Bacteria in Water
aerobes: 1.1 – 340 × 103/mL
anaerobes: 2 – 7.8 × 102/mL

3

Waste leachate, Trench 19S, Maxey Flats, KY

Bacteria: aerobes: 2.2 × 102 CFU/mL
anaerobes: 3.2 × 102 CFU/mL

4

Groundwater (5 M NaCl), Waste Isolation Pilot Plant (WIPP),
New Mexico, USA

Bacteria: Far-field: 1.02 ± 0.49 × 105 cells/mL
Near-field: 1.24 ± 0.13 × 105 cells/mL

5

Study Site Groundwater, Nevada Test Site, USA

Bacteria: 102 viable cells/mL

6

Granite fracture zone, Äspö Hard Rock Lab, Sweden

5

Bacteria: 0.44 – 9.3 × 10 cells/mL
3

7

Granite shear zone, Grimsel Test Site, Switzerland

Bacteria: 3.97 ± 0.37 × 10 cells/mL

12

Yucca Mountain tuff, Nevada, USA

0.32 – 2.0 × 105 cells/g

10

Cigar Lake uranium ore deposit, Saskatchewan, Canada– sandstone w/ clay-hosted U ore

Bacteria in groundwater: 4.7 × 102 – 4.4 × 104 cells/mL
Bacteria in ore:
1.4 ± 0.9 × 105 CFU/g viable cells

17aWyoming bentonite

5.32 ± 0.34 × 1011 cells/g; <1.07 ± 3.54 × 102 CFU/g viable cells

11

Avonlea bentonite

6.29 ± 0.75 × 1011 cells/g; 3.48 ± 0.56 × 104 CFU/g viable cells

11

Canadian sand and bentonite buffer material

16Bacteria: 10 to 10 viable cells/g

Microorganisms in a natural system are highly adaptable andsufficiently diverse for carrying out any specific metabolic reaction that the environment permits. The extent of each individual
microbial reaction will be limited by environmental constraints,
not by the types of microbes present. A specific group of
microbes will thrive only to the extent that the environment
allows. This argument is consistent with the modeling approachthat has been used successfully over the years for modelingbiogeochemical processes in aquatic sediments.18, 19 In thisapproach, microbial population is not explicitly included. A
theoretical basis for this has been provided.16 The environmental factors that can potentially affect microbial growth andactivity in a repository environment include moisture, temperature, pH, Eh, availability of organic and inorganic nutrients, andradiation. The key to evaluate the geochemical impact of
microbial activity on repository performance is to determine theextents of individual microbial metabolic pathways under theconstraints of these environmental factors.
Heterotrophs vs. autotrophs. The metabolic pathway thata given microorganism will use depends on the energy source(light or chemical); thus, a microorganism is described as eithera phototroph or chemotroph, respectively. In a deep geologic
repository, the activity of phototrophs can be excluded becauseof the lack of a light source after closure. Among chemotrophs,
heterotrophs use organic carbon as both carbon and energy
sources, whereas autotrophs derive energy from the oxidationof inorganic compounds and derive cell carbon from CO2. As
shown in Figure 1, because of their different nutritional requirements, the activities of these two groups of microorganisms
in a deep geologic repository are limited in different ways,
depending on the ambient chemical environment and the typeof wastes to be emplaced in the repository.
An autotrophic reaction is generally limited by the availability of inorganic electron donors such as H2, NH4+, NO2−,
Mn2+, Fe2+, H2S, and S. To support cellular growth, autotrophs
fix CO2 through a “Calvin cycle”and require a considerable
investment of energy.20 As a result, the direct utilization of
CO2 by autotrophs in subsurface systems is probably slow. In
contrast, heterotrophs show a great range of flexibility withrespect to carbon sources. Some bacteria will degrade almost

13

Figure 1. Systematics of Repository Biogeochemistry.

any reduced carbon source, whereas others will only catabolize
a few carbon compounds. Heterotrophic metabolism dominates over autotrophic metabolism in many subsurface environments, even in some pristine environments where theorganic carbon supply is relatively limited, since it is observedthat carbonate species, which are the end products ofheterotrophic metabolism, are accumulated in these natural
systems.20

Evaluation of Microbial Activity

Microbially mediated redox processes. Microbial groups
are known to use many redox pairs to derive their energy. Ifsuch chemical kinetic constraints exist that the rate of a given
chemical reaction is sufficiently slow, bacteria can compete;thus, almost any redox pair that yields energy could be exploited.
Microorganisms tend to accumulate at redox interfaces, where
both metabolic electron donors and electron acceptors are available. Numerous studies have shown that microbial metabolism
couples the inorganic redox chemistry of groundwaters to theoxidation of organic carbon.21 Heterotrophs derive their energy
from the oxidative breakdown of external organic substrates;
hence, microbial ecologies are frequently classified in terms ofdominant pathways. The principal pathways are aerobic respiration, denitrification, manganese reduction, iron reduction, sulfate
reduction, fermentation, and methanogenesis. From a biochemical point of view, the energy-yielding metabolic processes
involve complex electron transfer chains. From a geochemical
perspective, the significant reaction is the final electron transfer
to an external electron acceptor. The common electron acceptors in subsurface environments are O2, NO3−, MnO2, Fe(OH)3,
SO42−, and CO2. Fermentation does not rely on an external
electron acceptor; instead, it partially oxidizes and reduces anorganic substrate. The final fermentation products include
CO2, H2, alcohols, and organic acids.
The primary pathways of organic carbon oxidation tend tooccur in a predictable sequence. The most energetically favorable is aerobic respiration, followed, in order of decreasing
free energy yield, by denitrification, Mn(IV) reduction, Fe(III)
reduction, sulfate reduction, and methanogenesis. The differences in the energy yields of these reactions, plus the availability of electron acceptors, lead to physical (spatial or
temporal) separation of redox zones in which each pathway
dominates. Because aerobic respiration is the most energetically favorable reaction, aerobic bacteria have a competitive
advantage over anaerobic microorganisms when a certain levelof O2 is present. This redox sequence is well established fornatural systems including aquatic sediments, 19 groundwater
systems,20 and landfills.22 This sequence imposes an important
constraint on metabolic processes that can occur in a specificrepository environment, and it provides a technical basis formodeling biogeochemical processes in a repository.23, 24
Relevant Biogeochemical Processes in radioactive repositories. The relative importance of various microbial processes
in a specific repository greatly depends on the ambient environment of the repository, and the materials to be emplaced(Figure 1). A repository in unsaturated igneous rock formations (such as volcanic tuff rocks at Yucca Mountain) is generally expected to be oxidizing in its chemical environment; arepository in a hydrologically saturated zone, particularly in
sedimentary rocks, could be reducing. Sedimentary rocks
contain a certain amount of organic matter, which may stimulate significant microbial activities and, thus, maintain therepository and its surrounding areas in a reducing condition.
Both low-level and intermediate-level radioactive wastes
contain a large portion of organic materials (e.g. cellulosics)
and a significant amount of inorganic nutrients (e.g. NO3−).
Microbial degradation of these materials has been a major
concern for long-term repository performance assessment. An
example is the WIPP, which is located within a salt bed inSouthern New Mexico, and was designed for disposal of
defense-related transuranic wastes. Microbial degradation oforganic carbon-rich materials in the WIPP repository has beenstudied for its impact on repository pressurization and waterchemistry.24, 25 Unlike low-level and intermediate-level wastes,
high-level radioactive wastes generally contain no organic
materials and are thus not conducive to microbial activity.
As discussed above, autotrophs are limited by the supply ofelectron donors and inorganic carbon. Since carbon dioxide ispresent in most systems, the availability of reduced inorganic

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

45

species (e.g. H2) is expected to be a major limiting factor for
autotrophs. In a nuclear-waste repository, the electron donors
are derived from three sources: (1) The anaerobic oxidation oforganic compounds by heterotrophic bacteria can serve as theprimary source of both the reduced inorganic substrates andthe inorganic carbon for autotrophic metabolisms. For
instance, fermentation produces H2 and CO2, both of which areneeded by autotrophic methanogens for synthesizing methane.
(2) A large fraction of metals or metal alloys is always present
in repositories, either as waste package materials or as otherengineered components. Anoxic corrosion of these metals, Fe°+ 2H2O = Fe(OH) 2 + H2, generates hydrogen gas. And (3)reduced inorganic species may be brought in by groundwateror gas flows from the surrounding areas. Figure 1 also indicatesthe possibility that, through biomass recycling, a biomass
synthesized by autotrophic bacteria can be used by heterotrophs.
Mobilization or immobilization of radionuclides by theactivities of autotrophs (in the case of inorganic compounds)
and by heterotrophs (in mixed wastes containing organics)
could be significant.4 Autotrophic bacteria such as iron andsulfur oxidizers play a significant role in the solubilization of
uranium from ores and mill tailings. The biogeochemistry of
uranium has been extensively studied in light of the recovery
of U from ores. Microbial leaching of pyritic uranium ore is
primarily indirect, and confined to the generation of the oxidizing agent, ferric sulfate, and the solvent sulfuric acid. The
indirect involvement of Fe2+/Fe3+ in the process of cyclicallymediating the oxidation of the insoluble uranium oxide has
been documented: UO2 + Fe3+ → UO22+ + 2Fe2+. T. ferroxoxidans can also directly oxidize reduced compounds of uranium(uranous sulfate and UO2) to their hexavalent form without the
involvement of extraneous Fe3+/Fe2+ complex as the chemicalelectron carrier. Iron- and sulfur-oxidizing bacteria (T. ferrooxidans and T. thiooxidans) were isolated from several uranium ores. An increase in heterotrophic microbial activity due
to biodegradation of organic compounds can affect the solubility of radionuclides and metals. Heterotrophic bacteria andfungi are able to release metals from various materials, including copper-nickel concentrates, low-grade copper ore, uranium
and manganese ore. Several mechanisms for heterotrophic
aerobic microbial solubilization of insoluble metal have been
proposed. These include organic acid production, formation ofchelating agents, and metabolism of metal-associated anion.
Leaching by heterotrophic organisms is entirely due to chemical reaction of excreted microbial metabolites and decomposition products.
Figure 1 provides a theoretical framework for the evaluationof the microbial activity within the repository. Note that (inaddition to the processes described in the figure) temperature,
water availability, and radiation will play an important role in
the regulation of microbial activities within the repository
during relevant time periods.
3. Evaluation of Microbial Activity in the YM Repository
Microbial population distributions. The Yucca Mountain(YM) repository is located in an unsaturated zone of welded
volcanic tuff. Thus, the geochemical impact of microbialactivity within the repository should be considered in thecontext of physical, chemical, and hydrologic conditions in
vadose zones. Characterization of microbial communities has
principally focused on correlating the types, numbers, andactivities of present microorganisms to extant geologicalfeatures, and with water and carbon availability.10, 26−31 Thesestudies, along with those examining the activities of subsurface
isolates in pure cultures, have shown bacteria are present in all
vadose zones examined thus far.
The quantity of intact phospholipid fatty acids (PLFA) is adirect indicator of viable or potentially viable cells in an envi-

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Wang

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

ronmental sample. Thus, PLFA analysis permits the direct
quantification of microbial biomass in environmental samples
without the need for culturing microorganisms.32 Quantitative
analysis of the total number of bacteria present in a Yucca
Mountain rock sample aseptically collected from the
Exploratory Studies Facility (ESF) was undertaken using thistechnique.31 A single rock core was collected and cut into twopieces: one piece consists of the region facing the tunnel (proximal to the drift), and the other piece is the area reaching into
the tunnel wall (distal to the drift). Using this method, it was
deduced that the proximal sample contained 6 × 104 cells pergram of dry rock (3 pmole (pM) of PLFA/g dry rock); thedeeper-dwelling distal sample had 4 × 104 cells per gram ofdry rock (2 pM of PLFA/g dry rock), using a conversion factorof 2 × 104 cells/pM PLFA. No diglycerides, an indicator ofdead bacteria, were detected. The PLFA analysis showed a
preponderance of gram-negative organisms.
Subsurface environments have demonstrated high proportionsof heterotrophic bacteria,33 especially in studies of oxygenatedvolcanic tuff at the Nevada Test Site.6, 27 Microbial analyses
conducted in the YM Experimental Study Facility (ESF) havedetermined the existence of aerobic heterotrophs and autotrophs34
including iron oxidizing, sulfur oxidizing and nitrifying organisms. Cell counts for autotrophs range between 10 and 500
cells per gram dry weight and, for the heterotrophs, between
3.2 × 104 to 2 × 105 cells per gram of tuff. The relatively small
population of autotrophs reflects the oxidizing environment at
Yucca Mountain, which is not conducive to sustaining autotrophic
bacteria. Characterization of DNA extracted directly from YM
rock was undertaken to generate a more complete roster of theorganisms contained within the YM community.35 Four hundred
grams of Yucca Mountain rock were subjected to extraction.
The resulting DNA was purified and subjected to a series ofbiochemical and genetic manipulations that included amplification of microbial 16S rDNA genes and cloning amplified
genes. Two hundred rDNA clones were screened to obtain thosethat were unique based on restriction fragment length polymorphisms. After screening 200 rDNA clones, 65 unique clonal
types, representing 65 separate types of organisms, were distinguished. Identified YM organisms span a wide phylogenetic
range, and include groups of organisms known to reside in dry
environments (e.g. the High Guanine (G) + Cytosine (C) Gram
Positives). The great diversity of microorganisms detected at
the YM site further confirms our early assertion that microorganisms in a subsurface environment are highly adaptable andsufficiently diverse for carrying out any specific metabolic
reaction that the environment permits.
Temperature constraint. The temperature of the subsurface environment will limit the type of bacteria that are present,
based on the optimum growth temperatures of the organismsthat are present. Only a few of the identified isolates are known
to withstand elevated temperatures (e.g. P. stutzeri, R. pickettii,
B. natatorius). Only one isolate, M. mesophilicum, is capableof forming cysts that are desiccation resistant; M. mesophilicum
has also been found to be dominant in soils surrounding theChernobyl Atomic Power Station.36 Other bacteria, if they survive, will remain in a dormant state, without any contribution to
the geochemical processes of interest. Sulfate-reducing bacteria isolated from a mine in Britain and Thiobacillus ferrooxidans found in several sites in Europe have been subjected torepository conditions in order to establish their ability to survive under these conditions.15 These experiments have included
establishing growth curves, temperature tolerance tests, combined high pressure and temperature tolerance, and gammairradiation. The experimental data suggest that sulfate-reducing
bacteria are more tolerant to extreme environmental conditions
than the sulfur-oxidizing Thiobacillus ferroxidans.15 Sulfatereducing bacteria, strict anaerobes, are not expected to be activein the oxidizing Yucca Mountain environment. It is agreed that

Upper limit for
microbial activity

Lower limit for
microbial growth

Figure 2. Thermal and Relative Humidity Histories Predicted for YM
Repository and Their Constraints on Microbial Growth (modified
from BSC, 2004).38

the maximum temperature at which known microorganisms can
exist in an active state is ~120 ˚C.37
The in-drift thermal history in the YM repository has been
predicted by a thermal-hydrologic model that accounts for heat
conduction from the drift wall, into the rock matrix, resulting
in vaporization and boiling, with vapor migration out of matrix
blocks into fractures (Figure 2).38 During the period of thermal
perturbation resulting from waste package emplacement, thetemperature in the repository drifts could exceed 120 ˚C (theupper temperature limit for the presence of microorganisms)
and, for a waste package surface, could be as high as 170 ˚C.
Therefore, microbes initially present in the drift will beseverely limited in growth, if not totally eliminated, by heating
for a few hundred years at the early stage of the repository.
Microbes may migrate into the repository with infiltrating fracture flow, once temperatures decrease. Infiltrating organismsthat survived the heating period may be presumed to colonize
if conditions are favorable for growth. As shown in Figure 2,even after the peak temperature, the in-drift temperature willremain above 50 ˚C for the duration of the 10,000-year regulatory period. Therefore, the microbial population will be dominated by thermophiles and hyperthermophiles.
Relative humidity constraint. Water is essential for microbial growth. In order for microorganisms to grow, the relative
humidity in the environment needs to be 75 to 100%.37, 39 Manybacteria perish, sporulate, or become dormant when water
activity falls below approximately 0.90.39, 40 Some fungi andyeast can be active at lower water activity levels (on the orderof 0.7 to 0.85.37 However, these microorganisms have verysmall populations as compared to bacteria (Table 1) and aresolely dependent on the availability of organic carbon, which
will be very limited in the Yucca Mountain repository. In afull-scale test conducted at Atomic Energy Canada Ltd. (AECL),a simulated waste container (maximum heat output 85 ˚C) wasburied for 2.5 years, surrounded by compacted buffer materials.
Extensive microbial analysis of this test has shown that mostviable organisms in the buffer material disappear around amoisture content of 15 percent (corresponding to RH 95 to 96
percent).41
The activity and availability of water for microbial growthin the UZ at Yucca Mountain will be dependent upon thermalhydrologic conditions. The range of relative humidity on all
waste packages is shown in Figure 2. During the thermal
pulse, relative humidity on the waste package surface can belower than 10 percent. In some realizations, relative humidity
recovers (back to 100 percent) after approximately 1,000
years. In many other realizations, relative humidity remains
below 90 percent, and even below 70 percent in some cases,
throughout the regulatory period, thus limiting microbial
growth and activity. In addition, low water activity generally
corresponds to low liquid-water availability. Low saturation ofliquid water in fractures on the drift wall will limit the transport growth substrates to microbial cells and, therefore, theEvaluation of Microbial Activity

activity of the cells.
Constraint of oxidizing environment. Bacteria that areable to grow in the presence of molecular oxygen, either in agaseous or dissolved form, are termed aerobes; those that cangrow without oxygen are anaerobes. Wang and Van Cappellen19
have shown that a typical limiting concentration of O2 foraquatic sediments is 1 to 30 µM (approximately 0.4 to 10
percent atmospheric oxygen), above which anaerobic microbial reactions are completely inhibited.
During the thermal pulse, water vapor will drive air mass
away from the waste drift, thus resulting in depletion inoxygen concentration, which is proportional to the fraction ofremaining air mass in the gas phase. A thermo-hydrologicalcalculation shows that the air mass fraction will decrease toless than 10 percent during the thermal pulse, and will thenquickly recover (back to 100 percent) after the thermal pulse,
as does the oxygen concentration.42 However, the depletion inoxygen concentration is not expected to have an impact on
microbial reaction pathways, because microbial activity will beseverely limited, if not completely eliminated, due to both theheating and the desiccating effects, during the thermal pulse, as
discussed above. Therefore, within the time window permissive for microbial activity (i.e. with temperature less than 100
˚C), the oxygen concentration in the repository will be maintained above its limiting concentration (i.e. greater than 10
percent of atmospheric value). Therefore, the overall chemical
environment in the repository will be oxic, and a significant
anaerobic microbial activity will be unlikely.
The assertion of an overall oxic environment in the repository
is consistent with field observations.42a Iron and manganeseoxides/oxyhydroxides are the most common mineral phases
found in the fractures in both saturated and unsaturated zones.
The redox state of the unsaturated zone (UZ) waters is probably
dominated by the presence of oxygen in the UZ gas phase. At
the two locations where such measurements have been made, theUZ gas phase has atmospheric levels of oxygen. The measured
Eh values are typically high (400 to 600 mV). In addition to theoxic background environment, radiolysis of water inside thewaste drift will generate hydrogen peroxide, thus adding anotheroxidizing agent to the water.
The oxidizing environment will also prevent the generationor accumulation of reduced inorganic species (e.g. H2, NH4+,
NO2−, Mn2+, Fe2+, H2S, and S), which are a prerequisite substrate
for autotrophic microbial processes. Chemical analyses of YM
groundwaters from Nye County Early Warning Detection
Program wells show that the waters contain only trace amountsof dissolved iron (less than 2.34 ppm) and manganese (less than
0.58 ppm) (not necessarily all in Fe2+ and Mn2+), and NH4+ (lessthan 1 ppm). Fe 2+ ions may be present as an intermediate
product of waste package corrosion, but they are transient intime and localized in space to the corrosion front. Long-term
corrosion tests evaluated waste package material coupons invarious solutions under anticipated repository conditions. 35
Interestingly, even in this oligiotrophic and metallic alloy-dominated environment, the microbial population was dominated byhetrotrophic bacteria. Bacilli, known as heterotrophs, wereobserved to be the most numerous, representing 84 percent of
the screened clones. This indicates that metal corrosion under
Yucca Mountain-relevant conditions will not support any significant autotrophic microbial growth. This may be partly because
abiotic Fe2+ oxidation is kinetically favored over microbial
oxidation.
Constraints of nutrient supply. Experiments have been
performed to define conducive and inhibitory environmental
conditions that pertain to growth of Yucca Mountain microbialcommunities in modified Yucca Mountain groundwater. 31
Experimental data show that nitrogen and sulfate sources areapparently sufficient to support microbial growth, even in
unconcentrated Yucca Mountain groundwater. Both phosphate

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 200547

and organic carbon have been shown to have significant effecton microbial growth. Phosphate increased cell densities
approximately 1.5 orders of magnitude. The addition of acarbon source, glucose, resulted in increases of one order ofmagnitude in unconcentrated simulated groundwater medium.
Yucca Mountain groundwater, however, has extremely low
levels of organic carbon and phosphate. Only trace concentrations of organic carbon (up to 1.1 mg/L) have been reported inqualified measurements of Yucca Mountain groundwater. The
materials introduced into emplacement drifts will not containany organic carbon,43 except for a trace amount of reduced
carbon in metal or metal alloys. These carbons, like graphite,
are expected to be refractory and, thus, will not be biodegradable. Therefore, the extremely low organic-carbon supply inthe repository will limit heterotrophic microbial activity.
Radiation effect. Much of the information on the radiation
resistance of microorganisms comes from the food industry,
because ionizing radiation is used in many countries as a meansof preserving foods. Exposing foods to low doses of ionizing
radiation (less than 1 Mrad) substantially reduces the numberof those microorganisms responsible for food spoilage. Many
radioresistant strains of bacteria have been isolated from soil,
and studies of soil microflora irradiated with long-term γ-radiation (in an open-air facility at 15, 150, 800, and 1500 kradover 18 months) showed that radiation resistance increased at
all doses when compared with the radiation resistance of soil
microflora from soil shielded from irradiation with lead bricks.44
However, these microbes did not show significant growth underirradiation of higher than 5 Mrad. Barnhart et al.45 found thatapproximately 68 percent of the microbial isolates from radioactive shallow land burial site soil exhibited greater resistance to γand β-radioactivity than did the common soil bacterium Bacillus
subtilis to ionizing radiation. Approximately 25 percent of theisolates from nonradioactive soil were more resistant than B.
subtilis, but all these microbial isolates were less radioresistant
than Deinococcus radiodurans, which is resistant to γ-radiation(105 rad). However, radio-resistant bacteria are constantly being
enriched in an environment containing nanocurie levels of γand β-radioactivity.45 In the later study, the radio-resistance of
10 isolates of bacteria from a soil core (of a 40-cm-to-50-cm
depth, and containing 20 nCi to 30 nCi of radioactivity), takenfrom a shallow land burial site at Los Alamos National Laboratory,
was studied. Several of the isolates were resistant to 5 × 104
rad γ-radiation. At 7.5 × 104 rad, none of the isolates survived.
A time-course experiment was conducted to evaluate the effectsof γ radiation on the indigenous microbiota present in rockobtained from Yucca Mountain and the Nevada Test Site.46
Microcosms were constructed by placing pulverized Yucca
Mountain rock in polystyrene cylinders. Continuous exposure(96 h) at a dose rate of 1.63 Gray (Gy)/min was used to mimic
the near-field environment surrounding waste canisters. The
microbial communities were characterized after receiving cumulative doses of 0 kGy, 0.098 kGy, 0.58 kGy, 2.33 kGy, 4.67
kGy, 7.01 kGy, and 9.34 kGy. Radiation-resistant microorganisms in the pulverized rock became viable but nonculturable
after a cumulative dose of 2.33 kGy.
Based on the current repository loading design, the expected
maximum surface dose rate from one unbreached canister
designed to contain spent nuclear fuels is 1.7 Mrad/year.
Therefore, it is expected that radiation may inhibit microbial
growth in the repository. This assessment is consistent with an
AECL test that used a simulated waste container with amaximum heat output of 85 ˚C). The test has shown that thesurface of nuclear fuel waste containers is “sterilized” 9 to 33
days after emplacement.41
Overall impact of microbial activity on YM near-fieldchemistry. Microbial growth and activity at Yucca Mountainare limited by multiple factors: (1) The in-drift temperatures

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

during the thermal pulse will exceed the temperature toleranceof all known microbes for a significant portion of the repository time. (2) An oxic environment will prevail in the repository over the growth-permissive period and, therefore, preventthe generation and accumulation of reduced inorganic species
that are the prerequisite for autotrophic metabolism. (3) The
relative humidity and the liquid-water saturation in the repository are predicted to be low for a significant duration, thus
further limiting microbial activity. And (4) the extremely low
organic carbon supply in the repository will limit heterotrophic
microbial activity. Due to these environmental constraints, themicrobial activity in the repository is expected to be low, andits impacts on drift chemistry can be negligible.

Wang
300

CO2 Accumulated in Sample Headspace ( moles/g cellulose)48

Anaerobic, inundated samples
250

Inoculated+nutrients+nitrate

200

150

100

Inoculated+nutrients

50

Inoculated only
00

4. Evaluation of Microbial Activity in the WIPP Repository
Environmental constraints. The Waste Isolation Pilot
Plant (WIPP), located in a salt bed in southern New Mexico, isdesigned by U.S. Department of Energy for permanent
disposal of defense-related transuranic wastes. In this highsalinity environment, microbes present in the repository are
dominated by halophilic or halotolerent bacteria, with a population of 1.02 ± 0.49 × 105 cells/mL in the far field and 1.24 ±0.13 × 105 cells/mL in the near field.5 Microbes detected in the
WIPP include denitrifiers, fermenters, sulfate reducers, andmethanogens.5
Unlike the YM repository, the WIPP can be categorized as
an organic carbon-rich repository. Based on the inventory estimates,24 wastes to be emplaced to the WIPP contain a large
quantity of organic materials and various nutrients: ∼ 10 9moles of organic carbon, 2.6 × 107 moles of nitrate, and 6.6 ×
106 moles sulfate. The organic materials are dominated bycellulsics, rubbers and plastics. There is a concern for the
long-term performance assessment of the repository with apotential CO2 generation from biodegradation of organic materials, especially for the scenario in which a large volume ofbrine is introduced into the repository by human intrusions.
The generation of CO2 can potentially impact the mobility ofactinides and the closure of disposal room.
Incubation experiments. Long-term incubation experiments (~ 10 years) were started in January, 1992, to obtaindata on microbial gas generation under anticipated WIPP
conditions. 8, 25 The experiments performed with simulated
cellulose materials and G-seep brine collected underground in
the WIPP. The G-seep brine contains 10 4 – 10 6 bacterial
cells/mL.8 In the experiments, a certain amount of microbial
inoculum was added to each sample. This inoculum was
prepared from a mixrure of a variety of WIPP repository-relevant samples: sediment and brine from Nash Draw, brine fromthe WIPP underground workings, and inocula from a nonsterile laboratory environment. The data collected in theexperiments include quantification of total gas, H2, O2, CO2,N 2 , N 2 O, CH 4 and H 2 S produced in the headspace of eachsample.
The results of headspace gas analyses for anaerobic, brineinundated samples are shown in Figure 3. The amount of CO2
accumulated in nitrate-amended samples was systematicallyhigher than that in samples without excess nitrate amendment,
demonstrating the dependence of cellulose biodegradation on
the availability of electron acceptors and nutrients. Microbes
use nitrate both as an electron acceptor and as a nutrient forbiomass synthesis. Nutrient-amended samples initiallycontained 250 µmoles of nitrate/g cellulose (equivalent toNO3−/C ≈ 0.007), while nutrient-amended samples plus excess
nitrate contained 1240 µmoles of nitrate/g cellulose (equivalent to NO3−/C ≈ 0.033).8
Based on the waste inventory estimates,24 the wastes to be
emplaced in the WIPP contain 2.85 × 107 kg of total equivalentcellulose materials (or 1.05 × 109 moles of C) and 1.6 × 106 kg

500

1000

1500

2000

2500

3000

Time (days)

Figure 3. CO2 production in anaerobic, brine-inundated cellulose
samples. The accumulation of CO2 in each set of samples displays
two distinct gas generation phases: a rapid increase in the first 500
days followed by a long period of slow accumulation.

(or 2.6 × 107 moles of NO3−) of nitrate. Therefore, the average
molar ratio of NO3− to organic C in the waste will be 0.025,
slightly less than in the experiments amended with excess
nitrate. Therefore, the measurements on nitrate-amended
samples provide an upper bound for the microbial gas generation rate in actual wastes.
The accumulation of CO2 in each set of samples displays
two distinct gas-generation phases: a rapid transient rate in the
500 days followed by a long period of slow accumulation(Figure 3). The transition from the transient to the long-term
phase is unlikely to be caused by pH changes or the accumulation of metabolites. In fact, no significant pH changes weredetected during incubation.25 In addition, the occurrence of
methane (CH4) production in amended samples without excess
nitrate implies that the conditions in the experiment were not
detrimental to microbial activity, since methanogens are generally extremely sensitive to environmental changes. 47 The
continuous production of CO2 in the long-term phase provides
direct evidence that microbial activities in the experiments
were not inhibited by metabolite accumulation. There are two
possible reasons for the slow CO2 accumulation in the longterm phase of the experiments: (1) changes in microbialcommunities and metabolic reaction pathways due to nitrate
depletion and (2) depletion of labile constituents of thesubstrate.
Impact of microbial gas generation on WIPP performance. In order to mitigate the detrimental effect of microbial
CO2 generation on the WIPP performance, a sufficient amountof MgO (2 × 109 moles) will be added to the repository as a
backfill.24 MgO will react with brine and CO 2 through thefollowing reactions:
MgO + H2O → Mg(OH)2(1)

Mg(OH)2 + CO2 → MgCO3 + H2O.

(2)

Reaction (2) will buffer CO2 fugacity at ∼ 10−7 atm. This low
CO2 fugacity implies that Reaction (2) will practically remove
all CO2 from both gaseous and liquid phases. The mineralformation and brine pH resulting from MgO reactions havebeen calculated with computer code EQ3/6 v. 7.2a. The calculation shows that the addition of sufficient MgO into the repository will buffer pH within 9.2 to 9.9 for WIPP brines.24 Under
these chemical conditions, actinide solubility becomesminimal. Microbial gas generation has a significant impact on
the evolution of disposal room pressure. The pressure buildup
shown in Figure 4 is attributed to both microbial gas generation and anoxic metal corrosion.24

Evaluation of Microbial Activity

Figure 4. Evolution of Disposal Room Pressure in the WIPP
Repository. The pressure buildup in the repository is partly due tomicrobial degradation of organic materials in the wastes.

5. Conclusions
Microorganisms are ubiquitous in subsurface environments
and play a major role in the biogeochemical recycling ofvarious elements. In this paper, we have developed a generalapproach for a systematic evaluation of microbial impact on
the long-term performance of the repository. We have demonstrated that data on microbial population alone are not sufficient for the evaluation of microbial impact on repository
performance and a sensible approach for such evaluation must
be based on the consideration of environmental constraints on
microbial reaction pathways. We have applied our approach toboth the Yucca Mountain (YM) repository and the Waste
Isolation Pilot Plant (WIPP). We have demonstrated that theeffect of microbial activity on the near-field chemistry in the
Yucca Mountain repository is negligible because of limited
nutrient supply and harsh environmental conditions created bywaste emplacement. Whereas for the WIPP, we have shown
that, due to the presence of a large quantity of organic materials and nutrients in the wastes, a significant microbial activity
can potentially be stimulated and its impact on repository
performance can be evaluated with carefully designed incubation experiments coupled with performance assessment calculations.
Acknowledgement. This work was performed at Sandia
National Laboratories, which is a multiprogram laboratory
operated by Sandia Corporation, a Lockheed-Martin Company,
for the DOE under contract DE-AC04-94AL8500. The work
was partly supported by DOE Office of Civilian Radioactive
Waste Management (OCRWM) Yucca Mountain Program.
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