Microb Ecol (1987) 14:141-155
MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1987
Ecological Aspects of Microorganisms Inhabiting Uranium Mill Tailings Christine L. Miller, z Edward R. Landa, 2 and David M. Updegraff3 'US Geological Survey, Water Resources Division, Arvada, Colorado, USA; 2US Geological Survey, Water Resources Division, Reston, Virginia, USA; and 3Colorado School of Mines, Golden, Colorado, USA
Abstract. Numbers and types of microorganisms in uranium mill tailings were determined using culturing techniques. Arthrobacter were found to be the predominant microorganism inhabiting the sandy tailings, whereas Bacillus and fungi predominated in the slime railings. Sulfate-reducing bacteria, capable of leaching radium, were isolated in low numbers from tailings samples but were isolated in significantly high numbers from topsoil in contact with the railings. The results are placed in the context of the magnitude of uranium mill tailings in the United States, the hazards posed by the tailings, and how such hazards could be enhanced or diminished by microbial activities. Patterns in the composition of the microbial population are evaluated with respect to the ecological variables that influence microbial growth.
Introduction Uranium (U) mill tailings are the leached rock residues from the processing of ore for its uranium content. The present quantity of U-mill tailings in the United States is estimated to be about 1.56 x 108 cubic meters (m 3) and this quantity is expected to increase to about 3.5 • 10 s m 3 by the year 2000 [30]. Following extraction of uranium from the ore by either a sulfuric acid leach (the most commonly used method in the US) or alkaline leach 0NIa2CO3) process, the mill-railings slurry is generally discharged into an engineered impoundment. While about 90-95% of the uranium is extracted in the milling process, most uranium daughter products and some nonradioactive metals of potential environmental concern remain with the tailings. This voluminous solid material thus constitutes a low-level radioactive waste and as such has been the focus of research and regulatory attention in recent years. Reclamation of uranium mill tailings has generally involved drainage or evaporation of liquid from the tailings solids, followed by their stabilization with a vegetated earthen cover. In some cases, the fine fraction o f the tailings slurry (slime) has been discharged into a separate impoundment, and because of the higher conductivity, acidity, and metal content associated with fines, revegetation is difficult.
142
C.L. Miller et al.
Although much study has b e e n made of the physical and chemical stability o f U-mill railings, very little work has been done on the presence or potential effect o f microorganisms in the railings. Microbial activity can influence the geochemical cycling of elements out o f the tailings through a variety of processes. Partitioning of metals between mobile and immobile phases can be affected by metal adsorption to microbial cell walls or exopolymers, by microbial redox reactions that either directly or indirectly alter the mobility o f the metal, and by acid or base production resulting from microbial metabolism. Previous work has demonstrated that sulfate-reducing bacteria are capable o f mobilizing radium from radium-barium precipitates [25] and from sulfuric acid-leached U-mill railings [21] resulting from their reduction of SO42- to sulfide, thereby solubilizing the radium thought to exist primarily as Ra(Ba)SO4 precipitates in the mill tailings. Studies of metal binding to cell wall material [1, 2, 38] reveal that complexation can be an effective way to immobilize and concentrate metals (including radionuclides), although, in some cases low molccular weight microbial complexing agents such as organic acids [42] can act to mobilize metals. Production of organic acids during O2-1imited microbial decomposition o f remnant or added organic matter occurs in many environments and the potential exists for sulfuric acid production by sulfur oxidizers in pyritic railings exposed to air. Acid conditions can mobilize many metals, including the radionuclides 23~ and 226Ra. However, the type of acid produced is important, as an investigation of sulfuric acid production by Thiobacillus ferrooxidans in pyritic tailings [31] revealed that the dissolved 226Ra concentration was lowered presumably as a result of the higher sulfate concentration. Mill tailing management practices can strongly influence the character and abundance o f the indigenous microbial flora. The purpose of this study was to characterize generally the microbial populations at three U-mill tailings sites with different types o f cover, and to enumerate specific bacteria that could influence the migration of radium. Measurements o f in situ activity were not carried out since enrichment methods provided more valuable information for an initial study of this kind. Microbial activities at isolated points in time do not necessarily relate to past or future activities, especially in soil systems where fluctuations in moisture content can cause frequent transitions between dorm ant and active states. Enrichment media facilitate the growth o f dor m ant organisms by providing nutrients and water that might be absent in the soil. This allows a more timeaveraged assessment o f the past microbial growth that led to established populations and indicates the potential for future growth under favorable environmental conditions. The genera o f microorganisms isolated in enrichment media point to specific metabolic pathways that would be worthy o f in situ measurement in the future.
Materials and M e t h o d s
Sandy and slime (wet, acid-fines < 325 mesh) tailings samples were obtained from Edgemont, South Dakota, and Rifle and Maybell, Colorado(Fig. 1). The sandy tailings site at Edgemonthas
Microbes in Mill Tailings
143 Edgemont, S Dakota
E
l
1
~
~ Slime railings ponds Old
R16 ~._.
\~=~
.~-Sandytailings pile
Colorado River
Maybell, Colorado
i oear road
Mill effluent~ " Mill to
wes,
e, Colorado
~ " Mi 9 "k.
MI~ /
)I
\ f
~
Sandy and slime tailings basin area
Fig. 1. A schematic map of the sample sites.
been revegetated with topsoil and wheatgrass and fertilized with a m m o n i u m nitrate and, in some areas, heavy applications of manure. At the time of sampling, there was no irrigation. The slime ponds at Edgemont were uncovered and free of vegetation. The Rifle site was revegetated with topsoil and wheatgrass, and this cover was maintained with a daily schedule of irrigation with sprinklers. The Maybell site was revegetated with topsoil and wheatgrass, but received no irrigation. All topsoil samples (i.e., topsoil overlying the tailings or A-horizon topsoil from soils adjacent to the railings) were collected at a depth of 3-51/2 inches, and all tailings and B-horizon soils were collected at a depth of 15-20 inches. Sample numbers 1 through 11 were collected at Edgemont (coded E in Tables 1-4), numbers 12 through 17 were collected at Rifle (R), and numbers 18 through 23 were collected at Maybell (M). The uraniferous peat sample was obtained from the vicinity of the U-mining operations at the Flodelle Creek bog in Washington State (for a description of this site see ref. [29]).
144
C.L. Miller et al.
All of the sandy tailings and slime tailings of this study were enriched in sulfate as a result of the sulfuric acid milling process. The pH of the railings and topsoil samples was measured by allowing a 1 g (wet wt.) sample/1 ml deionized H20 mixture to equilibrate for 1/2hour after mixing, then mixing again, and then reading with the pH electrode. Total recoverable metals of interest in the solids were released by H F digestion and analyzed by inductively coupled argon plasma-atomic emission spectrometry [7]. Hg was determined by cold-vapor atomic absorption after hot digestion with KMnO4 and K2S2Os in acid solution [35]. Organic carbon was determined by difference between total carbon (dry combustion) and inorganic carbon (acid digestion and titrimetry [10]). Sterile technique was observed throughout sample collection and sample processing. All incubations were carried out in the dark at a room temperature maintained from between 24-30"C. To achieve homogenization, 1 g (wet weight) of the sample was ground with a pestle in a mortar containing 9 ml 0.8% NaC1 solution [32] for 1.5 min and then vortex-mixed for 10 see in a culture tube. Serial 1:10 dilutions were then made from this well-mixed solution into tubes containing 0.8% NaCI solution, and used for inoculations into selected media. For liquid media methods, M P N (most probable number) enumerations were carried out. Colony counts were made on a solid agar medium.
Medium for Enumerating Sulfate-Reducing Bacteria
(adapted
from
Postgate [32])
Compound FeCI2.4H20 MgCIz-6H20 Na lactate Na ascorbate Yeast extract NH4CI KI-I2PO4 NaSO4 CaCI2.6H20
g/liter 0.36 1.83 3.5 0.5 1.0 1.0 1.0 1.0 1.0
adjusted to pH 7.6 before autoclaving. The medium was dispensed to within 1 ml of the top of 13 x 100 m m culture tubes, autoclaved with the caps on tight, inoculated with serial dilutions of the sample, and incubated for 90 days. Growth was considered positive if 100% of the sediment turned black.
Medium for Enumerating Sulfur-Oxidizing Bacteria, S-Medium $6 medium
of Hutchinson
e t al. [ 1 5 ] )
Compound KHzPO4 MgC12 96H20 (NH4)2SO4 CaC12' 2H20 FeC13" 6H20 MnC13' 4H20 Yeast extract adjusted to pH 7.0 before autoclaving.
gliter 0.5 0.17 0.1 0.04 0.02 0.02 0.01
(modified
Microbes in Mill Tailings
145
Six milliliters of this medium were autoclaved in 16 • 125 m m culture tubes. Powdered sulfur, autoclaved for 1 hour on 2 consecutive days, was then added to each tube in approximately 0.01 g aliquots, using the volume of sulfur held in a sterilized pipette tip as a measure of weight. One milliliter of each sample dilution was added to S-medium tubes and incubated for 95 days. Growth was considered positive if the pH of the medium fell below 4.0. This level was chosen as that which would indicate autotrophic, energy-generating oxidation o f sulfur rather than incidental sulfur oxidation accompanying heterotrophic processes. An additional consideration in the selection of this pH was the fact that a sterile control (sulfur medium without sample), incubated for the same period of time as the samples (95 days), fell to a pH of 6.5, indicating that some slight inorganic oxidation of the sulfur had occurred. The initial pH of the medium inoculated with the samples (in some cases somewhat acid) was tested and ranged from a low of 5.1 to a high of 6.4. Thus, a final growth pH of less than 4.0 appeared to be a level that would be generated only by substantial biological oxidation of sulfur.
Method for Enumerating and Identifying Aerobic Heterotrophs Nutrient agar was used for these purposes. The inoculation procedure involved pipetting 0.5 ml of the sample suspension onto a 100 • 15 m m nutrient agar plate, swirling to distribute the liquid evenly, and allowing the liquid to dry on upright plates for at least 24 hours before inverting. Distinct and well-distributed colonies result from this procedure. Bacterial genera were identified based on colony morphology, bacterial morphology, gram reaction [6], and in some cases bacterial physiology. Arthrobacter were distinguished by their grampositive reaction and pleomorphic morphology. Biochemical characterization of the Arthrobacter was also carried out, as described by D. M. Updegraff, C. L. Miller, and E. R. Landa (1986) Characterization o f Arthrobacter isolated from uranium mill tailings, U.S. Geological Survey Open File Report 86-527.
Method for Determining Specific Metal Resistance (modified from the techniques of J. Watterson (1981) Reaction of 12 strains of soil baceria to 15 metals, U.S. Geological Survey Open File Report #81-1082:12 and Novick and Roth [28]) Pour plates of bacterial isolates were prepared in 150 • 15 m m Petri dishes using a salt-supplementcd nutricnt agar o f the following composition: Compound
g/liter
KzHPO 4 MgCI2-6H20 CaC12" 2H20 NH4C1 NaSO4 Nutrient agar
0.10 0.55 0.25 0.40 0.05 20.0
adjusted to pH 7.0. The presence of the cationic species in the medium should tend to minimize complexation of the metals by organics. Immediately upon agar solidification, discs impregnated with metal salts (Certified Atomic Absorption Standard Reference Solutions, Fisher Scientific Company) were placed on the surface of the agar in a predetermined pattern. Diffusion of a metal salt from a disc depends on its solubility in the agar medium. Bioavailability further depends on the metal speciation, which is not known for such a complex organic matrix; however, this is not a reason for concern since the test is used to compare the resistance of several bacterial isolates to a given metal, and not to compare the resistance o f one bacterial isolate to different metals. The degree of inhibition of a bacterium by a given metal is proportional to the width of the clear zone (free of growth) which develops around a disc. Since these zones are symmetrical for the most part, diffusion rates are fairly uniform.
146
C.L. Miller et al.
The following metals were tested at the specified amount o f metal per disc (present in the disc as a soluble salt): Metal Amount (gg) per disc
Hg
Ag
Ni
Mn Mo Co
V
Sn
Pb
5
100
I0
25
20
25
100 20
40
25
Cu
Cd
As
Ba
Cr
7
15
25
20
The concentration used for each metal was determined to be a level which, for numerous other bacterial isolates, has caused measurable inhibition of growth using these methods O. Watterson (1981) Reaction of 12 strains of soil bacteria to 15 metals, U.S. Geological Survey Report #811082:12).
Method for Determining the Capability o f B a c i l l u s and A r t h r o b a c t e r spp. to Leach 226Ra The inoculation of a mixed culture of anaerobic bacteria (isolated from uranium mill tailings) in medium containing sterile uranium mill railings to test for radium solubilization resulted in the overwhelming dominance of the culture by an unidentified species of Bacillus. The medium was not filled to the top of the culture flask, leaving a small air space to promote mixed culture growth (facultative anaerobes followed by strict anaerobes, theoretically), and then tightly sealed. The composition of the medium was as follows (modified from medium for enumerating sulfatereducing bacteria to encourage mixed-culture growth): Compound FeCI2'4H20 MgC12.6HzO Na lactate Dextrose Na ascorbate Yeast extract KC1 NH4CI KI-12PO4
g/liter 0.2 1.83 3.5 0.7 0.5 1.0 0.28 1.0 0.02
adjusted to pH 7.0 after autoclaving. Phosphate was added in such trace amounts (0.02 g KH2PO4)to minimize phosphate precipitates that might interfere with leaching. Calcium and sulfate were supplied in adequate amounts by the railings (150 mg/liter and 380 rag/liter, respectively, in a 1 hour slurry of a 1:10 dilution o f tailings in deionized water). Thirty g of sandy Edgemont tailings were added to 50 ml o f distilled water, autoclaved on 2 consecutive days at 120 psig for 1 hour and then added to 500 ml of this medium. After inoculation with the mixed culture o f anaerobic bacteria, the solution was incubated for 11 days. A sterile control (railings plus medium) was included in this study. An Arthrobacter isolate from the Maybell railings was inoculated into a medium of the following composition that also contained 30 g o f sterile tailings (sterilized as above; modified from medium for enumerating sulfate-reducing bacteria to encourage Arthrobacter growth): Compound FeC12.4H20 MgClz.6H20 KH2PO 4 NH4C1 Yeast extract Dextrose
g/liter 0.2 1.83 0.05 1.0 1.0 2.0
Microbes in Mill Tailings
147
Table 1. Organic carbon content and sulfate-reducing bacteria~
Sample group High organic carbon, high sulfate-reducing bacteria Low organic carbon, low sulfate-reducing bacteria
Sample site
Sample type
R12 El E3 M21 R13 M22
Topsoil Topsoil Topsoil Topsoil Topsoil Topsoil
pH 7.6 6.6 6.6 8.2 7.5 7.7
Number of Organic sulfate% carbon reducing Moisture content (%) bacteriaJg 6.2 1.8 4.6 4.0 8.0 4.6
0.91 0.29 0.21 0.03
MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1987
Ecological Aspects of Microorganisms Inhabiting Uranium Mill Tailings Christine L. Miller, z Edward R. Landa, 2 and David M. Updegraff3 'US Geological Survey, Water Resources Division, Arvada, Colorado, USA; 2US Geological Survey, Water Resources Division, Reston, Virginia, USA; and 3Colorado School of Mines, Golden, Colorado, USA
Abstract. Numbers and types of microorganisms in uranium mill tailings were determined using culturing techniques. Arthrobacter were found to be the predominant microorganism inhabiting the sandy tailings, whereas Bacillus and fungi predominated in the slime railings. Sulfate-reducing bacteria, capable of leaching radium, were isolated in low numbers from tailings samples but were isolated in significantly high numbers from topsoil in contact with the railings. The results are placed in the context of the magnitude of uranium mill tailings in the United States, the hazards posed by the tailings, and how such hazards could be enhanced or diminished by microbial activities. Patterns in the composition of the microbial population are evaluated with respect to the ecological variables that influence microbial growth.
Introduction Uranium (U) mill tailings are the leached rock residues from the processing of ore for its uranium content. The present quantity of U-mill tailings in the United States is estimated to be about 1.56 x 108 cubic meters (m 3) and this quantity is expected to increase to about 3.5 • 10 s m 3 by the year 2000 [30]. Following extraction of uranium from the ore by either a sulfuric acid leach (the most commonly used method in the US) or alkaline leach 0NIa2CO3) process, the mill-railings slurry is generally discharged into an engineered impoundment. While about 90-95% of the uranium is extracted in the milling process, most uranium daughter products and some nonradioactive metals of potential environmental concern remain with the tailings. This voluminous solid material thus constitutes a low-level radioactive waste and as such has been the focus of research and regulatory attention in recent years. Reclamation of uranium mill tailings has generally involved drainage or evaporation of liquid from the tailings solids, followed by their stabilization with a vegetated earthen cover. In some cases, the fine fraction o f the tailings slurry (slime) has been discharged into a separate impoundment, and because of the higher conductivity, acidity, and metal content associated with fines, revegetation is difficult.
142
C.L. Miller et al.
Although much study has b e e n made of the physical and chemical stability o f U-mill railings, very little work has been done on the presence or potential effect o f microorganisms in the railings. Microbial activity can influence the geochemical cycling of elements out o f the tailings through a variety of processes. Partitioning of metals between mobile and immobile phases can be affected by metal adsorption to microbial cell walls or exopolymers, by microbial redox reactions that either directly or indirectly alter the mobility o f the metal, and by acid or base production resulting from microbial metabolism. Previous work has demonstrated that sulfate-reducing bacteria are capable o f mobilizing radium from radium-barium precipitates [25] and from sulfuric acid-leached U-mill railings [21] resulting from their reduction of SO42- to sulfide, thereby solubilizing the radium thought to exist primarily as Ra(Ba)SO4 precipitates in the mill tailings. Studies of metal binding to cell wall material [1, 2, 38] reveal that complexation can be an effective way to immobilize and concentrate metals (including radionuclides), although, in some cases low molccular weight microbial complexing agents such as organic acids [42] can act to mobilize metals. Production of organic acids during O2-1imited microbial decomposition o f remnant or added organic matter occurs in many environments and the potential exists for sulfuric acid production by sulfur oxidizers in pyritic railings exposed to air. Acid conditions can mobilize many metals, including the radionuclides 23~ and 226Ra. However, the type of acid produced is important, as an investigation of sulfuric acid production by Thiobacillus ferrooxidans in pyritic tailings [31] revealed that the dissolved 226Ra concentration was lowered presumably as a result of the higher sulfate concentration. Mill tailing management practices can strongly influence the character and abundance o f the indigenous microbial flora. The purpose of this study was to characterize generally the microbial populations at three U-mill tailings sites with different types o f cover, and to enumerate specific bacteria that could influence the migration of radium. Measurements o f in situ activity were not carried out since enrichment methods provided more valuable information for an initial study of this kind. Microbial activities at isolated points in time do not necessarily relate to past or future activities, especially in soil systems where fluctuations in moisture content can cause frequent transitions between dorm ant and active states. Enrichment media facilitate the growth o f dor m ant organisms by providing nutrients and water that might be absent in the soil. This allows a more timeaveraged assessment o f the past microbial growth that led to established populations and indicates the potential for future growth under favorable environmental conditions. The genera o f microorganisms isolated in enrichment media point to specific metabolic pathways that would be worthy o f in situ measurement in the future.
Materials and M e t h o d s
Sandy and slime (wet, acid-fines < 325 mesh) tailings samples were obtained from Edgemont, South Dakota, and Rifle and Maybell, Colorado(Fig. 1). The sandy tailings site at Edgemonthas
Microbes in Mill Tailings
143 Edgemont, S Dakota
E
l
1
~
~ Slime railings ponds Old
R16 ~._.
\~=~
.~-Sandytailings pile
Colorado River
Maybell, Colorado
i oear road
Mill effluent~ " Mill to
wes,
e, Colorado
~ " Mi 9 "k.
MI~ /
)I
\ f
~
Sandy and slime tailings basin area
Fig. 1. A schematic map of the sample sites.
been revegetated with topsoil and wheatgrass and fertilized with a m m o n i u m nitrate and, in some areas, heavy applications of manure. At the time of sampling, there was no irrigation. The slime ponds at Edgemont were uncovered and free of vegetation. The Rifle site was revegetated with topsoil and wheatgrass, and this cover was maintained with a daily schedule of irrigation with sprinklers. The Maybell site was revegetated with topsoil and wheatgrass, but received no irrigation. All topsoil samples (i.e., topsoil overlying the tailings or A-horizon topsoil from soils adjacent to the railings) were collected at a depth of 3-51/2 inches, and all tailings and B-horizon soils were collected at a depth of 15-20 inches. Sample numbers 1 through 11 were collected at Edgemont (coded E in Tables 1-4), numbers 12 through 17 were collected at Rifle (R), and numbers 18 through 23 were collected at Maybell (M). The uraniferous peat sample was obtained from the vicinity of the U-mining operations at the Flodelle Creek bog in Washington State (for a description of this site see ref. [29]).
144
C.L. Miller et al.
All of the sandy tailings and slime tailings of this study were enriched in sulfate as a result of the sulfuric acid milling process. The pH of the railings and topsoil samples was measured by allowing a 1 g (wet wt.) sample/1 ml deionized H20 mixture to equilibrate for 1/2hour after mixing, then mixing again, and then reading with the pH electrode. Total recoverable metals of interest in the solids were released by H F digestion and analyzed by inductively coupled argon plasma-atomic emission spectrometry [7]. Hg was determined by cold-vapor atomic absorption after hot digestion with KMnO4 and K2S2Os in acid solution [35]. Organic carbon was determined by difference between total carbon (dry combustion) and inorganic carbon (acid digestion and titrimetry [10]). Sterile technique was observed throughout sample collection and sample processing. All incubations were carried out in the dark at a room temperature maintained from between 24-30"C. To achieve homogenization, 1 g (wet weight) of the sample was ground with a pestle in a mortar containing 9 ml 0.8% NaC1 solution [32] for 1.5 min and then vortex-mixed for 10 see in a culture tube. Serial 1:10 dilutions were then made from this well-mixed solution into tubes containing 0.8% NaCI solution, and used for inoculations into selected media. For liquid media methods, M P N (most probable number) enumerations were carried out. Colony counts were made on a solid agar medium.
Medium for Enumerating Sulfate-Reducing Bacteria
(adapted
from
Postgate [32])
Compound FeCI2.4H20 MgCIz-6H20 Na lactate Na ascorbate Yeast extract NH4CI KI-I2PO4 NaSO4 CaCI2.6H20
g/liter 0.36 1.83 3.5 0.5 1.0 1.0 1.0 1.0 1.0
adjusted to pH 7.6 before autoclaving. The medium was dispensed to within 1 ml of the top of 13 x 100 m m culture tubes, autoclaved with the caps on tight, inoculated with serial dilutions of the sample, and incubated for 90 days. Growth was considered positive if 100% of the sediment turned black.
Medium for Enumerating Sulfur-Oxidizing Bacteria, S-Medium $6 medium
of Hutchinson
e t al. [ 1 5 ] )
Compound KHzPO4 MgC12 96H20 (NH4)2SO4 CaC12' 2H20 FeC13" 6H20 MnC13' 4H20 Yeast extract adjusted to pH 7.0 before autoclaving.
gliter 0.5 0.17 0.1 0.04 0.02 0.02 0.01
(modified
Microbes in Mill Tailings
145
Six milliliters of this medium were autoclaved in 16 • 125 m m culture tubes. Powdered sulfur, autoclaved for 1 hour on 2 consecutive days, was then added to each tube in approximately 0.01 g aliquots, using the volume of sulfur held in a sterilized pipette tip as a measure of weight. One milliliter of each sample dilution was added to S-medium tubes and incubated for 95 days. Growth was considered positive if the pH of the medium fell below 4.0. This level was chosen as that which would indicate autotrophic, energy-generating oxidation o f sulfur rather than incidental sulfur oxidation accompanying heterotrophic processes. An additional consideration in the selection of this pH was the fact that a sterile control (sulfur medium without sample), incubated for the same period of time as the samples (95 days), fell to a pH of 6.5, indicating that some slight inorganic oxidation of the sulfur had occurred. The initial pH of the medium inoculated with the samples (in some cases somewhat acid) was tested and ranged from a low of 5.1 to a high of 6.4. Thus, a final growth pH of less than 4.0 appeared to be a level that would be generated only by substantial biological oxidation of sulfur.
Method for Enumerating and Identifying Aerobic Heterotrophs Nutrient agar was used for these purposes. The inoculation procedure involved pipetting 0.5 ml of the sample suspension onto a 100 • 15 m m nutrient agar plate, swirling to distribute the liquid evenly, and allowing the liquid to dry on upright plates for at least 24 hours before inverting. Distinct and well-distributed colonies result from this procedure. Bacterial genera were identified based on colony morphology, bacterial morphology, gram reaction [6], and in some cases bacterial physiology. Arthrobacter were distinguished by their grampositive reaction and pleomorphic morphology. Biochemical characterization of the Arthrobacter was also carried out, as described by D. M. Updegraff, C. L. Miller, and E. R. Landa (1986) Characterization o f Arthrobacter isolated from uranium mill tailings, U.S. Geological Survey Open File Report 86-527.
Method for Determining Specific Metal Resistance (modified from the techniques of J. Watterson (1981) Reaction of 12 strains of soil baceria to 15 metals, U.S. Geological Survey Open File Report #81-1082:12 and Novick and Roth [28]) Pour plates of bacterial isolates were prepared in 150 • 15 m m Petri dishes using a salt-supplementcd nutricnt agar o f the following composition: Compound
g/liter
KzHPO 4 MgCI2-6H20 CaC12" 2H20 NH4C1 NaSO4 Nutrient agar
0.10 0.55 0.25 0.40 0.05 20.0
adjusted to pH 7.0. The presence of the cationic species in the medium should tend to minimize complexation of the metals by organics. Immediately upon agar solidification, discs impregnated with metal salts (Certified Atomic Absorption Standard Reference Solutions, Fisher Scientific Company) were placed on the surface of the agar in a predetermined pattern. Diffusion of a metal salt from a disc depends on its solubility in the agar medium. Bioavailability further depends on the metal speciation, which is not known for such a complex organic matrix; however, this is not a reason for concern since the test is used to compare the resistance of several bacterial isolates to a given metal, and not to compare the resistance o f one bacterial isolate to different metals. The degree of inhibition of a bacterium by a given metal is proportional to the width of the clear zone (free of growth) which develops around a disc. Since these zones are symmetrical for the most part, diffusion rates are fairly uniform.
146
C.L. Miller et al.
The following metals were tested at the specified amount o f metal per disc (present in the disc as a soluble salt): Metal Amount (gg) per disc
Hg
Ag
Ni
Mn Mo Co
V
Sn
Pb
5
100
I0
25
20
25
100 20
40
25
Cu
Cd
As
Ba
Cr
7
15
25
20
The concentration used for each metal was determined to be a level which, for numerous other bacterial isolates, has caused measurable inhibition of growth using these methods O. Watterson (1981) Reaction of 12 strains of soil bacteria to 15 metals, U.S. Geological Survey Report #811082:12).
Method for Determining the Capability o f B a c i l l u s and A r t h r o b a c t e r spp. to Leach 226Ra The inoculation of a mixed culture of anaerobic bacteria (isolated from uranium mill tailings) in medium containing sterile uranium mill railings to test for radium solubilization resulted in the overwhelming dominance of the culture by an unidentified species of Bacillus. The medium was not filled to the top of the culture flask, leaving a small air space to promote mixed culture growth (facultative anaerobes followed by strict anaerobes, theoretically), and then tightly sealed. The composition of the medium was as follows (modified from medium for enumerating sulfatereducing bacteria to encourage mixed-culture growth): Compound FeCI2'4H20 MgC12.6HzO Na lactate Dextrose Na ascorbate Yeast extract KC1 NH4CI KI-12PO4
g/liter 0.2 1.83 3.5 0.7 0.5 1.0 0.28 1.0 0.02
adjusted to pH 7.0 after autoclaving. Phosphate was added in such trace amounts (0.02 g KH2PO4)to minimize phosphate precipitates that might interfere with leaching. Calcium and sulfate were supplied in adequate amounts by the railings (150 mg/liter and 380 rag/liter, respectively, in a 1 hour slurry of a 1:10 dilution o f tailings in deionized water). Thirty g of sandy Edgemont tailings were added to 50 ml o f distilled water, autoclaved on 2 consecutive days at 120 psig for 1 hour and then added to 500 ml of this medium. After inoculation with the mixed culture o f anaerobic bacteria, the solution was incubated for 11 days. A sterile control (railings plus medium) was included in this study. An Arthrobacter isolate from the Maybell railings was inoculated into a medium of the following composition that also contained 30 g o f sterile tailings (sterilized as above; modified from medium for enumerating sulfate-reducing bacteria to encourage Arthrobacter growth): Compound FeC12.4H20 MgClz.6H20 KH2PO 4 NH4C1 Yeast extract Dextrose
g/liter 0.2 1.83 0.05 1.0 1.0 2.0
Microbes in Mill Tailings
147
Table 1. Organic carbon content and sulfate-reducing bacteria~
Sample group High organic carbon, high sulfate-reducing bacteria Low organic carbon, low sulfate-reducing bacteria
Sample site
Sample type
R12 El E3 M21 R13 M22
Topsoil Topsoil Topsoil Topsoil Topsoil Topsoil
pH 7.6 6.6 6.6 8.2 7.5 7.7
Number of Organic sulfate% carbon reducing Moisture content (%) bacteriaJg 6.2 1.8 4.6 4.0 8.0 4.6
0.91 0.29 0.21 0.03
