Environ. Sci. Technol. 1986, 20,243-248
Microbiological Treatment of Uranium Mine Waters Jim W. Kauffman," Willlam C. Laughlln, and Roger A. Baldwln Kerr-McGee Corporation, P.O. Box 25861, Oklahoma Clty, Oklahoma 73125
Percolation of uranium mine discharge water through Ambrosia Lake, NM, soil is shown to be an effective method for lowering selenium, uranium, molybdenum, and sulfate concentrations in the mine water. Selenium concentrations were lowered from -1.6 to C0.05 mg/L by reduction of soluble selenate and selenite to insoluble selenium metal. This reaction is most likely performed by bacteria belonging to the genus Clostridium. In addition, sulfate-reducing bacteria in the soil, such as Desulfouibrio bacteria, metabolize sulfate to hydrogen sulfide, which reacts with uranyl and molybdate ions to form insoluble uranium and molybdenum species. The concentrations of sulfate, uranium, and molybdenum were reduced to less than 600,0.1, and 0.05 mg/L, respectively. A qualitative understanding of the effects of mine water temperature, flow rate, and nutrients on metals removal is provided. The process was successfully field tested for 7 months in a soil column 1.5 m deep.
Introduction Some uranium mines in the seleniferous Ambrosia Lake region of New Mexico interface with water-bearing rock and must be continuously drained in order to remain operational. The mine water can contain concentrations of selenium, uranium, molybdenum, and sulfate that exceed the environmental limits of 0.05,5.0, 1.0, and 600 mg/L, respectively. Ion-exchange treatment of the mine water is used to recover most of the uranium and lowers the uranium content to about 1 mg/L. However, this treatment does not significantly reduce the selenium, molybdenum, and sulfate concentrations. This paper presents our findings on a technique that utilizes soil bacteria, e.g., Clostridium, to effectively remove selenium from discharge mine water to below regulatory limits. In addition to selenium removal, uranium, molybdenum, and sulfate concentrations were also substantially reduced by this process. This work focused primarily on selenium, existing as 80-95% selenate and the rest selenite (1-3), because it was not amenable to conventional water treatment removal techniques. Alum and iron coagulation, lime softening, and other similar treatments were found to be ineffective in lowering the mine water selenium concentration. Sorg (4-6) encountered similar problems when he tried to remove selenium from river waters. Experimental Section Soil columns, 2.5 cm in diameter, were used for the laboratory selenium removal tests. The glass tubes were loaded with 100 or 200 g of either 100% soil or a 5050 wt % soil/sand mixture to a depth of 15 or 30 cm, respectively. The soil density was approximately 1450 kg/m3. Mine water from the Ambrosia Lake mining district was pumped either upflow or downflow through the columns at 70-900 L/m2/day using a peristaltic pump. Six soil samples were obtained by grabbing -0.5-kg samples from the top 30 cm of soil at random locations in the Ambrosia Lake, NM, mining district. The soils from the bottom of a mine water pond and an alluvial surface soil were found to be the most and least active based on preliminary soil column tests. That is to say, the soils 0013-936X/86/0920-0243$01.50/0
removed selenium to less than 0.050 mg/L within 8 and 26 days, respectively. The pond soil total organic carbon (TOC) content was 1.1% and had a strong "grassy" odor suggesting a relatively large concentration of microflora, plants, etc. In contrast, the surface soil was light brown, contained only 0.4% TOC, and had no significant odor. These two soils were chosen for additional laboratory testing and the field test because of their contrasting behaviors, Soils were used without modification except in the test with sterile soil. This soil was steam sterilized and certified free of all detectable microorganisms by Langston Laboratories, Inc., Leawood, KS. The mine water used in this experiment was also sterilized. The sterile soil was inoculated with 10 mL of liquid withdrawn from the middle of the fertile soil column using a sterile syringe. The procedure for selenium speciation and sample collection was previously reported (3). Radiometric analysis employing Na275Se04was used to measure the water selenium concentrations for the day-to-day analyses of soil column effluents. The radiometric values were periodically checked against atomic absorption spectrometry (AAS) analyses. The correlation coefficient between the analytical procedures was 0.96. Water samples analyzed for U, Mo, Sod2-, and HC03- were filtered through a 0.45-pm filter and stored in linear polyethylene bottles. The field test soil columns measured 0.91 m in diameter and were initially filled to a depth of 0.9 m with gravel. A 1:l mixture of soil and sand was next loaded into the column giving a soil bed depth of 1.5 m with a soil density of approximately 1500 kg/m3. A 1 cm diameter pipe, located 0.3 m above the soil, was used for an overflow and sampling the influent. A 1 cm diameter pipe located at the bottom of the column and 90" apart from the top pipe was used for the column water discharge and effluent sampling. The percolation rate through the soil was controlled with a peristaltic pump attached to this port. Culturing and identification of bacteria were performed by Langston Laboratories, Inc., and E. A. Grula, Department of Microbiology,Oklahoma State University. Aerobic microorganisms were cultured on Standard Methods agar (tryptone-glucose yeast agar), with and without selenium, and incubated at 37 "C for 48 h. Both 1.0- and 0.1-mL aliquots of a 1:lOO dilution of each soil sample were plated in duplicate using the pour plate method. Isolated colonies were transferred to Standard Plate Count agar without selenium for identification. Detection of anaerobic microorganisms involved diluting soil samples in pH 7 phosphate buffer to give a final concentration of 0.05 g/ mL. Aliquots of 1.0 and 0.1 mL were taken from the phosphate buffer dilution and plated on anaerobic agar (pH 7.2))with and without selenium, by pour plate techniques. The plates were incubated under COz at 37 "C for 48 h. The procedure for isolating sulfate-reducing microorganisms was similar to that for anaerobic microorganisms, except Postgates Medium B containing FeS04-7H20 was used.
Results and Discussion Laboratory Soil Column Tests. Analysis of a soil sample from a pond, used to hold uranium mine water before being discharged, showed an unusually high amount
0 1986 American Chemical Society
Environ. Sci. Technol., Vol. 20, No. 3, 1986
243
Table I. Comparison of Microorganism Characteristics characteristic
Cornyeform group
soil isolate
Gram reaction
Gram-positive, Gram-positive, older cultures older cultures gram negative gram negative morphology irregular rods, irregular rods pleomorphic, occurring singly may show or in short coccoid shapes in chains, older cultures pleomorphic form with older cultures showing high numbers of ovoid cells colonies on agar little or no cream to medium pigmentation translucent colonies with thick pellicle motility variable motile selenium/sulfur species isolated selenite reduced to metabolism capable of selenium metal selenium reduction culturing aerobic aerobic environment spore former non-spore forming non-spore forming
Clostridium group
Environ. Sci. Technol., Vol. 20,
No. 3, 1986
soil isolate
Gram-positive
Gram-positive
Gram-negative Gram-negative
single rods
single rods
curved rods
curved rods
little or no pigmentation
nonpigmented mucoid colonies
motile species isolated capable of selenium reduction anaerobic
motile selenite and selenate reduced to selenium metal anaerobic
sulfate reduced to sulfide
iron sulfate reduced to sulfide
anaerobic
anaerobic
spore forming
spore forming
non-spore formine
non-spore formine
of selenium. The selenium concentration in this sample was -100 ppm compared to core samples from just outside the pond that assayed 400 L/m2 per day, which caused the sharp increase in effluent selenium concentration on this day. Although a comparable flow rate of 80-140 L/m2 per day was used
1.15 1.13 0.64 0.032 0.12 0.43 0.14 0.73 0.19 0.016 0.011 0.018 0.024 0.031 0.026 0.020 0.022 0.012 0.001 0.015 0.003 0.001 0.001 0.002 0.001 0.001 0.016 0.016 0.001 0.001
-
Stopped after 137 days of operation.
when these materials were used compared to the control column, which had no additive. Apparently even complex compounds such as lignosulfonate and cellulose can act as a source of carbon, in addition to sugars and starches. These complex compounds are typically degraded slowly in comparison to sugar and starches. However, this is not apparent from the soil column tests, which show the selenium concentration being lowered over similar time intervals. As a check on the soil activity, the sludge was not added to that soil column until the 26th day to allow sufficient time for the column to activate. The sludge was put into a column upstream of the soil column with the
Table 111. Utilization of Different Nutrient Sources for Selenium Removal effluent selenium concn, mg/L days operating
control (no additives)
0 4
1.30 1.30
11
1.15
17 18 21 24 25 31 39
sucrose (0.7 g/L)
1.30
1.60
1.60
1.37
1.60
1.60
1.25 1.32
1.58 1.60 b
1.68
0.90
0.053
0.047
1.00
0.024
1.30e 0.051 0.021
0.010 0.007 d
1.60 0.093
0.045 0.86 0.001 0.46 0.31 0.23 d
Stopped after 39 days of operation. 246
sludge" (20 g)
sodium lignosulfonate (0.7 g/L)
0.074 1.20 1.20
13 14
1.30 0.088
6
7 9 10
cellulose (20 g)
potato4 starch (0.7 g/L)
0.002 d
* Sucrose added.
Environ. Sci. Technoi., Vol. 20, No. 3, 1986
Starch added.
Stopped.
e Sludge
C
0.061 d
0.025
added after day 25 of operation.
Table IV. Ambrosia Lake Field Test Data days operated when sampled 10 26 40 76 97 132 160 172* 188
13 49
porta
Se
U
1 2 1 2 1 2 1 2 1 2 1 2 1 2
1.70 1.40 1.40 0.78 1.20 0.034 1.20 0.014 1.10 0.011 1.30 0.28 0.005
0.90 5.10
1 2 1 2 1
71b 105 133 161 (I
so:-
HCOC
Algae Pond Soil Column 0.800 1110 0.900 900
effluent water temp, OC
980
20.0
980
20.0
100
21.1
160
20.6
100
18.9
>400
17.8
180 200
1.60
0.600 1.300 0.700 1.100 0.580 1.200 0.450 0.730 0.760
780 1010 870 940 990 loo0 800 820 740
190 220 120 200 190 190 170 190 160 200
1.30 0.016
0.40 0.50
0.710 0.043
830 630
190 630
175
14.4
0.49 0.75 0.61 0.56 0.85 0.76
Surface Soil Column 0.600 780 0.600 970 0.680 850 960 0.780 0.620 960 0.830 930
190 210 160 200 780 200
110
21.0
140
20.6
1 2
1.20 1.00 1.10 0.38 0.96 0.037
0
18.9
1 2
1.30 0.018
0.94 1.00
170 600
110
17.4
-
-
0.015 1.30 0.005
0.05 0.33 0.07
2 70
Mo
flow rate, L/m2/day
1 2 1 2
0.49 4.00 0.50 3.50 0.90 2.70 0.94 2.20
-
0.450 0.270 0.046 0.680 0.0031
800
770
-
-
520 950 520
820 210 1340
17.8
17.4 80 110
14.4
Port 1, influent concentrations (mg/L); port 2, effluent concentrations (mg/L) *Sucrose first added.
in the surface soil column, the effluent selenium concentration never fell below 0.38 mg/L through the 49th day. This behavior was expected for a soil with a relatively Iow nutrient content. The anomalous 0.037 mg/L reading on the 70th day resulted from a period of zero flow rate and indicated selenium was being removed, but not at a high enough rate to be effective at 8Ck140 L/m2 per day. In order to improve the rate of selenium removal, sucrose was added to the feed water beginning on the 172nd and 71st days for the pond and surface soil column tests, respectively. Sucrose served as a convenient nutrient since it could be added to the water in specific amounts. It was initiaIly added in batch quantities; however, the concentration was continuously diluted due to a constant mixing of fresh mine water with the head water. This procedure was changed on the 179th and 96th days for the pond and surface soil columns, respectively. During the rest of the test, fresh mine water was added to each column in batch quantities to maintain a constant sucrose concentration. The nutrient additions were extremely beneficial in decreasing the effluent selenium concentration down to less than 0.050 mg/L for both columns, Table IV. After addition of sucrose, hydrogen sulfide was detected in the surface soil column effluent on the 105th day at a level of 37 ppm sulfide. The occurrence of sulfide in the effluent indicated the mine water sulfate was being reduced. This reduction was verified by a decreasing sulfate concentration from about 800 ppm to less than 600 ppm by the 133rd day. Previous to sucrose addition, the effluent sulfate concentration was about the same or slightly higher than the influent concentration, indicating some soil leaching in the latter case. Sulfate-reducing bacteria
of the genus Desulfovibrio have been identified in these soils, Table I, and possibly with other sulfate-reducing microorganisms such as Desulfotomaculum (II), would account for the production of hydrogen sulfide. Addition of sucrose also caused a sharp rise in bicarbonate concentration from 200 to 1340 ppm, which results from metabolism of the sucrose to carbon dioxide. The pond soil initially contained 183 ppm uranium compared to 5.6 ppm in the surface soil. A significant amount of the uranium in the former soil was apparently water soluble since the effluent uranium concentration was higher than the influent concentration,Table IV. Leaching of uranium continued through the 160th day, but gradually decreased. There was no significant difference between influent and effluent uranium concentration in the surface soil column until about 2 months after sucrose was initially added. At this time, the effluent uranium concentration was reduced to 0.05 mg/L and remained near this level through the end of the field test. Compared to the surface soil test there was not sufficient time between sucrose addition and the end of the pond soil test to have observed a lowering of uranium in its effluent. Since the decrease in uranium concentration occurred only after hydrogen sulfide was generated, it is believed the water-soluble uranium was precipitated when it reacted with the hydrogen sulfide undergoing a reduction to insoluble uranium dioxide, U02, (11-13). The molybdenum effluent concentration in the surface soil remained essentially unchanged from the influent concentration except for some slight leaching through the 105th day. After addition of sucrose, there was a sharp reduction of the effluent mine water molybdenum, from Environ. Sci. Technol., Vol. 20,
No. 3, 1986 247
as much as 0.7 mg/L to less than 0.050 mg/L. The molybdenum removal mechanism is believed to involve reaction of the molybdate anion, MOO:-, which exists in aerated water at pH 8, with hydrogen sulfide to form the water-insoluble molybdenum disulfide (11). The effluent molybdenum concentration in the pond sail test was also initially higher than the influent concentration, indicating molybdenum was being leached from the soil. However, analogous to the surface soil test, the effluent molybdenum concentration decreased after sucrose addition to less than 0.050 mg/L. Two 10-cm-diameter soil columns run under laboratory conditions were used for comparison with the field test columns. These columns were each charged with a composite soil sample from the corresponding field soil column and run in a similar manner as the field test. The laboratory columns performed better since they were maintained at a room temperature of 24-32 “C compared to the field test temperatures of 14-21 “C. The effluent selenium concentration was reduced to less than 0.050 mg/L within 10 and 17 days compared to 40 and 70 days for the corresponding field soil columns. In addition, higher feed rates of 280-375 L/m2 per day could be used in these soil columns while maintaining the effluent selenium concentration below 0.050 mg/L. At the completion of the field test, the columns were drained and three 1.5-m-deep vertical core samples were taken, one at the column center and one on each side of the column. The highest selenium concentration was in the top 2.5-cm soil layer closest to the influent end. The selenium concentration decreased rapidly from 220 and 130 ppm to the background levels of 7 and 21 ppm in the surface and pond soil columns, respectively. Selenium was more broadly distributed through the pond soil column due to the higher flow rate. Profiles of laboratory columns showed similar results. These selenium depth profiles indicated there was no selenium breakthrough at flow rates of 100-175 L/m2 per day and suggested that higher flow rates could have been tolerated. Summary A process to reduce uranium discharge mine water contaminants to environmentally safe levels using soilsupported bacteria has been developed and demonstrated. Clostridium and Desulfovibrio bacteria metabolize the mine water soluble selenium and sulfate, respectively. Metallic selenium and hydrogen sulfide are the products of this metabolism. The hydrogen sulfide reacts with the soluble uranium and molybdenum to form insoluble uranium dioxide and molybdenum disulfide. Small-scale laboratory column tests indicated that mine water temperature, flow rate, and nutrient concentration affect selenium removal. The interrelationship of these variables was found to be complex and only a qualitative description as follows was developed. (1) During column startup, minor variations in either temperature or flow rate impacted selenium removal. However, similar variations had virtually no effect on columns that had continuously removed selenium to less than 0.050 mg/L for at least several days.
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(2) Selenium removal could be affected at flow rates up to the mechanical failure of the column bed (900 L/m2 per day) provided sufficient nutrients were available. (3) Uranium and molybdenum removals were not observed until nutrients in addition to ones present in the soil were introduced. (4) The nutrient concentration had the greatest impact on column performance. Acceptable contaminant removal could be achieved even with major excursion of other process variables provided an adequate supply of nutrients was available. Numerous low-cost materials such as sucrose, cellulose, lignosulfonates, and refining sludge were effective nutrients.
Acknowledgments We thank Homestake Mining Co. for its financial assistance and especially Edward Kennedy for his many helpful suggestions throughout this project. We are grateful to W. E. Swartz, Department of Chemistry, University of South Florida, for the ESCA analyses. Registry No. Se, 7782-49-2; U, 7440-61-1;Mo, 7439-98-7; glucose, 50-99-7;sucrose, 57-50-1;potato starch, 9005-25-8;sodium lignosulfonate, 8061-51-6. Literature Cited (1) Muylder, J. V.; Pourbaix, M. “Atlas of Electrochemical Equilibria in Aqueous Solutions”,1st ed.; Pourbaix, M., Ed.; Pergamon: Oxford, 1966; pp 554-559. (2) Anderson et al. “Selenium in Agriculture”; Agriculture Handbook No. 200, US. Dept. of Agriculture: Washington, DC, 1961. (3) Ward, D. A. “ProblemsAssociated with the Determination of Soluble Selenium in Ground Water Samples by Graphite Furnace Atomic Absorption Spectroscopy”;39th Southwest American Chemical Society Regional Meeting, Tulsa, OK, 1983. (4) Sorg, T. J.; Love, 0. T., Jr.; Logsdon, G. S.EPA Report NO.EPA-6001 8-77-005, 1977. ( 5 ) Sorg, T. J.; Logsdon, G. S.J. Am. Water Works Assoc. 1978, 70, 379-393. (6) Sorg, T. J.; Logsdon, G. S. “Removal of Selenium from Water-State of the Art”; Proceedings of the Symposium
on Selenium-Tellurium in the Environment: University of Notre Dame, South Bend, IN, 1976; pp 114-128. (7) Alexander, M. “Introductionto Soil Microbiology”; Wiley: New York, 1961; p 416. (8) Zingaro, R. A.; Cooper, W. C. “Selenium”;Van Nostrand Reinhold: New York, 1974; p 553. (9) Ehrlich, H. L. “Geomicrobiology”;Marcel Dekker, Inc.: New
York, 1981; pp 299-302. (10) Alexander, M. “Introductionto Soil Microbiology”;Wiley: New York, 1961; pp 127-292. (11) Brierly, C. L.; Brierly, J. A. “Contamination of Ground and Surface Waters by Uranium Mining and Milling”; U.S. Bureau of Mines: Washington, DC, 1981. (12) Zajic, J. E. “MicrobialBiogeochemistry”;Academic Press: New York, 1969; p 193. (13) Brierly, J. A.; Brierly, C. L.; Dreher, K. T. “Uranium Mine Water Disposal”;Society of Mining Engineers: New York, 1980. Received for review August 30, 1984. Revised manuscript received August 2, 1985. Accepted September 25, 1985.
