Mobilization of Bi, Cd, Pb, Th, and U Ions from Contaminated Soil and

Chapter 5

Mobilization of Bi, Cd, Pb, Th, and U Ions from Contaminated Soil and the Influence of Bacteria on the Process 1-3

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Downloaded by PENNSYLVANIA STATE UNIV on September 9, 2012 | http://pubs.acs.org Publication Date: March 10, 1994 | doi: 10.1021/bk-1994-0554.ch005

K. W. Tsang , P. R. Dugan , and R. M. Pfister 1

Idaho National Engineering Laboratory, EG&G Idaho, Inc., P.O. Box 1625, Idaho Falls, ID 83415-2203 The Ohio State University, Columbus, OH 43210 2

Sterile or nonsterile soil experimentally contaminated with bismuth, cadmium, lead, thorium, and uranium as uranyl then incubated with sterile water showed negligible release of metals from either sample. 10 mM cysteine solution mixed with metal-amended soil under each condition: (a) nonsterile; (b) sterile; (c) sterile then inoculated with pure cultures of soil bacterial isolates; indicated that 90.5% Bi, 4.3% Cd, and 25.9% Pb were released from the nonsterile mix within 24 hours. With uranium, 57.9% was released gradually over eight days. The same pattern was observed with sterile soil, but in smaller amounts, 30.6%, 2.6%, 5.2% and 28.3% respectively. Sterile soil containing cysteine then reinoculated with any of four bacteria isolated from the original soil resulted in release of metal greater than from sterile soil. Nonsterile soil released more Bi and U than any of the sterilized reinoculated soils tested. In the case of Cd and Pb some sterilized reinoculated soils released more Cd and Pb than the nonsterile soil while others released less. In all cases extraction of Th was negligible. The results indicated that (a) active microorganisms influence the ability of soil to retain or release metals and (b) cysteine is an effective agent for the release of some metals from soil. 10 mM glycine removed 40.7% and 5.1% of U from nonsterile and sterile soil, respectively. Commercially prepared thioglycollate culture medium resulted in significant release of both Cd and Th from of nonsterile soil. Background

Among the various environmental concerns, soil and sediment remediation has received considerable attention in recent years because soils and sediments are the 3

Current address: Reno Research Center, U.S. Bureau of Mines, 1605 Evans Avenue, Reno, N V 89512 0097-6156/94/0554-0078$08.00/0 © 1994 American Chemical Society

In Emerging Technologies in Hazardous Waste Management IV; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Bi, Cd, Pb, Th, and U Ions in Contaminated Soil 79

TSANG ET A L .

ultimate repositories for many metals that cycle in the environment as a result of activities such as mining, electroplating, and various manufacturing and industrial processes. There is considerable interest in the remediation of contaminated soils and sediments by so-called soil-cleaning techniques and in the prevention of future contamination via removal of hazardous metals from processing streams prior to deposition into receiving waters. Present methods of metal recovery from waste waters include the use of ion-exchange resins (1), biosorption (2,3,4), chemical precipitation, electrolysis, reverse osmosis and membrane filtration. A variety of chemical technologies may be of value in the extraction of heavy metals from soils and sediments including washing with: water, salts, complexing agents such as ethylenediaminetetraacetate (EDTA) or nitrilotriacetic acid (NTA), mineral acids, strong bases, and some organic acids, e.g. critic acid, that are also complexing agents (5). An understanding of the complex associations and interactions among soil components (e.g., clay, silt, sand, etc.), the natural microflora (e.g., bacteria and other organisms) and both metal and organic contaminants is fundamental to our ability to accurately model or predict the behavior (bioavailability, toxicity, migration velocity, immobilization, etc.) of contaminants in subsurface environments. There is a body of evidence indicating that radioactive contaminants in soils are associated primarily with fine soil particles such as clays and microorganisms (6,7,8). It is also known that both clays and microorganisms are capable of concentrating large amounts of metals from solution and that bioconcentration by microorganisms is capable of exceeding both rate and total uptake of certain clays (7,9). With respect to the behavior of metal contaminants in soil, some indication of relative migration velocities of mixtures of heavy metals (e.g., Bi , Cd , Pb , Th , and U0 ) in contaminated clay soil can be gleaned from the report of Tsang et dl.(10). All of the above ions added as nitrate salts to a clay type soil column strongly sorbed to the soil. The maximum amount of each ion capable of being sorbed by the soil varied as did the relative rate of migration. The rate of migration of these metal ions occurred in the following order: Cd > U0 , Pb > Bi , Th , while the maximum amount of these ions sorbed to the soil was in the reverse order. Bismuth and thorium were not observed to migrate once they were sorbed to the soil. There was also an indication of interaction between Cd , the most mobile and U0 , the second most mobile of the ions examined. Continued addition of U0 solution to the soil column resulted in release of Cd suggesting that either a common binding site exists for Cd and U0 and that there may be a cation exchange occurring or that Cd is not tightly sorbed and the equilibrium shifts toward solution in the presence of Cd free water. These kinds of interactions and relative migration rates need to be considered when modeling the behavior of metal contaminants in the subsurface environments. +3

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Bioremediation also appears to have value because of its potential economic advantage (11). The purpose of this report is to further demonstrate the role of In Emerging Technologies in Hazardous Waste Management IV; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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EMERGING TECHNOLOGIES FOR HAZARDOUS WASTE MANAGEMENT


bacteria as agents to effect the mobilization of hazardous metals from contaminated soil under relatively high and relatively low Eh (i.e., approaching anaerobic conditions). Further, the effectiveness of the amino acid cysteine, a reducing agent as well as a metal complexing agent and a nutrient for many microorganisms (12), for the removal of several hazardous metals from soil is shown in comparison to: (a) a non-reducing but metal complexing amino acid microbial nutrient (glycine) (b) the reducing agent sodium thioglycollate and (c) the presence or absence of microorganisms. Experimental

Source and characteristics of soil. The soil used was obtained from the Snake River Plain about one mile south of the Radioactive Waste Management Complex (RWMC), the waste storage facility for the Idaho National Engineering Laboratory located between Idaho Falls and Arco, Idaho. The soil sample area has not been used for waste storage and represents soil native to the area. The surficial deposits in the vicinity of this soil have been previously described as: "eolian and alluvial which range in thickness from less than 0.6 to more than 7.3 m. Alluvial material was transported to the location in times of high runoff and contains gravel. The eolian material probably derived from the finer fraction of alluvial deposits located to the southwest, in the windward direction. Surficial sedimentary materials and interflow sedimentary bed samples have an average median grain size of less than 1 mm with between 70 and 95% material less than 62 μm~predominantly silt and clay sized. The bulk mineralogy indicates that clay and quartz are the dominant minerals. Accessory minerals include potassium feldspar, plagioclase feldspar and a pyroxene. Clay content ranged from 25 to 40% of the samples analyzed and contained 30 to 36% illite, 13 to 26% smectite, 6 to 12% kaolinite, 0 to 26% carbonate and a cation exchange capacity between 11 and 27 milliequivalents per 100 gm soil" (13). The soil moisture content was 14% by weight at the time of sampling. Washedriversand was mixed with the soil in 1:1 ratio by weight to facilitate percolation of the metal solutions used to experimentally contaminate the soil. It has been determined that the sand used in this study did not sorb any of the metals under investigation in significant amounts. Preparation of metal-amended soil. A 3.5 kg soil/sand mix was placed in a lucite column (76 mm I.D. χ 914 mm) equipped with a drain on the bottom. Based on the metal sorption capacity of the soil/sand mix determined previously, the following, in the form of nitrate salts, in a total of 20 1 of H 0, acidified to pH 3 with dilute HN0 , were added to the column: 3.54 gm Bi; 1.42 gm Cd; 3.54 gm Pb: 3.54 gm Th; 2.83 gm U. The flow rate was measured at 15 ml per min. No attempt was made to alter the natural flow rate of the metal solution. The column was allowed to stand for one week until effluence stopped. The resultant contaminated soil/sand was then mixed thoroughly in a stainless steel container. 2

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The actual metal concentration in the experimentally contaminated soil was determined to establish a baseline. One hundred grams (wet weight) of the metalamended soil/sand mix was dried to constant weight at 85°C. The dry mix was In Emerging Technologies in Hazardous Waste Management IV; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

5. TSANG ET AL.

81 Bi, Cd, Pb, Th, and U Ions in Contaminated Soil

weighed and then mixed thoroughly. A 1 gm portion (in triplicate) was digested according to procedures described in EPA SW-846 Method 3050 (14). The concentration of Bi, Cd, Pb, Th, and U in the resultant aqueous sample was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The total amount of each metal in 100 gm (wet weight) of the metal-amended soil/sand mix was then calculated.


Pure Cultures of Bacteria from Metal-Amended Soil Five hundred milliliters of sterile 10 mM cysteine solution was mixed with 100 gm of metal-amended soil in a 1 L sterile flask. With a sterile pipet, a 1 ml sample was transferred into a test-tube containing 9 ml of sterile trypticase soy broth (TSB) (30 g/L, Becton Dickinson Microbiology Systems, Cockeysville, MD 21030) and mixed thoroughly. A 1 ml aliquot was then transferred into another 9 ml TSB tube and mixed. From this tube 0.1 ml aliquot was dispensed into a sterile petri dish containing -20 ml of sterile, solidified trypticase soy agar (TSA) (40 g/L) and spread over the entire surface of the agar plate with a sterile L-shaped glass rod. The plate was incubated for 4 d at 22 i 3°C. Bacterial colonies showing different morphologies on the agar plate were transferred individually to separate TSA plates and streaked over the surface of the agar with a sterile inoculating loop. The plates were incubated for 4 d at 22 ± 3°C. With proper aseptic techniques, each plate should contain colonies with the same morphology, i.e., pure culture. The pure cultures were subcultured onto tubes of solidified TSA with slanting surface. The inoculated agar slants were numbered #011, 012, 013, etc. and incubated at 22 ± 3°C for 4 d and then kept at 4°C for stock culture. To inoculate autoclaved sterile soil with a pure culture isolated from the metal-amended soil, 250 ml of TSB was inoculated with a loopful of pure culture from the stock culture and incubated overnight at 22 +. 3°C on a shaker table at 150 rpm. The cells were harvested by centrifugation at 10,000 χ g for 10 min. The supernatant was discarded and the pellet was resuspended in 250 ml of sterile phosphate buffer and again centrifuged at 10,000 χ g for 10 min. The supernatant was discarded and the pellet of bacterial cells was transferred to the sterile soil. Soil washing with water. The efficacy of distilled water on removal of metals from soil, either with or without indigenous microorganisms, was evaluated by mixing 200 gm (wet weight) of nonsterile or sterile metal-amended soil with 500 ml of sterile distilled water in a 1 L flask. The mixture was agitated by stirrer motor at 150 rpm for four days at ambient temperature. The content was allowed to settle for 30 minutes. A 6 ml aliquot of the wash water was withdrawn, centrifuged at 3000 χ g for 20 min, and then filtered through a 0.45 μτη filter disk. A 5 mL sample of the filtrate was diluted to 25 ml with 1 % HN0 and assayed for Bi, Cd, Pb, Th, and U by ICP-AES. 3

Effect of cysteine and microorganisms on release of metals from metal-contaminated soil. To investigate the effect of cysteine on the release of metals and the microbial influence on the process, a metal-amended soil/sand sample was pre-washed by filtering 2 L of distilled water through a soil/sand column as previously described. In Emerging Technologies in Hazardous Waste Management IV; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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EMERGING TECHNOLOGIES FOR HAZARDOUS WASTE MANAGEMENT

The pre-washed soil/sand sample was then removed from the column and a 500 ml 10 mM sterile cysteine solution was mixed with 100 gm of the metal-amended soil/sand by stirrer motor at 150 rpm for 8 d under each of the following conditions: (a) nonsterile soil/sand; (b) sterile soil/sand; and (c) autoclaved sterile soil/sand then inoculated with pure cultures of four bacteria that were isolated from the metalamended soil, designated with an isolate number and later identified as the following


organisms: #011, Arthrobacter oxydans; #012, Micrococcus luteus; #014, Bacillu

megaterium; and #016, Arthrobacter aurescens. Viable microbes were enumerated daily by spread plating 0.1 ml of a series of diluted soil and cysteine mixtures (i.e., 1/10,1/100, 1/1000, etc.) (14) on trypticase soy agar which was incubated for 4 d at 22 +. 3°C. Eh and pH of the soil/sand and cysteine mixture were measured daily. Eh measurements were accomplished by using a Pt/AgCl redox electrode, and pH was measured with a standard pH electrode. A 6 ml aliquot of the cysteine solution was withdrawn after 30 min settling, centrifuged at 3000 χ g for 20 min, and filtered through a 0.45 μπι filter disk. Five milliliters of the filtrate was diluted to 25 ml with 1% HN0 for metal analysis by ICP-AES. 3

Effect of glycine and microorganisms on release of metals from metal-contaminated soil. The effect of glycine, an amino acid known to complex some metals and to serve as a nutrient for many microorganisms, was evaluated on release of metals from metal-amended soil. Experimental procedures were similar to that for cysteine extraction, except that 10 mM glycine was substituted for cysteine and only sterile soil and nonsterile soil with indigenous microorganisms were used. Comparison of sodium thioglycollate solution and nutrient-supplemented sodium thioglycollate medium on release of metals from metal-contaminated soil. The effect of 0.05% sodium thioglycollate solution on release of metals from soil was compared with that of thioglycollate culture medium (29.8 g/L, Difco Laboratory, Detroit, Michigan) which contained bacterial nutrients such as casitone, yeast extract, dextrose, sodium chloride and L-cystine in addition to 0.05% sodium thioglycollate. One hundred grams of nonsterile metal-amended soil was placed in 500 ml of either solution and allowed to incubate for four days at 22 ± 3°C with continuous stirring. Metal concentration in solution was assayed as described previously, To ensure the autoclaving process did not change the chemistry and texture of the soil or cause permanent binding of metal ions to the soil particles, a similar study using thioglycollate broth and sterile soil reinoculated with bacteria was performed for comparison. Results and Discussion Effect of water washing on release of metals from sterile and nonsterile soils. Soilwashing with distilled water removed only negligible amounts of metals from either sterile or nonsterile metal-amended soil (Table I). The change in pH for nonsterile soil dropped slightly from 7.40 to 7.27 while a slight increase from pH 7.65 to 7.68 was observed with sterile soil suggesting that microorganisms produced a slight

In Emerging Technologies in Hazardous Waste Management IV; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

5. TSANG ET AL.

Bi, Cd, Pb, Th, and U Ions in Contaminated Soil S3

Table I. Metal released from water-washed nonsterile or sterile soil by distilled water after four days with continuous stirring at 22 ± 3°C Bi


Amount of metal (mg) in 200 gm (wet weight) of amended soil/sand mix Nonsterile soil

mg %

Sterile soil

mg %

Cd

Pb

Th

U

152.00 54.00 162.00 86.00

102.00

0.10 0.07

0.15 0.28

0.05 0.03

Mobilization of Bi, Cd, Pb, Th, and U Ions from Contaminated Soil and

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