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In situ bioremediation of soils contaminated with radioactive elements and toxic heavy metals
Int. J. Miner. Process. 62 Ž2001. 301–308 www.elsevier.nlrlocaterijminpro
In situ bioremediation of soils contaminated with radioactive elements and toxic heavy metals S.N. Groudev ) , I.I. Spasova, P.S. Georgiev Department of Engineering Geoecology, UniÕersity of Mining and Geology, Studentski grad - DurÕenitza, Sofia 1100, Bulgaria Received 15 August 1999; accepted 17 July 2000
Abstract Two experimental plots of an agricultural land contaminated with radioactive elements Žuranium, radium thorium. and toxic heavy metals Žcopper, zinc, cadmium. were treated by two different biotechnological in situ methods. The soil in this land was characterized by a negative net neutralization potential, and the soil pH was in a slightly acidic pH range Žfrom 4 to 5.. The contaminants were located mainly in the upper soil layers Žmainly in the horizon A.. Both methods were connected with the initial solubilization of the contaminants. Water acidified with sulphuric acid was used as a leach solution. The solubilization was mainly a result of the activity of the indigenous soil microflora. This activity was enhanced by suitable changes in the levels of some essential environmental factors, such as water, oxygen and nutrient content of the soil. The first method was then connected with the removal of the dissolved contaminants from the soil through the soil effluents. The second method was based on the transfer of the contaminants into the deeply located soil horizon B 2 , where they were immobilized mainly as a result of the activity of the indigenous sulphate-reducing bacteria. Their activity was enhanced by injecting water solutions of organic compounds into the horizon B 2 through boreholes located in the relevant experimental plot. q 2001 Elsevier Science B.V. All rights reserved. Keywords: chemolithotrophic bacteria; sulphate-reducing bacteria; uranium; soil flushing; metal mobilization; metal precipitation
1. Introduction Some agricultural land located near the uranium deposit ACB in Central Bulgaria has been contaminated with radioactive elements Žuranium, radium, thorium. and toxic )
Corresponding author. Fax: q359-2-9624-940. E-mail address: [email protected] ŽS.N. Groudev..
0301-7516r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 7 5 1 6 Ž 0 0 . 0 0 0 6 1 - 2
302
S.N. GroudeÕ et al.r Int. J. Miner. Process. 62 (2001) 301–308
heavy metals Žcopper, zinc, cadmium. as a result of mining and in situ leaching activities carried out in the deposit for a long period of time. Laboratory experiments carried out with soil samples from this land revealed that an efficient remediation of the soils was achieved by means of two different biotechnological in situ methods, which had already done good results with other soils contaminated with heavy metals ŽGroudev, 1996; Groudev and Spasova, 1997.. Both methods were connected with the solubilization of the contaminants Žlocated mainly in the upper soil layers. as a result of the activity of the indigenous soil microflora. This activity was enhanced by suitable changes in the levels of some essential environmental factors such as water, oxygen and nutrient content of the soil. The first method was then connected with the flushing of the soil by means of acidified leach solutions. The pregnant soil effluents containing the dissolved contaminants were then treated by a passive system of the type of constructed wetlands and the depleted solutions were recycled to the soil. The second method was connected with the transfer of the dissolved contaminants to the deeply located soil horizon B 2 , where the contaminants were immobilized as a result of the activity of the indigenous sulphate-reducing bacteria inhabiting this anoxic soil horizon. The activity of these bacteria was enhanced by injecting water solutions of organic compounds into the horizon B 2 . In 1997 the above-mentioned methods were applied under real field conditions in two experimental plots located near the uranium deposit. Some data about this study are shown in this paper.
2. Materials and methods A detailed sampling procedure was carried out to characterize the soil and the subsurface geologic and hydrogeologic conditions of the site. Surface and bulk soil samples up to a depth of 2 m were collected by an excavator. Drill hole samples were collected up to a depth of 10 m. Elemental analysis in the samples was performed by digestion and measurement of the ion concentration in solution by atomic absorption spectrometry and induced coupled plasma spectrometry. Mineralogical analysis was carried out by X-ray diffraction techniques. The main geotechnical characteristics of the site, such as permeability and wet bulk density, were measured in situ using the sand-core method ŽU.S. Environmental Protection Agency, 1991.. True density measurements were carried out in the laboratory using undisturbed core samples. Such samples were also used for determination of their acid generation and net neutralization potentials using static acid-base accounting tests. The bioavailable fractions of the pollutants were determined by leaching the samples with DTPA and EDTA ŽSobek et al., 1978.. The mobility of the pollutants was determined by the sequential extraction procedure ŽTessier et al., 1979.. The toxicity of soil samples was determined by the EPA Toxicity Characteristics Leaching Procedure ŽU.S. Environmental Protection Agency, 1990.. The isolation, identification and enumeration of soil microorganisms were carried out by methods described previously ŽKaravaiko et al., 1988; Groudeva et al., 1993..
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The experimental plots had a rectangular shape and were 200 m2 in size Ž20 = 10 m. each. Water acidified with sulphuric acid to pH of 3.5–4.5 was used as leach solution. The upper soil layers were ploughed up periodically to enhance the natural aeration. In the experimental plot No. 2, water solutions of dissolved organic compounds and ammonium phosphate were injected through vertical boreholes into the deeply located soil layers Žhorizon B 2 . to enhance the activity of anaerobic sulphate-reducing bacteria. The flowsheet included also a system to collect the soil drainage solutions and to avoid their seepage and the distribution of contaminants into the environment. The system consisted of several ditches and wells located in suitable sites in the experimental plots. The soil effluents collected by this system were then treated by a constructed wetland located near the experimental plots to remove the residual amounts of toxic elements, nonprecipitated in the deeply located soil horizons.
3. Results and discussion The soil profile was approximately 100 cm deep Žhorizon A, 30 cm; horizon B, 50 cm; horizon C, 20 cm.. The soil profile was underlined by intrusive rocks consisting of dense diorite and gabbro. The filtration properties of these intrusives were very low. The filtration coefficient was approximately 6 = 10y8 mrs, and the deeply located rocks were even more impermeable.
Table 1 Characteristics of the soil used in this study Parameters Chemical composition, % SiO 2 Al 2 O 3 Fe 2 O 3 CaO MgO K 2O Na 2 O S total S sulphidic humus Bulk density, grcm3 Specific density, grcm3 Porosity, % Moisture capacity, % Permeability, cmrs pH ŽH 2 O. Net neutralization potential, kg CaCO 3 rt
Horizon A Ž0–30 cm.
Horizon B Ž31–80 cm.
63.5 14.5 10.7 0.37 0.44 1.70 0.17 0.95 0.77 2.8 1.50 2.84 48 46 8=10y2 4.4 y145
67.1 14.0 8.0 0.32 0.28 1.40 0.32 0.82 0.77 1.2 1.55 3.05 44 41 6=10y2 4.6 y159
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S.N. GroudeÕ et al.r Int. J. Miner. Process. 62 (2001) 301–308
The ground water level was located generally 5 to 10 m below the surface. The surface and ground waters in the site were well separated by the impermeable rocks. It was assumed that both the geologic and hydrogeologic conditions in the site were suitable for the application of in situ methods for soil remediation. Data about the chemical composition and some essential geotechnical parameters of the soil are shown in Table 1. The concentrations of contaminants were higher in the upper soil layers Žmainly in the horizon A. ŽTables 2 and 3.. The contaminants were present mainly in forms susceptible to biological andror chemical leaching but considerable portions were refractory to solubilization. The primary sulphide minerals were essential components of these inert fractions. The soil exhibited negative net neutralization potential, indicating a relatively high potential of acid generation. The permeability of the soil was high and rainwater infiltrated into and created conditions favorable for the dissolution of elements. The pore water quality was poor with high concentrations of contaminants such as radioactive elements Žuranium, radium, and thorium., some toxic heavy metals Žcopper, zinc, cadmium., free sulphuric acid and sulphates. Prior laboratory experiments with soil samples from the experimental plots treated in this study had shown that the dissolution of contaminants was connected with the activity of the indigenous soil microflora, mainly with the activity of acidophilic chemolithotrophic bacteria. These bacteria were able to oxidize sulphide minerals and to solubilize their metal components. Table 2 Toxic elements in the horizon A of the soil before and after the bioremediation Parameters
U
Content of toxic elements, ppm Before treatment 82 After treatment 23 BioaÕailable fraction, ppm Ža. By DTPA leaching Before treatment 12 After treatment 3.2 Žb. By EDTA leaching Before treatment 2.8 After treatment 0.5
Ra
Cu
Zn
Cd
640 190
230 68
170 51
3.5 1.2
120 25
41 2.8
35 2.4
0.7 0.02
45 10
17 1.2
14 1.2
0.3 0.01
Easily leachable fractions-exchangeableq carbonate, ppm Before treatment 14 140 After treatment 3.0 25
59 3.7
48 5.0
1.0 0.03
Inert fraction, ppm Before treatment After treatment
91 59
82 41
1.9 0.9
24 16
260 170
Toxic elements solubilized during the toxicity test, ppm Before treatment 1.45 0.35 After treatment 0.28 0.10 The contents of radium are shown in Bqrkg dry soil or Bqrl.
6.40 0.32
7.10 0.28
0.12 0.01
S.N. GroudeÕ et al.r Int. J. Miner. Process. 62 (2001) 301–308
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Table 3 Removal of contaminants from the polluted soil Parameters Content of contaminants in the soil before treatment Horizon A Horizon B Content of contaminants in the horizon A after treatment Content of contaminants in the horizon B after treatment By flushing By detoxification Removal of contaminants from the horizon A, % Removal of contaminants from the horizon B, % By flushing By detoxification Removal of contaminants from the whole soil profile Žhorizon Aqhorizon B., % By flushing By detoxification
U
Ra
Cu
Zn
Cd
82 37 23
640 300 190
230 90 68
170 120 51
49 65 71.9
410 530 70.3
87 166 70.4
93 172 69.9
1.4 3.1 65.7
23.0 9.7
20.8 7.0
41.1 11.2
44.2 9.9
47.8 6.1
27.1 8.2
23.4 6.2
43.9 9.5
44.3 8.6
46.2 5.1
3.5 1.9 1.2
The content of Ra is shown in Bqrkg dry soil; the content of all other contaminants is shown in mgrkg dry soil.
The analysis of the soil microflora revealed that it included a rich variety of microorganisms ŽTable 4.. The mesophilic, acidophilic bacteria related to the species Thiobacillus ferrooxidans, T. thiooxidans and Leptospirillum ferrooxidans were the prevalent microorganisms in the top soil layers, but some basophilic chemolithotrophic species Žmainly T. thioparus and T. denitrificans. and some heterotrophs were also present in high numbers. In the soil horizon B 2 , the total number of microorganisms was much lower than in the horizon A but various anaerobes were well present. It was found that under natural conditions the bacterial oxidation of sulphide minerals in the soil proceeded continuously but at relatively low rates. The number and activity of the acidophilic chemolithotrophs in the soil were limited by some essential environmental factors such as the relatively high soil pH, shortage of oxygen inside the soil horizons, insufficient soil moisture during relatively long periods of time, absence of some important nutrients such as nitrogen and phosphorus sources. The treatment of the contaminated soil was connected with increasing the number and activity of the indigenous microorganisms by suitable changes in the levels of the above-mentioned environmental factors. This was achieved by regular ploughing up and irrigation of the soil and by addition of some essential nutrients. The optimum soil humidity was about 50% of the moisture capacity of the soil, but periodic flushing with slightly acidified water ŽpH about 3.5–4.5. was needed to remove the soil contaminants. Zeolite saturated with ammonium phosphate was added to the soil Žin amounts of 2–5
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Table 4 Concentration of microorganisms related to different physiological groups in the soil during the treatment Microorganisms
Horizon A Ž0–30 cm.
Horizon B Ž31–80 cm. Treatment by
Aerobic heterotrophic bacteria Oligocarbophiles Cellulose-degrading microorganisms Nitrogen-fixing bacteria Nitrifying bacteria S 2 O 32y-oxidising chemolithotrophs Žat pH 7. S o-oxidising chemolithotrophs Žat pH 2. Fe 2q-oxidising chemolithotrophs Anaerobic heterotrophic bacteria Bacteria fermenting carbohydrates with gas production Denitrifying bacteria Sulphate-reducing bacteria Fe 3q-reducing bacteria Mn4q-reducing bacteria Methanogenic bacteria Streptomycetes Fungi Total cell numbers
Flushing
Detoxification
Cellsrg dry soil 10 5 –10 7 10 4 –10 6 10 2 –10 6 10 2 –10 5 10 2 –10 4 10 4 –10 7
10 4 –10 5 10 2 –10 4 10 2 –10 4 10 2 –10 4 10 1 –10 3 10 3 –10 5
10 4 –10 6 10 2 –10 5 10 2 –10 5 10 2 –10 4 10 1 –10 3 10 3 –10 5
10 5 –10 7
10 3 –10 5
10 2 –10 5
10 4 –10 7 10 3 –10 5 10 2 –10 3
10 2 –10 5 10 3 –10 5 10 2 –10 4
10 2 –10 4 10 4 –10 7 10 3 –10 5
10 2 –10 3 10 3 –10 4 10 2 –10 4 10 2 –10 4 0–10 2 10 2 –10 4 10 3 –10 6 1=10 7 –3=10 8
10 1 –10 3 10 2 –10 4 10 2 –10 4 10 2 –10 4 1–10 3 10 1 –10 3 10 2 –10 4 5=10 5 –1=10 7
10 3 –10 5 10 4 –10 7 10 3 –10 5 10 2 –10 5 10 1 –10 4 10 2 –10 4 10 3 –10 5 2=10 6 –6=10 7
The quantitative determination of the different physiological groups of microorganisms was carried out by the spread plate technique on solid nutrient media or by the most probable number method using end-point dilutions. The total number of microbial cells was determined by epifluorescence microscopy.
kgrt dry soil. to provide the microorganisms with ammonium and phosphate ions and to improve the physico-mechanical properties of the soil. The treatment of the soil was started in the middle of March 1997, and at the middle of November 1997, it was found that considerable portions of the contaminants were removed from the upper soil horizon A and their residual concentrations were lower or, at least, very close to the relevant permissible levels. The treatment caused some changes in the composition of the soil microflora increasing the number of the acidophilic chemolithotrophic bacteria and slightly decreasing the number of heterotrophs. The chemical composition, structure and main physical and water properties of the soil were altered to a small extent. In the experimental plot No. 1 treated by means of the flushing system, considerable portions of the contaminants were washed out from the soil profile by the soil effluents ŽTable 3.. However, the larger portions of the contaminants were accumulated in the horizon B 2 . Portions of the toxic heavy metals were present in this horizon as the relevant insoluble sulphides. A portion of the uranium was present as the mineral
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Table 5 Sulphate-reducing bacteria in the experimental plots Sulphate-reducing bacteria
Plot No. 1
Plot No. 2
Desulfovibrio Desulfobulbus Desulfococcus Desulfobacter Desulfobacterium Desulfotomaculum Desulfosarcina
Cellsrg dry soil 3=10 2 –1=10 4 8=10 1 –3=10 3 5=10 1 –1=10 2 3=10 1 –5=10 2 – 1=10 1 –4=10 1 1=10 1 –6=10 1
1=10 4 –8=10 6 3=10 3 –8=10 5 3=10 2 –5=10 5 7=10 2 –7=10 4 8=10 1 –2=10 2 1=10 2 –5=10 3 5=10 2 –1=10 4
uraninite ŽUO 2 .. Both the sulphides and uraninite were formed as a result of microbial dissimilatory sulphate reduction taking place in this anoxic zone regardless of the low content of dissolved organic compounds serving as electron donors. The above-mentioned forms of the contaminants are relatively stable and refractory to leaching, especially under anaerobic conditions. Portions of the nonferrous metals and uranium as well as most of the radium were adsorbed on the clay minerals present in the horizon B2 . In the experimental plot No. 2 treated by means of the detoxification system, only small amounts of the contaminants were washed out from the soil profile by the soil effluents ŽTable 3.. The larger portions of the contaminants were retained in the horizon B 2 as a result of the very active process of microbial dissimilatory sulphate reduction, which was artificially enhanced by providing the indigenous sulphate-reducing bacteria with high concentrations of suitable electron donors. The population density of these bacteria in the experimental plot No. 2 was much higher than that in the experimental plot No. 1 ŽTables 4 and 5.. The concentrations of dissolved contaminants in the soil effluents from the experimental plot No. 2 were much lower than those from the experimental plot No. 1. Both types of effluents were treated efficiently by the constructed wetland. In 1998, the experimental plots were subjected to some conventional remediation procedures such as grassing of the treated soil, addition of suitable fertilizers and animal manure as well as with periodical ploughing up, liming and irrigation. As a result of this, the quality of the soil was completely restored. No soluble forms of the above-mentioned contaminants in concentrations higher than the relevant permissible levels were detected so far ŽApril 1999. in the soil pore and drainage waters after rainfall.
Acknowledgements A part of this study was funded by the National Science Fund ŽResearch Contracts No. TH-803r98, VRP-TH-9r99 and MY-X-09r96..
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References Groudev, S.N., 1996. Microbial detoxification of heavy metals in soil. Miner. Slovaca 28, 335–338. Groudev, S.N., Spasova, I.I., 1997. Microbial treatment of soil contaminated with heavy metals and arsenic. Paper Presented at the International Biohydrometallurgy Symposium, Sydney, August 25–30. Groudeva, V.I., Ivanova, I.A., Groudev, S.N., Uzunov, G.C., 1993. Enhanced oil recovery by stimulating the activity of the indigenous microflora of soil reservoirs. In: Torma, A.E., Apel, M.L., Brierley, C.L. ŽEds.., Biohydrometall. Technol., vol. II. TMS Minerals, Metals and Materials Society, Warrendale, PA, pp. 349–356. Karavaiko, G.I., Rossi, G., Agate, A.D., Groudev, S.N., Avakyan, Z.A., 1988. Biogeotechnology of Metals, Manual Center for International Projects GKNT Moscow. Sobek, A.A., Schuller, W.A., Freeman, J.R., Smith, R.M., 1978. Field and laboratory methods applicable to overburden and mine soils. US EPA Report 600r2-78-054. Tessier, A., Campbell, P.G.C., Bisson, M., 1979. Sequential extraction procedure for speciation of particulate trace metals. Anal. Chem. 51 Ž7., 844–851. U.S. Environmental Protection Agency, 1990. Characteristics of EP Toxicity, Paragraph 261.24, Federal Register 45 Ž98.. U.S. Environmental Protection Agency, 1991. Description and sampling of contaminated soils — A field pocket guide EPAr625r12-91r002 Technology Transfer, Centre for Environmental Research Information, United States Environmental Protection Agency, Cincinnati.
In situ bioremediation of soils contaminated with radioactive elements and toxic heavy metals S.N. Groudev ) , I.I. Spasova, P.S. Georgiev Department of Engineering Geoecology, UniÕersity of Mining and Geology, Studentski grad - DurÕenitza, Sofia 1100, Bulgaria Received 15 August 1999; accepted 17 July 2000
Abstract Two experimental plots of an agricultural land contaminated with radioactive elements Žuranium, radium thorium. and toxic heavy metals Žcopper, zinc, cadmium. were treated by two different biotechnological in situ methods. The soil in this land was characterized by a negative net neutralization potential, and the soil pH was in a slightly acidic pH range Žfrom 4 to 5.. The contaminants were located mainly in the upper soil layers Žmainly in the horizon A.. Both methods were connected with the initial solubilization of the contaminants. Water acidified with sulphuric acid was used as a leach solution. The solubilization was mainly a result of the activity of the indigenous soil microflora. This activity was enhanced by suitable changes in the levels of some essential environmental factors, such as water, oxygen and nutrient content of the soil. The first method was then connected with the removal of the dissolved contaminants from the soil through the soil effluents. The second method was based on the transfer of the contaminants into the deeply located soil horizon B 2 , where they were immobilized mainly as a result of the activity of the indigenous sulphate-reducing bacteria. Their activity was enhanced by injecting water solutions of organic compounds into the horizon B 2 through boreholes located in the relevant experimental plot. q 2001 Elsevier Science B.V. All rights reserved. Keywords: chemolithotrophic bacteria; sulphate-reducing bacteria; uranium; soil flushing; metal mobilization; metal precipitation
1. Introduction Some agricultural land located near the uranium deposit ACB in Central Bulgaria has been contaminated with radioactive elements Žuranium, radium, thorium. and toxic )
Corresponding author. Fax: q359-2-9624-940. E-mail address: [email protected] ŽS.N. Groudev..
0301-7516r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 7 5 1 6 Ž 0 0 . 0 0 0 6 1 - 2
302
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heavy metals Žcopper, zinc, cadmium. as a result of mining and in situ leaching activities carried out in the deposit for a long period of time. Laboratory experiments carried out with soil samples from this land revealed that an efficient remediation of the soils was achieved by means of two different biotechnological in situ methods, which had already done good results with other soils contaminated with heavy metals ŽGroudev, 1996; Groudev and Spasova, 1997.. Both methods were connected with the solubilization of the contaminants Žlocated mainly in the upper soil layers. as a result of the activity of the indigenous soil microflora. This activity was enhanced by suitable changes in the levels of some essential environmental factors such as water, oxygen and nutrient content of the soil. The first method was then connected with the flushing of the soil by means of acidified leach solutions. The pregnant soil effluents containing the dissolved contaminants were then treated by a passive system of the type of constructed wetlands and the depleted solutions were recycled to the soil. The second method was connected with the transfer of the dissolved contaminants to the deeply located soil horizon B 2 , where the contaminants were immobilized as a result of the activity of the indigenous sulphate-reducing bacteria inhabiting this anoxic soil horizon. The activity of these bacteria was enhanced by injecting water solutions of organic compounds into the horizon B 2 . In 1997 the above-mentioned methods were applied under real field conditions in two experimental plots located near the uranium deposit. Some data about this study are shown in this paper.
2. Materials and methods A detailed sampling procedure was carried out to characterize the soil and the subsurface geologic and hydrogeologic conditions of the site. Surface and bulk soil samples up to a depth of 2 m were collected by an excavator. Drill hole samples were collected up to a depth of 10 m. Elemental analysis in the samples was performed by digestion and measurement of the ion concentration in solution by atomic absorption spectrometry and induced coupled plasma spectrometry. Mineralogical analysis was carried out by X-ray diffraction techniques. The main geotechnical characteristics of the site, such as permeability and wet bulk density, were measured in situ using the sand-core method ŽU.S. Environmental Protection Agency, 1991.. True density measurements were carried out in the laboratory using undisturbed core samples. Such samples were also used for determination of their acid generation and net neutralization potentials using static acid-base accounting tests. The bioavailable fractions of the pollutants were determined by leaching the samples with DTPA and EDTA ŽSobek et al., 1978.. The mobility of the pollutants was determined by the sequential extraction procedure ŽTessier et al., 1979.. The toxicity of soil samples was determined by the EPA Toxicity Characteristics Leaching Procedure ŽU.S. Environmental Protection Agency, 1990.. The isolation, identification and enumeration of soil microorganisms were carried out by methods described previously ŽKaravaiko et al., 1988; Groudeva et al., 1993..
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The experimental plots had a rectangular shape and were 200 m2 in size Ž20 = 10 m. each. Water acidified with sulphuric acid to pH of 3.5–4.5 was used as leach solution. The upper soil layers were ploughed up periodically to enhance the natural aeration. In the experimental plot No. 2, water solutions of dissolved organic compounds and ammonium phosphate were injected through vertical boreholes into the deeply located soil layers Žhorizon B 2 . to enhance the activity of anaerobic sulphate-reducing bacteria. The flowsheet included also a system to collect the soil drainage solutions and to avoid their seepage and the distribution of contaminants into the environment. The system consisted of several ditches and wells located in suitable sites in the experimental plots. The soil effluents collected by this system were then treated by a constructed wetland located near the experimental plots to remove the residual amounts of toxic elements, nonprecipitated in the deeply located soil horizons.
3. Results and discussion The soil profile was approximately 100 cm deep Žhorizon A, 30 cm; horizon B, 50 cm; horizon C, 20 cm.. The soil profile was underlined by intrusive rocks consisting of dense diorite and gabbro. The filtration properties of these intrusives were very low. The filtration coefficient was approximately 6 = 10y8 mrs, and the deeply located rocks were even more impermeable.
Table 1 Characteristics of the soil used in this study Parameters Chemical composition, % SiO 2 Al 2 O 3 Fe 2 O 3 CaO MgO K 2O Na 2 O S total S sulphidic humus Bulk density, grcm3 Specific density, grcm3 Porosity, % Moisture capacity, % Permeability, cmrs pH ŽH 2 O. Net neutralization potential, kg CaCO 3 rt
Horizon A Ž0–30 cm.
Horizon B Ž31–80 cm.
63.5 14.5 10.7 0.37 0.44 1.70 0.17 0.95 0.77 2.8 1.50 2.84 48 46 8=10y2 4.4 y145
67.1 14.0 8.0 0.32 0.28 1.40 0.32 0.82 0.77 1.2 1.55 3.05 44 41 6=10y2 4.6 y159
304
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The ground water level was located generally 5 to 10 m below the surface. The surface and ground waters in the site were well separated by the impermeable rocks. It was assumed that both the geologic and hydrogeologic conditions in the site were suitable for the application of in situ methods for soil remediation. Data about the chemical composition and some essential geotechnical parameters of the soil are shown in Table 1. The concentrations of contaminants were higher in the upper soil layers Žmainly in the horizon A. ŽTables 2 and 3.. The contaminants were present mainly in forms susceptible to biological andror chemical leaching but considerable portions were refractory to solubilization. The primary sulphide minerals were essential components of these inert fractions. The soil exhibited negative net neutralization potential, indicating a relatively high potential of acid generation. The permeability of the soil was high and rainwater infiltrated into and created conditions favorable for the dissolution of elements. The pore water quality was poor with high concentrations of contaminants such as radioactive elements Žuranium, radium, and thorium., some toxic heavy metals Žcopper, zinc, cadmium., free sulphuric acid and sulphates. Prior laboratory experiments with soil samples from the experimental plots treated in this study had shown that the dissolution of contaminants was connected with the activity of the indigenous soil microflora, mainly with the activity of acidophilic chemolithotrophic bacteria. These bacteria were able to oxidize sulphide minerals and to solubilize their metal components. Table 2 Toxic elements in the horizon A of the soil before and after the bioremediation Parameters
U
Content of toxic elements, ppm Before treatment 82 After treatment 23 BioaÕailable fraction, ppm Ža. By DTPA leaching Before treatment 12 After treatment 3.2 Žb. By EDTA leaching Before treatment 2.8 After treatment 0.5
Ra
Cu
Zn
Cd
640 190
230 68
170 51
3.5 1.2
120 25
41 2.8
35 2.4
0.7 0.02
45 10
17 1.2
14 1.2
0.3 0.01
Easily leachable fractions-exchangeableq carbonate, ppm Before treatment 14 140 After treatment 3.0 25
59 3.7
48 5.0
1.0 0.03
Inert fraction, ppm Before treatment After treatment
91 59
82 41
1.9 0.9
24 16
260 170
Toxic elements solubilized during the toxicity test, ppm Before treatment 1.45 0.35 After treatment 0.28 0.10 The contents of radium are shown in Bqrkg dry soil or Bqrl.
6.40 0.32
7.10 0.28
0.12 0.01
S.N. GroudeÕ et al.r Int. J. Miner. Process. 62 (2001) 301–308
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Table 3 Removal of contaminants from the polluted soil Parameters Content of contaminants in the soil before treatment Horizon A Horizon B Content of contaminants in the horizon A after treatment Content of contaminants in the horizon B after treatment By flushing By detoxification Removal of contaminants from the horizon A, % Removal of contaminants from the horizon B, % By flushing By detoxification Removal of contaminants from the whole soil profile Žhorizon Aqhorizon B., % By flushing By detoxification
U
Ra
Cu
Zn
Cd
82 37 23
640 300 190
230 90 68
170 120 51
49 65 71.9
410 530 70.3
87 166 70.4
93 172 69.9
1.4 3.1 65.7
23.0 9.7
20.8 7.0
41.1 11.2
44.2 9.9
47.8 6.1
27.1 8.2
23.4 6.2
43.9 9.5
44.3 8.6
46.2 5.1
3.5 1.9 1.2
The content of Ra is shown in Bqrkg dry soil; the content of all other contaminants is shown in mgrkg dry soil.
The analysis of the soil microflora revealed that it included a rich variety of microorganisms ŽTable 4.. The mesophilic, acidophilic bacteria related to the species Thiobacillus ferrooxidans, T. thiooxidans and Leptospirillum ferrooxidans were the prevalent microorganisms in the top soil layers, but some basophilic chemolithotrophic species Žmainly T. thioparus and T. denitrificans. and some heterotrophs were also present in high numbers. In the soil horizon B 2 , the total number of microorganisms was much lower than in the horizon A but various anaerobes were well present. It was found that under natural conditions the bacterial oxidation of sulphide minerals in the soil proceeded continuously but at relatively low rates. The number and activity of the acidophilic chemolithotrophs in the soil were limited by some essential environmental factors such as the relatively high soil pH, shortage of oxygen inside the soil horizons, insufficient soil moisture during relatively long periods of time, absence of some important nutrients such as nitrogen and phosphorus sources. The treatment of the contaminated soil was connected with increasing the number and activity of the indigenous microorganisms by suitable changes in the levels of the above-mentioned environmental factors. This was achieved by regular ploughing up and irrigation of the soil and by addition of some essential nutrients. The optimum soil humidity was about 50% of the moisture capacity of the soil, but periodic flushing with slightly acidified water ŽpH about 3.5–4.5. was needed to remove the soil contaminants. Zeolite saturated with ammonium phosphate was added to the soil Žin amounts of 2–5
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Table 4 Concentration of microorganisms related to different physiological groups in the soil during the treatment Microorganisms
Horizon A Ž0–30 cm.
Horizon B Ž31–80 cm. Treatment by
Aerobic heterotrophic bacteria Oligocarbophiles Cellulose-degrading microorganisms Nitrogen-fixing bacteria Nitrifying bacteria S 2 O 32y-oxidising chemolithotrophs Žat pH 7. S o-oxidising chemolithotrophs Žat pH 2. Fe 2q-oxidising chemolithotrophs Anaerobic heterotrophic bacteria Bacteria fermenting carbohydrates with gas production Denitrifying bacteria Sulphate-reducing bacteria Fe 3q-reducing bacteria Mn4q-reducing bacteria Methanogenic bacteria Streptomycetes Fungi Total cell numbers
Flushing
Detoxification
Cellsrg dry soil 10 5 –10 7 10 4 –10 6 10 2 –10 6 10 2 –10 5 10 2 –10 4 10 4 –10 7
10 4 –10 5 10 2 –10 4 10 2 –10 4 10 2 –10 4 10 1 –10 3 10 3 –10 5
10 4 –10 6 10 2 –10 5 10 2 –10 5 10 2 –10 4 10 1 –10 3 10 3 –10 5
10 5 –10 7
10 3 –10 5
10 2 –10 5
10 4 –10 7 10 3 –10 5 10 2 –10 3
10 2 –10 5 10 3 –10 5 10 2 –10 4
10 2 –10 4 10 4 –10 7 10 3 –10 5
10 2 –10 3 10 3 –10 4 10 2 –10 4 10 2 –10 4 0–10 2 10 2 –10 4 10 3 –10 6 1=10 7 –3=10 8
10 1 –10 3 10 2 –10 4 10 2 –10 4 10 2 –10 4 1–10 3 10 1 –10 3 10 2 –10 4 5=10 5 –1=10 7
10 3 –10 5 10 4 –10 7 10 3 –10 5 10 2 –10 5 10 1 –10 4 10 2 –10 4 10 3 –10 5 2=10 6 –6=10 7
The quantitative determination of the different physiological groups of microorganisms was carried out by the spread plate technique on solid nutrient media or by the most probable number method using end-point dilutions. The total number of microbial cells was determined by epifluorescence microscopy.
kgrt dry soil. to provide the microorganisms with ammonium and phosphate ions and to improve the physico-mechanical properties of the soil. The treatment of the soil was started in the middle of March 1997, and at the middle of November 1997, it was found that considerable portions of the contaminants were removed from the upper soil horizon A and their residual concentrations were lower or, at least, very close to the relevant permissible levels. The treatment caused some changes in the composition of the soil microflora increasing the number of the acidophilic chemolithotrophic bacteria and slightly decreasing the number of heterotrophs. The chemical composition, structure and main physical and water properties of the soil were altered to a small extent. In the experimental plot No. 1 treated by means of the flushing system, considerable portions of the contaminants were washed out from the soil profile by the soil effluents ŽTable 3.. However, the larger portions of the contaminants were accumulated in the horizon B 2 . Portions of the toxic heavy metals were present in this horizon as the relevant insoluble sulphides. A portion of the uranium was present as the mineral
S.N. GroudeÕ et al.r Int. J. Miner. Process. 62 (2001) 301–308
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Table 5 Sulphate-reducing bacteria in the experimental plots Sulphate-reducing bacteria
Plot No. 1
Plot No. 2
Desulfovibrio Desulfobulbus Desulfococcus Desulfobacter Desulfobacterium Desulfotomaculum Desulfosarcina
Cellsrg dry soil 3=10 2 –1=10 4 8=10 1 –3=10 3 5=10 1 –1=10 2 3=10 1 –5=10 2 – 1=10 1 –4=10 1 1=10 1 –6=10 1
1=10 4 –8=10 6 3=10 3 –8=10 5 3=10 2 –5=10 5 7=10 2 –7=10 4 8=10 1 –2=10 2 1=10 2 –5=10 3 5=10 2 –1=10 4
uraninite ŽUO 2 .. Both the sulphides and uraninite were formed as a result of microbial dissimilatory sulphate reduction taking place in this anoxic zone regardless of the low content of dissolved organic compounds serving as electron donors. The above-mentioned forms of the contaminants are relatively stable and refractory to leaching, especially under anaerobic conditions. Portions of the nonferrous metals and uranium as well as most of the radium were adsorbed on the clay minerals present in the horizon B2 . In the experimental plot No. 2 treated by means of the detoxification system, only small amounts of the contaminants were washed out from the soil profile by the soil effluents ŽTable 3.. The larger portions of the contaminants were retained in the horizon B 2 as a result of the very active process of microbial dissimilatory sulphate reduction, which was artificially enhanced by providing the indigenous sulphate-reducing bacteria with high concentrations of suitable electron donors. The population density of these bacteria in the experimental plot No. 2 was much higher than that in the experimental plot No. 1 ŽTables 4 and 5.. The concentrations of dissolved contaminants in the soil effluents from the experimental plot No. 2 were much lower than those from the experimental plot No. 1. Both types of effluents were treated efficiently by the constructed wetland. In 1998, the experimental plots were subjected to some conventional remediation procedures such as grassing of the treated soil, addition of suitable fertilizers and animal manure as well as with periodical ploughing up, liming and irrigation. As a result of this, the quality of the soil was completely restored. No soluble forms of the above-mentioned contaminants in concentrations higher than the relevant permissible levels were detected so far ŽApril 1999. in the soil pore and drainage waters after rainfall.
Acknowledgements A part of this study was funded by the National Science Fund ŽResearch Contracts No. TH-803r98, VRP-TH-9r99 and MY-X-09r96..
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