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desorption processes in soil amended with sewage sludge
Chemosphere 67 (2007) 724–730 www.elsevier.com/locate/chemosphere
Microbially mediated cadmium sorption/desorption processes in soil amended with sewage sludge Małgorzata Majewska a, Ewa Kurek a
a,*
, Jerzy Rogalski
b
Department of Environmental Microbiology, Institute of Microbiology and Biotechnology, University of Maria Curie-Sklodowska, Akademicka 19, 20-033 Lublin, Poland b Department of Biochemistry, University of Maria Curie-Sklodowska, Pl. Marii Curie-Sklodowskiej 3, 20-031 Lublin, Poland Received 7 April 2006; received in revised form 17 October 2006; accepted 19 October 2006 Available online 19 December 2006
Abstract A multi-compartment system was used to study the importance of microorganisms for Cd desorption from soil amended with sewage sludge and simultaneous resorption of the mobilized metal by soil constituents. Using this system made it possible to study the participation of microorganisms (Arthrobacter, Trichoderma), montmorillonite, humic acids, and iron oxides in resorption of the released Cd. A filter-sterilized water extract of root-free soil of pH 6.7 (RF) or RF supplemented with glucose (RFG) were used to mobilize Cd from soil at 14 C in 48 h. Cadmium found in those extracts after 48-h incubation was recognized as bioavailable. Changes in pH values and enrichment of soil extracts with organic acids and siderophores resulted from microbial growth. RFG with lower pH and a higher content of ligands mobilized, on average, 40% of Cd introduced with sewage sludge amended soil, whereas RF mobilized only 20% of it. Sequential extractions of Cd at time 0 and Cd remaining in soil showed that RFG had mobilized Cd mostly from the fraction bound with Fe and Mn oxides. Microbial biomass accounted for only up to 3.4% (w/w) of the soil constituents used in the experiments but resorbed 25% of mobilized Cd. The chemical composition of mobilizing soil extracts and the solid-to-mobilizing-extracts volume ratio had a significant effect on the amount of bioavailable Cd. The results of the study suggest that microbial metabolites were involved in Cd mobilization, while the biomass of microorganisms was involved in Cd resorption as a biosorbent. 2006 Elsevier Ltd. All rights reserved. Keywords: LMWOAs; Siderophores; Cd speciation; Mobilization; Resorption; Soil constituents
1. Introduction Adsorption and desorption processes occurring in natural soil simultaneously control the mobility and the bioavailability of a metal (Christensen and Huang, 1999). The properties influencing the concentration of Cd in the aqueous phase of a soil include the soil’s pH values, its redox potential, soil texture, its mineral composition (including the proportion of clays, iron, and manganese oxides), its cation-exchange capacity, the amount and type
*
Corresponding author. Tel.: +48 81 5375920; fax: +48 81 537 5959. E-mail address: [email protected] (E. Kurek).
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.10.051
of organic compounds present in the soil and its aqueous phase, heavy metals composition (other heavy metals may compete for adsorption sites), the temperature, the moisture content, and the numbers and types of microorganisms inhabiting the soil (Ledin, 2000; Kabata-Pendias and Sadurski, 2004). Microorganisms are very efficient heavy metal sorbents and their growth can also significantly affect many of the physico-chemical soil properties. The largest number and highest physiological activity of microorganisms in soil occur in rhizosphere enriched with root exudates, so their effect on heavy metal bioavailability can be robust (Huang and Germina, 2002). Studies have been conducted to determine the kinetics of microbial transformation of Cd added as a defined chemical
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compound to liquid media (Ledin et al., 1996, 1999), soil suspensions (Chanmugathas and Bollag, 1987), soil (Kurek and Bollag, 2004), and a continuously leached acid sandy soil [pH 4.0] (Chanmugathas and Bollag, 1988). All these experiments, however, have been conducted under conditions unsuitable for obtaining information about the influence of microorganisms on changes in (1) the composition of soil solution, (2) chemical speciation of Cd occurring in soil, and (3) distribution of Cd between constituents of the soil solid phase. Meanwhile, these are chemical speciation and affinity for individual soil components that play an important role in the transfer of the metal between the solid phase and soil solution (Shrivastava and Banerjee, 2004). The aim of the present study was to determine the effect of microbial activity on a bioavailable pool of cadmium in sewage sludge amended soil (SS soil). The experiments were conducted under conditions mimicking the soil system, using a multi-compartment system PIGS (Partitioning in Geobiochemical Systems) (Calmano et al., 1992; Ledin et al., 1996). The use of PIGS made it possible to study, for the first time, the influence of microbial activity on the pH and solute composition, the factors affecting the mobility of Cd. Also, simultaneous distribution of Cd mobilized from the SS soil among the soil solid phase components (fungal and bacterial biomasses, humic acids, iron hydroxides, goethite, and montmorillonite) was studied and the interaction effect between the different soil factors was estimated. 2. Materials and methods
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water, re-suspended in water, and stored at 4 C prior to use in the experiments. The experiments used sterile suspensions of abiotic components. 2.2. Soils used in the experiments A sandy loam soil (SL soil) was used in the studies. Prior to the experiments, the soil was air-dried at room temperature and passed through a 2-mm sieve. The SL soil was amended with sewage sludge from the Municipal Plant ‘‘Hajdo´w’’ in Lublin (Poland) in an amount corresponding to a dose of 200 t per ha (SS soil). Before it was used in the experiments, the SS soil was incubated for six months at natural field temperatures in the Lublin region (from September 15th to March 15th). The properties of both the soils, presented in Table 1, were analyzed by standard methods described by Alef and Nannipieri (1995). The sludge used in the experiments contained an average of 62 ± 16 mg Cd kg 1 and other heavy metals as follows (mg kg 1): Cr 127; Zn 2379; Cu 232; Ni 127; Pb 69. The results of a sequential extraction of the sewage sludge used to amend the soil indicated that 3% of its total Cd was in the exchangeable fraction, 70% was bound to Fe and Mn oxides, 21% was in the organic fraction, and 6% was residual. The sewage sludge was very heterogeneous with respect to the heavy metals content so the total cadmium Table 1 Characteristics of the SL soil and the SS soil Characteristic
SL soil
SS soil
pH (H2O)
6.5 ± 0.1
6.7 ± 0.1
0.28 0.62 0.42 3.51
0.13 0.38 0.96 6.93
Deionized Milli-Q water (Millipore, Billerica, Mass., USA) was used throughout the study. All glassware and plastic containers were soaked for at least 2 h in 7.5 M HNO3 and rinsed thoroughly with deionized water before use.
CEC (meq 100 g Na+ K+ Mg2+ Ca2+
2.1. Soil components
Total C (g kg 1of soil) Total N (g kg 1of soil)
8.0 0.7
13.0 1.6
Soil texture (%) Sand (1.0–0.1 mm) Silt (0.1–0.02 mm) Clay (<0.02 mm)
58 24 18
55 27 18
1.0 ± 0.3
5.2 ± 1.0
Characteristics of Arthrobacter LII and Trichoderma koningii 3Ag0 strains, methods of their cultivation, harvesting of biomass, and determination of the number of colony-forming unit (cfu) had been described earlier by Kurek and Majewska (1998, 2004). Montmorillonite (bentonite containing 75% of montmorillonite) was obtained from Mining and Metallurgical Works in Ze˛biec near Ostrowiec S´wie˛tokrzyski (Poland) and used in the experiments in a form homoionic to Na. Humic acids were obtained from Aldrich. They were initially dissolved in deionized water and, then, the solution was acidified by means of 0.5 M HCl to pH 2. The sediment of humic acids was rinsed three times with deionized water, separated by filtration, and dried at 60 C. A commercially available preparation of goethite (a-FeOOH, Aldrich) was used in the experiments. Fe(OH)3 was prepared according to the procedure described by Jones et al. (1994). Fe(OH)3 was rinsed three times with deionized
a
Total Cd (mg kg
1
1
of soil)
of soil)
Contents of Cd fractions (% of total Cd) Soluble and exchangeable 4 ± 4.0 Bound to Fe and Mn 26 ± 9.2 oxides Bound to organics 43 ± 12.1 Residual 27 ± 24.4 Number of microorganisms (cfu g 1 of soil) Oligotrophs 2.5 · 105 ± 0.8 · 105 Copiotrophs 2.4 · 106 ± 0.9 · 106 Fungi 1.3 · 103 ± 0.6 · 103
9 ± 5.6 73 ± 3.7 9 ± 1.5 9 ± 1.5 1.1 · 107 ± 0.3 · 107 4.7 · 107 ± 0.3 · 105 9.1 · 104 ± 3.0 · 104
Results are mean values of three replicates ± standard deviation. a Cation-exchange capacity.
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concentration in SS soil samples was determined each time before the experiment started.
The RF extract (soil:water ratio, 1:1 w/v) was prepared according to the procedure described by Kurek and Majewska (2004). Chemical analysis indicated that this extract contained an average of 2 ± 0.6 lg ml 1 of reducing sugars, 43 ± 18.7 lg ml 1 of Fe(III)-chelators, 4 ± 0.8 lg ml 1 of hydroxamate siderophores, 8 ± 0.1 lg ml 1 of catechole siderophores, 14 ± 1.0 lg ml 1 of citric acid, and 2 ± 0.6 lg ml 1 of acetic acid. Before use in the experiments, RF was sterilized by filtration (membrane filter pore of 0.22 lm). The amount of glucose (13.7 g l 1) added to RF to get RFG corresponded to the sum of all the sugars (root exudates) per kg of soil contained in a 1 mm zone of the rhizosphere of maize (Krafftczyk et al., 1984).
by the plate method. Changes were also determined in the pH and in the concentration of citric and acetic acids as well as Fe(III)-chelators present in the soil extracts during the incubation period. The changes in pools of Cd during the incubation period were expressed as changes in the percent of total metal concentration in the SS soil at time 0. A system consisting of the central chamber and two side chambers (PIGS II) was used in some experiments to simulate bidirectional interaction between soil constituents and microorganisms occurring in natural soil (Chenu and Stotzky, 2002). RF or RFG was introduced to the central chamber. SS soil suspension in an appropriate soil extract was introduced to one side chamber while the other chamber was filled with a suspension of a mixture of all the tested soil constituents in an appropriate soil extract. All other experimental conditions were the same as for PIGS I.
2.4. Cadmium sorption and desorption experiments
2.5. Sequential extraction of total Cd
The multi-compartment system (PIGS I) used in the study consisted of one central chamber and six side chambers. Membrane filters (Isopore, Millipores; pore size 0.4 lm) were placed between the central and the side chambers to separate the solid phases while allowing an exchange of the aqueous phase. This made it possible to determine the simultaneous distribution of Cd released from the SS soil (introduced to one of the side chambers) between various soil constituents placed in other side chambers. Forty milliliter of SS soil suspension or a soil constituent in soil extracts (RF or RFG) was placed in each of the side chambers, and 100 ml of RF or RFG was introduced into the central chamber. The content of each chamber was stirred (250 rpm) with teflon-coated magnetic stirring bars. The complete mixing and exchange of solutions between the compartments of the system was achieved after 24 h of stirring; however, the contact of RF and RFG with the solids in PIGS I was extended up to 48 h. The pH values of soil extracts at time 0 were adjusted to the pH representing the values of the soil. The amounts of the tested solid phase constituents were as follows: montmorillonite – 2000 mg; humic acids – 800 mg; a mixture of goethite and Fe(OH)3 in a 1:1 ratio, (GFe) – 400 mg; bacterial biomass – 34 mg; and fungal biomass – 78 mg. The relative proportion between soil constituents was in the range of that found in sandy loam soils (Kurek et al., 1982; Paul and Clark, 1998). The amount of soil (3.31 g) used in the experiment was the sum of the amounts of all soil components introduced to PIGS I. Changes in the total Cd concentration were determined in the SS soil, in the circulating RF or RFG, and in individual soil constituents after 48 h of incubation at 14 C (an average temperature for the spring– summer period in central-eastern Poland). Changes in the cfu number of bacteria and fungi colonizing the SS soil, as well as those used as soil constituents (Arthrobacter and Trichoderma) during the incubation were estimated
The SS soil, before and after incubation in the PIGS systems, and the mixture of soil constituents used in the experiments with PIGS II were subjected to sequential extraction according to the procedure described by Keller and Vedy (1994).
2.3. Soil extracts
2.6. Cd determination The procedure used for determining the total amount of Cd in RF or RFG, as well as Cd immobilized by individual sorbents and Cd fractions released by sequential extraction from the SS soil and from the mixture of soil constituents (PIGS II) by an atomic absorption spectrophotometer (Unicam 939AA Spectrometer) had been described by Kurek and Majewska (2004). The Cd standards were prepared from a stock solution of Cd(NO3)2 in 5% of HNO3. 2.7. Microbial products Concentrations of Fe(III)-chelators and organic acids were determined in RF or RFG introduced to the system at time 0 and at the end of the experiments. 2.7.1. The Fe(III)-chelators The total amount of Fe(III)-chelators was measured in reaction with FeCl3 using desferrioxamine B as the standard (Gibson and Magrath, 1986). Hydroxamate siderophores were determined by the Csaky (1948) method with NH2OH Æ HCl as the standard. The concentration of catechol siderophores was determined by the Arnow (1937) method with 3,4-dihydrohybenzoic acid as the standard. 2.7.2. Organic acids The samples of RF or RFG were filtered through a 0.2 lm HPLC filter (Millipore, Billeria, Mass, USA) before determination of low molecular weight organic acids
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(LMWOAs) such as citric and acetic acid. The measurement was done by the HPLC method on a VP chromatographic system (Shimadzu, Tokio, Japan) composed of a LC-10AD pump, a RID-10A refractive index detector, a SCL-10A controller, a CTO-10AS oven (all of which were controlled by Class VP 5.03 Workstation Software; Shimadzu, 1999) and a Model 7725 sampling valve (Rheodyne, Berkeley, USA) with a 20 ll loop. The mobile phase (5 mM H2SO4 in Milli-Q water) was run at a flow rate of 0.5 ml min 1 at 55 C through a Rezex Organic Acid column (8 lm; 300 · 7.8 mm, Phenomenex, Torrence, USA). Calibration of the column was carried out using organic acids standards (Sigma. St. Louis, USA). 2.8. Statistical data analysis Statistical analysis was performed on three replicates from each treatment. Standard deviations and analysis of variance were determined using Microsoft Excel 2000 (Armitage and Berry, 1987). 3. Results and discussion 3.1. The effect of amending soil with sewage sludge In agreement with earlier reports (Moreno et al., 1999; Shrivastava and Banerjee, 2004), amendment of soil with sewage sludge resulted in changes of all the tested physico-chemical properties of the SL soil, increased the cfu number of soil microorganisms, and also affected distribution of Cd between operational fractions (Table 1). The Cd fraction bound to Fe and Mn oxides dominated in sludge and in the SS soil, and amounted to 70% and 73%, respectively. Iron (hydr)oxides are known to accumulate heavy metals by adsorption and/or coprecipitation in rivers and their estuaries. Studies of metal mobility in lake sediments strongly suggest that cadmium binds directly to hydroxyl surface groups of iron and manganese oxyhydroxides such as ferrihydrite and lepidocrocite under circumneutral conditions (Randall et al., 1999). 3.2. Growth of microorganisms Proliferation of bacteria inhabiting the SS soil and those introduced to the PIGS systems as soil constituents occurred during the 48-h incubation with RF. While no increase in the cfu number of soil fungi was observed, T. koningii introduced to the systems as a soil constituent was multiplied very intensively (from 7 · 102 ± 0.9 · 102 cfu ml 1 up to 9 · 104 ± 5.6 · 104 cfu ml 1). Enrichment of the soil extract with glucose had no effect on the cfu number of microorganisms introduced to the systems as soil constituents and soil fungi. Addition of glucose to RF resulted in an increase in the cfu number of soil bacteria from 1 · 107 ± 7.4 · 105 to 3 · 107 ± 2.1 · 106 cfu ml 1 in PIGS I; and, conversely, a decrease from 8 · 107 ± 3.1 · 106 to 1 · 107 ± 3.8 · 106 cfu ml 1 in PIGS II.
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Chemical analyses of RFG at time 0 and after incubation indicated that the content of glucose (or reducing sugars) decreased by 40%. 3.3. Cd mobilization in PIGS Soil extracts circulating in the PIGS systems mobilized from 20% to 43% of total Cd from the SS soil (Fig. 1a). The amounts of Cd mobilized from the SS soil by RFG were significantly larger than those with RF. There were no significant differences in the amounts of Cd mobilized from the SS soil by RF when soil constituents were placed in the chambers individually (PIGS I) or all together as a mixture (PIGS II). However, the disposal of soil constituents in the multi-chamber system affected the amounts of Cd mobilized from the SS soil by RFG (Fig. 1a). More Cd was mobilized when soil constituents were introduced to the chambers one by one [PIGS I] (43% ± 3.7%) than when they were introduced as a mixture [PIGS II] (36% ± 2.0%). Chemical analysis of soil extracts circulating in PIGS after incubation indicated that enrichment of RF with glucose and the disposal of soil constituents had an effect on their pH values (Fig. 1c) and content of other tested compounds such as citric and acetic acids, and Fe(III)-chelators. The experimental conditions in PIGS I and PIGS II were different with respect to total weight of solid-to-circulating-extracts volume ratio. The value of this ratio was 20 g l 1 in PIGS I but 47 g l 1 in PIGS II. This fact was one of the reasons for higher concentrations of the tested metabolites in extracts circulating in PIGS II than in PIGS I (date not shown). An addition of glucose to RF resulted in an enhanced release of metabolites to the aqueous phase, e.g., in PIGS II concentrations of citric acid increased from 24 to 72 lg ml 1, acetic acid from 0 to 28 lg ml 1, total Fe(III)-chelators from 100 to 200 lg ml 1, and catechole siderophores from 5 to 20 lg ml 1. Glucose was added to RF in order to stimulate microbial growth and activity. Enrichment of soil solution with low molecular nutrient substrates originating from root exudates is natural to plant rhizosphere (Huang and Germina, 2002). The enhanced Cd-mobilizing efficiency of RFG could be related to its lower pH and higher contents of microbial products synthesized during incubation compared to RF. The more effective release of Cd in PIGS I in comparison with PIGS II was probably connected with a lower solid/solution ratio in PIGS I which guaranteed an easier contact between the sorbed metal and the metabolites present in the solution. Sequential extraction of Cd remaining in the SS soil after its mobilization by soil extracts indicated that the mobilizing efficiency of RFG was twice as high as that of RF; and RFG mobilized Cd mostly from the operational fraction bound to Fe and Mn oxides (Fig. 2). The experiments indicated that RF, and even more so, RFG circulating in the PIGS systems were rich in metal complexing compounds including siderophores and organic acids. Both LMWOAs identified in RF and RFG, besides lowering the
M. Majewska et al. / Chemosphere 67 (2007) 724–730
% of total Cd in SS soil
728
100 PIGS I
PIGS II
80 60 40 20 0 RF
RF
RFG remaining in soil mobilized
[
RFG in aqueous phase
+
]
resorbed
% of mobilized Cd
70 60
RF
50 40
RFG
30 20 10 0
Bacterial biomass
Fungal biomass
Humic acids
Montmorillonite
GFe
8 PIGS I
7
PIGS II
6
pH
5 4 3 RF
RFG
RF
RFG
Fig. 1. Cadmium mobilization from the SS soil and total resorption by soil constituents expressed as percent of Cd introduced with the SS soil (a); resorption of mobilized Cd by individual soil constituents (b); changes in pH (c) after 48-h incubation with RF or RFG.
pH of the soil solution, are also known to be able to form complexes with metals. Siderophores – highly specific chelators of Fe3+ – were produced by microorganisms and released into the soil extract during incubation. This was the probable cause of the dissolution of soil iron minerals mobilizing Cd bound to them. Dissolution of iron oxides in the presence of siderophores is well documented (Violante et al., 2002). 3.4. Resorption of mobilized Cd Cadmium released from the SS soil to the aqueous phase was in part resorbed by soil constituents introduced into side chambers (from 15% to 36%), and some of it (from 0% to 7%) remained soluble in soil extracts and can be considered bioavailable (Fig. 1a). The metal released from the SS soil by RF (pH 6.4) was resorbed in PIGS I mostly by montmorillonite (66%) and bacterial biomass (16%). Less Cd was resorbed by other abiotic components: GFe (9%) and humic acids (6%). Fungal biomass was a less efficient sorbent than bacterial biomass (Fig. 1b). Cadmium sorption efficiency of some of the tested soil constituents
changed dramatically when Cd was released from the SS soil by RFG (pH 5.0): the percentage of Cd resorbed by humic acids increased to 46%, while the percentage sorbed by montmorillonite decreased to 19%. The resorption efficiency of fungal biomass was also significantly altered. Biomass of T. koningii sorbed 19% of Cd immobilized by soil constituents compared to only 2% sorbed from RF (Fig. 1b). Participation of bacterial biomass and GFe in resorption of Cd released from the SS soil was independent of the soil extract used (RF or RFG). The simultaneous sorption of Cd by soil constituents from a solution containing Zn, Cd, and Hg as chlorides had been studied by Ledin et al. (1996, 1999). These authors used Pseudomonas putida or Bacillus subtilis and Trichoderma harzianum biomass as biotic soil constituents, kaolinite, Al2O3, quartz and goethite as inorganic soil constituents, and peat as an abiotic organic soil component. The results of both of those works, and the present study alike, indicate that the pH, the chemical composition of the medium circulating in PIGS, as well as the type and proportions of the soil constituents used in a study can significantly affect metal sorption efficiency by individual soil
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Fig. 2. Changes of Cd speciation in the SS soil incubated for 48-h in the PIGS systems with RF (a) or RFG (b). Inserts represent speciation of Cd mobilized from the SS soil by RF or RFG and resorbed by the mixture of soil constituents. Bars marked with the same letters are not significantly different (P > 0.05).
constituents. However, while Ledin et al. (1999) found a significant increase in sorption efficiency of Zn and Hg, but not Cd, by T. harzianum in response to the lowering of the pH of the medium circulating in PIGS, the present study indicates that also immobilization of Cd by proliferating fungal biomass increases when RF (pH 6.4) is replaced by RFG (pH 5.0). The differing effects of low pH on Cd sorption efficiency by fungal biomass found in the present study and that by Ledin et al. (1999) may be connected with differences in the composition of the solution circulating in PIGS, as well as the physiological state and species of fungus used in the studies. In spite of the experimental differences between the study by Ledin et al. (1999) and the study by the present authors, the importance of microorganisms for sorption of Cd at various pH values, despite their much lesser content in soil as compared to other solid components, is clearly evidenced. Microbial biomass resorbed 19% of Cd at pH 6.4 and 26% at pH 5.0 (Fig. 1b). The two microorganisms, T. harzianum and B. subtilis used by Ledin et al. (1999), which together constituted 1.7% of solid soil mass, accumulated under various conditions from 1% to 16% of added Cd. PIGS II, in which all the tested soil constituents were placed together as a mixture in one chamber, was also used in the experiments. It was found that the metal mobilized
from the SS soil was resorbed evenly into all operational fractions separated from the mixture of soil constituents; however, the mixture resorbed three times more Cd mobilized by RFG than that mobilized by RF (Fig. 2). In natural soil, all solid phase constituents are in contact. Interactions between soil constituents and microorganisms are bidirectional. Soil particles influence the survival and biological activity of microorganisms while microorganisms affect soil particles by modifying their arrangement and producing a new surface with specific properties (Chenu and Stotzky, 2002). The composition of the soil extract circulating in PIGS and the disposal of soil constituents in the system affected the size of the bioavailable Cd pool. The percentage of Cd remaining in the RFG circulating in PIGS I (bioavailable) was significantly higher than those with RF. Yet in PIGS II, the percentage of bioavailable Cd decreased significantly when RFG was used instead of RF (Fig. 1a). The results of the present studies indicate that low pH values and high contents of metal chelators in RFG circulating in PIGS II do not inhibit resorption of Cd mobilized from the SS soil by soil constituents. This finding contradicts the generally accepted opinion that both these factors enhance mobilization of heavy metals from soil and prevent their immobilization (Huang and Germina, 2002). We did not determine Cd forms present
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in RF or RFG after incubation with SS soil or the fate of elements other than cadmium, such as Zn, Ni, Pb, Cr or Cu, which were introduced to the soil with sewage sludge. However, interactions between these heavy metals, microbial metabolites synthesized during incubation, and soil constituents probably affected Cd sorption and desorption processes. In conclusion, the present study indicates that microorganisms (biomass and activity) can strongly affect the size of the bioavailable Cd pool in a multimetallic environment created by introduction of sewage sludge to the soil. This phenomenon may be pronounced in rhizosphere with the highest number and activity of microorganisms in the soil. Acknowledgements This work was supported by grant 6 P04C 045 21 from the State Committee for Scientific Research. References Alef, K., Nannipieri, P., 1995. Method in Applied Soil Microbiology and Biochemistry. Academic Press, London. Armitage, P., Berry, G., 1987. Statistical Methods in Medical Research. Blackwell, Oxford. Arnow, L.E., 1937. Colorimetric determination of the components of 3,4dihydroksyphenylalanine – tyrosine mixtures. J. Biol. Chem. 228, 531– 537. Calmano, W., Ahlf, W., Bening, J.-C., 1992. Chemical mobility and bioavailabilty of sediment-bound heavy metals influenced by salinity. Hydrologia (235/236), 605–610. Chanmugathas, P., Bollag, J.-M., 1987. Microbial role in immobilization and subsequent mobilization of cadmium in soil suspensions. Soil Sci. Soc. Am. J. 51, 1184–1191. Chanmugathas, P., Bollag, J.-M., 1988. A column study of the biological mobilization and speciation of cadmium in soil. Arch. Environ. Contam. Toxicol. 17, 229–237. Chenu, C., Stotzky, G., 2002. Interaction between microorganisms and soil particles: an overview. In: Interactions between Soil Particles and Microorganisms. Impact on the Terrestial Ecosystem. In: Huang, P.M., Bollag, J.-M., Senessi, N. (Eds.), IUPAC Series on Analytical and Physical Chemistry of Environmental Systems, vol. 8. John Wiley & Sons, Ltd., Chichester, West Sussex, England, pp. 3–40. Christensen, T.H., Huang, P.M., 1999. Solid phase cadmium and the reaction of aqueous cadmium with soil surface. In: McLaughlin, M.J., Singh, B.R. (Eds.), Cadmium in Soil and Plants. Kluwer Academic Publishing, Dordrecht, The Netherlands, pp. 65–96. Csaky, T.Z., 1948. On the estimation of bound hydroxylamine in biological materials. Acta Chem. Scand. 2, 370–386. Gibson, F., Magrath, D.I., 1986. The isolation and characterization of hyroxamic acid (aerobactin) formed by Aerobacter aerogenes. Biochim. Biophys. Acta 192, 164–175.
Huang, P.M., Germina, J.J., 2002. Chemical and biological processes in the rhizosphere: metal pollutants. In: Interactions between Soil Particles and Microorganisms. Impact on the Terrestial Ecosystem. In: Huang, P.M., Bollag, J.-M., Senessi, N. (Eds.), IUPAC Series on Analytical and Physical Chemistry of Environmental Systems, vol. 8. John Wiley & Sons, Ltd., Chichester, West Sussex, England, pp. 381– 438. Jones, D.L., Edwards, A.C., Donachie, K., Darrah, P.R., 1994. Role of proteinaceous amino acids released in root exudates in nutrient acquisition from the rhizosphere. Plant Soil 158, 183–192. Kabata-Pendias, A., Sadurski, W., 2004. Trace elements and compounds in soil. In: Merian, E., Anke, M., Ihnat, M., Stoeppler, M. (Eds.), Elements and their Compounds in the Environment: Occurrence. Analysis and Biological Relevance, General Aspects, vol. 1. John Wiley & Sons, Ltd Chichester, West Sussex, England, pp. 79–100. Keller, C., Vedy, J.-C., 1994. Heavy metals in the environmental. Distribution of copper and cadmium fractions in two forest soils. J. Environ. Qual. 23, 987–999. Krafftczyk, I., Trolldenier, G., Beringer, H., 1984. Soluble root exudates of maize: influence of potassium supply and rhizosphere microorganisms. Soil Biol. Biochem. 16, 315–322. Kurek, E., Bollag, J.-M., 2004. Microbial immobilization of cadmium released from CdO in the soil. Biogeochemistry 69, 227–239. Kurek, E., Majewska, M., 1998. Release of Cd immobilized by soil constituents and its bioavailibility. Toxicol. Environ. Chem. 67, 237– 249. Kurek, E., Majewska, M., 2004. In vitro remobilization of Cd immobilized by fungal biomass. Geoderma 122, 235–246. Kurek, E., Czaban, J., Bollag, J.-M., 1982. Sorption of cadmium by microorganisms in competition with other soil constituents. Appl. Environ. Mircobiol. 43, 1011–1015. Ledin, M., 2000. Accumulation of metals by microorganisms – processes and importance for soil systems. Earth-Sci. Rev. 51, 1–31. Ledin, M., Krantz-Ru¨lcker, C., Allard, B., 1996. Zn, Cd and Hg accumulation by microorganisms, organic and inorganic soil component in multi-compartment systems. Soil Biol. Biochem. 28, 791–799. Ledin, M., Krantz-Ru¨lcker, C., Allard, B., 1999. Microorganisms as metal sorbents: comparison with other soil constituents in multi-compartment systems. Soil Biol. Biochem. 31, 1639–1648. Moreno, J.L., Herna´ndez, T., Garcia, C., 1999. Effects of a cadmiumcontaminated sewage sludge compost on dynamics of organic matter and microbial activity in an arid soil. Biol. Fertil. Soils 28, 230–237. Paul, E.A., Clark, F.E., 1998. Soil Microbiology and Biochemistry. Academic Press, USA. Randall, S.R., Sherman, D.M., Ragmarsdottir, K.V., Collins, C.R., 1999. The mechanism of cadmium surface complexation on iron oxyhydroxide minerals. Geochim. Cosmochim. Acta 63, 2971–2987. Shrivastava, S.K., Banerjee, D.K., 2004. Speciation of metals in sewage sludge and sludge-amended soils. Water Air Soil Pollut. 152, 219–232. Violante, A., Krishnamurti, G.S.R., Huang, P.M., 2002. Impact of organic substances on the formation and transformation of metal oxides in soil environments. In: Interactions between Soil Particles and Microorganisms. Impact on the Terrestial Ecosystem. In: Huang, P.M., Bollag, J.-M., Senessi, N. (Eds.), IUPAC Series on Analytical and Physical Chemistry of Environmental Systems, vol. 8. John Wiley & Sons, Ltd., Chichester, West Sussex, England, pp. 133–188.
Microbially mediated cadmium sorption/desorption processes in soil amended with sewage sludge Małgorzata Majewska a, Ewa Kurek a
a,*
, Jerzy Rogalski
b
Department of Environmental Microbiology, Institute of Microbiology and Biotechnology, University of Maria Curie-Sklodowska, Akademicka 19, 20-033 Lublin, Poland b Department of Biochemistry, University of Maria Curie-Sklodowska, Pl. Marii Curie-Sklodowskiej 3, 20-031 Lublin, Poland Received 7 April 2006; received in revised form 17 October 2006; accepted 19 October 2006 Available online 19 December 2006
Abstract A multi-compartment system was used to study the importance of microorganisms for Cd desorption from soil amended with sewage sludge and simultaneous resorption of the mobilized metal by soil constituents. Using this system made it possible to study the participation of microorganisms (Arthrobacter, Trichoderma), montmorillonite, humic acids, and iron oxides in resorption of the released Cd. A filter-sterilized water extract of root-free soil of pH 6.7 (RF) or RF supplemented with glucose (RFG) were used to mobilize Cd from soil at 14 C in 48 h. Cadmium found in those extracts after 48-h incubation was recognized as bioavailable. Changes in pH values and enrichment of soil extracts with organic acids and siderophores resulted from microbial growth. RFG with lower pH and a higher content of ligands mobilized, on average, 40% of Cd introduced with sewage sludge amended soil, whereas RF mobilized only 20% of it. Sequential extractions of Cd at time 0 and Cd remaining in soil showed that RFG had mobilized Cd mostly from the fraction bound with Fe and Mn oxides. Microbial biomass accounted for only up to 3.4% (w/w) of the soil constituents used in the experiments but resorbed 25% of mobilized Cd. The chemical composition of mobilizing soil extracts and the solid-to-mobilizing-extracts volume ratio had a significant effect on the amount of bioavailable Cd. The results of the study suggest that microbial metabolites were involved in Cd mobilization, while the biomass of microorganisms was involved in Cd resorption as a biosorbent. 2006 Elsevier Ltd. All rights reserved. Keywords: LMWOAs; Siderophores; Cd speciation; Mobilization; Resorption; Soil constituents
1. Introduction Adsorption and desorption processes occurring in natural soil simultaneously control the mobility and the bioavailability of a metal (Christensen and Huang, 1999). The properties influencing the concentration of Cd in the aqueous phase of a soil include the soil’s pH values, its redox potential, soil texture, its mineral composition (including the proportion of clays, iron, and manganese oxides), its cation-exchange capacity, the amount and type
*
Corresponding author. Tel.: +48 81 5375920; fax: +48 81 537 5959. E-mail address: [email protected] (E. Kurek).
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.10.051
of organic compounds present in the soil and its aqueous phase, heavy metals composition (other heavy metals may compete for adsorption sites), the temperature, the moisture content, and the numbers and types of microorganisms inhabiting the soil (Ledin, 2000; Kabata-Pendias and Sadurski, 2004). Microorganisms are very efficient heavy metal sorbents and their growth can also significantly affect many of the physico-chemical soil properties. The largest number and highest physiological activity of microorganisms in soil occur in rhizosphere enriched with root exudates, so their effect on heavy metal bioavailability can be robust (Huang and Germina, 2002). Studies have been conducted to determine the kinetics of microbial transformation of Cd added as a defined chemical
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compound to liquid media (Ledin et al., 1996, 1999), soil suspensions (Chanmugathas and Bollag, 1987), soil (Kurek and Bollag, 2004), and a continuously leached acid sandy soil [pH 4.0] (Chanmugathas and Bollag, 1988). All these experiments, however, have been conducted under conditions unsuitable for obtaining information about the influence of microorganisms on changes in (1) the composition of soil solution, (2) chemical speciation of Cd occurring in soil, and (3) distribution of Cd between constituents of the soil solid phase. Meanwhile, these are chemical speciation and affinity for individual soil components that play an important role in the transfer of the metal between the solid phase and soil solution (Shrivastava and Banerjee, 2004). The aim of the present study was to determine the effect of microbial activity on a bioavailable pool of cadmium in sewage sludge amended soil (SS soil). The experiments were conducted under conditions mimicking the soil system, using a multi-compartment system PIGS (Partitioning in Geobiochemical Systems) (Calmano et al., 1992; Ledin et al., 1996). The use of PIGS made it possible to study, for the first time, the influence of microbial activity on the pH and solute composition, the factors affecting the mobility of Cd. Also, simultaneous distribution of Cd mobilized from the SS soil among the soil solid phase components (fungal and bacterial biomasses, humic acids, iron hydroxides, goethite, and montmorillonite) was studied and the interaction effect between the different soil factors was estimated. 2. Materials and methods
725
water, re-suspended in water, and stored at 4 C prior to use in the experiments. The experiments used sterile suspensions of abiotic components. 2.2. Soils used in the experiments A sandy loam soil (SL soil) was used in the studies. Prior to the experiments, the soil was air-dried at room temperature and passed through a 2-mm sieve. The SL soil was amended with sewage sludge from the Municipal Plant ‘‘Hajdo´w’’ in Lublin (Poland) in an amount corresponding to a dose of 200 t per ha (SS soil). Before it was used in the experiments, the SS soil was incubated for six months at natural field temperatures in the Lublin region (from September 15th to March 15th). The properties of both the soils, presented in Table 1, were analyzed by standard methods described by Alef and Nannipieri (1995). The sludge used in the experiments contained an average of 62 ± 16 mg Cd kg 1 and other heavy metals as follows (mg kg 1): Cr 127; Zn 2379; Cu 232; Ni 127; Pb 69. The results of a sequential extraction of the sewage sludge used to amend the soil indicated that 3% of its total Cd was in the exchangeable fraction, 70% was bound to Fe and Mn oxides, 21% was in the organic fraction, and 6% was residual. The sewage sludge was very heterogeneous with respect to the heavy metals content so the total cadmium Table 1 Characteristics of the SL soil and the SS soil Characteristic
SL soil
SS soil
pH (H2O)
6.5 ± 0.1
6.7 ± 0.1
0.28 0.62 0.42 3.51
0.13 0.38 0.96 6.93
Deionized Milli-Q water (Millipore, Billerica, Mass., USA) was used throughout the study. All glassware and plastic containers were soaked for at least 2 h in 7.5 M HNO3 and rinsed thoroughly with deionized water before use.
CEC (meq 100 g Na+ K+ Mg2+ Ca2+
2.1. Soil components
Total C (g kg 1of soil) Total N (g kg 1of soil)
8.0 0.7
13.0 1.6
Soil texture (%) Sand (1.0–0.1 mm) Silt (0.1–0.02 mm) Clay (<0.02 mm)
58 24 18
55 27 18
1.0 ± 0.3
5.2 ± 1.0
Characteristics of Arthrobacter LII and Trichoderma koningii 3Ag0 strains, methods of their cultivation, harvesting of biomass, and determination of the number of colony-forming unit (cfu) had been described earlier by Kurek and Majewska (1998, 2004). Montmorillonite (bentonite containing 75% of montmorillonite) was obtained from Mining and Metallurgical Works in Ze˛biec near Ostrowiec S´wie˛tokrzyski (Poland) and used in the experiments in a form homoionic to Na. Humic acids were obtained from Aldrich. They were initially dissolved in deionized water and, then, the solution was acidified by means of 0.5 M HCl to pH 2. The sediment of humic acids was rinsed three times with deionized water, separated by filtration, and dried at 60 C. A commercially available preparation of goethite (a-FeOOH, Aldrich) was used in the experiments. Fe(OH)3 was prepared according to the procedure described by Jones et al. (1994). Fe(OH)3 was rinsed three times with deionized
a
Total Cd (mg kg
1
1
of soil)
of soil)
Contents of Cd fractions (% of total Cd) Soluble and exchangeable 4 ± 4.0 Bound to Fe and Mn 26 ± 9.2 oxides Bound to organics 43 ± 12.1 Residual 27 ± 24.4 Number of microorganisms (cfu g 1 of soil) Oligotrophs 2.5 · 105 ± 0.8 · 105 Copiotrophs 2.4 · 106 ± 0.9 · 106 Fungi 1.3 · 103 ± 0.6 · 103
9 ± 5.6 73 ± 3.7 9 ± 1.5 9 ± 1.5 1.1 · 107 ± 0.3 · 107 4.7 · 107 ± 0.3 · 105 9.1 · 104 ± 3.0 · 104
Results are mean values of three replicates ± standard deviation. a Cation-exchange capacity.
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concentration in SS soil samples was determined each time before the experiment started.
The RF extract (soil:water ratio, 1:1 w/v) was prepared according to the procedure described by Kurek and Majewska (2004). Chemical analysis indicated that this extract contained an average of 2 ± 0.6 lg ml 1 of reducing sugars, 43 ± 18.7 lg ml 1 of Fe(III)-chelators, 4 ± 0.8 lg ml 1 of hydroxamate siderophores, 8 ± 0.1 lg ml 1 of catechole siderophores, 14 ± 1.0 lg ml 1 of citric acid, and 2 ± 0.6 lg ml 1 of acetic acid. Before use in the experiments, RF was sterilized by filtration (membrane filter pore of 0.22 lm). The amount of glucose (13.7 g l 1) added to RF to get RFG corresponded to the sum of all the sugars (root exudates) per kg of soil contained in a 1 mm zone of the rhizosphere of maize (Krafftczyk et al., 1984).
by the plate method. Changes were also determined in the pH and in the concentration of citric and acetic acids as well as Fe(III)-chelators present in the soil extracts during the incubation period. The changes in pools of Cd during the incubation period were expressed as changes in the percent of total metal concentration in the SS soil at time 0. A system consisting of the central chamber and two side chambers (PIGS II) was used in some experiments to simulate bidirectional interaction between soil constituents and microorganisms occurring in natural soil (Chenu and Stotzky, 2002). RF or RFG was introduced to the central chamber. SS soil suspension in an appropriate soil extract was introduced to one side chamber while the other chamber was filled with a suspension of a mixture of all the tested soil constituents in an appropriate soil extract. All other experimental conditions were the same as for PIGS I.
2.4. Cadmium sorption and desorption experiments
2.5. Sequential extraction of total Cd
The multi-compartment system (PIGS I) used in the study consisted of one central chamber and six side chambers. Membrane filters (Isopore, Millipores; pore size 0.4 lm) were placed between the central and the side chambers to separate the solid phases while allowing an exchange of the aqueous phase. This made it possible to determine the simultaneous distribution of Cd released from the SS soil (introduced to one of the side chambers) between various soil constituents placed in other side chambers. Forty milliliter of SS soil suspension or a soil constituent in soil extracts (RF or RFG) was placed in each of the side chambers, and 100 ml of RF or RFG was introduced into the central chamber. The content of each chamber was stirred (250 rpm) with teflon-coated magnetic stirring bars. The complete mixing and exchange of solutions between the compartments of the system was achieved after 24 h of stirring; however, the contact of RF and RFG with the solids in PIGS I was extended up to 48 h. The pH values of soil extracts at time 0 were adjusted to the pH representing the values of the soil. The amounts of the tested solid phase constituents were as follows: montmorillonite – 2000 mg; humic acids – 800 mg; a mixture of goethite and Fe(OH)3 in a 1:1 ratio, (GFe) – 400 mg; bacterial biomass – 34 mg; and fungal biomass – 78 mg. The relative proportion between soil constituents was in the range of that found in sandy loam soils (Kurek et al., 1982; Paul and Clark, 1998). The amount of soil (3.31 g) used in the experiment was the sum of the amounts of all soil components introduced to PIGS I. Changes in the total Cd concentration were determined in the SS soil, in the circulating RF or RFG, and in individual soil constituents after 48 h of incubation at 14 C (an average temperature for the spring– summer period in central-eastern Poland). Changes in the cfu number of bacteria and fungi colonizing the SS soil, as well as those used as soil constituents (Arthrobacter and Trichoderma) during the incubation were estimated
The SS soil, before and after incubation in the PIGS systems, and the mixture of soil constituents used in the experiments with PIGS II were subjected to sequential extraction according to the procedure described by Keller and Vedy (1994).
2.3. Soil extracts
2.6. Cd determination The procedure used for determining the total amount of Cd in RF or RFG, as well as Cd immobilized by individual sorbents and Cd fractions released by sequential extraction from the SS soil and from the mixture of soil constituents (PIGS II) by an atomic absorption spectrophotometer (Unicam 939AA Spectrometer) had been described by Kurek and Majewska (2004). The Cd standards were prepared from a stock solution of Cd(NO3)2 in 5% of HNO3. 2.7. Microbial products Concentrations of Fe(III)-chelators and organic acids were determined in RF or RFG introduced to the system at time 0 and at the end of the experiments. 2.7.1. The Fe(III)-chelators The total amount of Fe(III)-chelators was measured in reaction with FeCl3 using desferrioxamine B as the standard (Gibson and Magrath, 1986). Hydroxamate siderophores were determined by the Csaky (1948) method with NH2OH Æ HCl as the standard. The concentration of catechol siderophores was determined by the Arnow (1937) method with 3,4-dihydrohybenzoic acid as the standard. 2.7.2. Organic acids The samples of RF or RFG were filtered through a 0.2 lm HPLC filter (Millipore, Billeria, Mass, USA) before determination of low molecular weight organic acids
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(LMWOAs) such as citric and acetic acid. The measurement was done by the HPLC method on a VP chromatographic system (Shimadzu, Tokio, Japan) composed of a LC-10AD pump, a RID-10A refractive index detector, a SCL-10A controller, a CTO-10AS oven (all of which were controlled by Class VP 5.03 Workstation Software; Shimadzu, 1999) and a Model 7725 sampling valve (Rheodyne, Berkeley, USA) with a 20 ll loop. The mobile phase (5 mM H2SO4 in Milli-Q water) was run at a flow rate of 0.5 ml min 1 at 55 C through a Rezex Organic Acid column (8 lm; 300 · 7.8 mm, Phenomenex, Torrence, USA). Calibration of the column was carried out using organic acids standards (Sigma. St. Louis, USA). 2.8. Statistical data analysis Statistical analysis was performed on three replicates from each treatment. Standard deviations and analysis of variance were determined using Microsoft Excel 2000 (Armitage and Berry, 1987). 3. Results and discussion 3.1. The effect of amending soil with sewage sludge In agreement with earlier reports (Moreno et al., 1999; Shrivastava and Banerjee, 2004), amendment of soil with sewage sludge resulted in changes of all the tested physico-chemical properties of the SL soil, increased the cfu number of soil microorganisms, and also affected distribution of Cd between operational fractions (Table 1). The Cd fraction bound to Fe and Mn oxides dominated in sludge and in the SS soil, and amounted to 70% and 73%, respectively. Iron (hydr)oxides are known to accumulate heavy metals by adsorption and/or coprecipitation in rivers and their estuaries. Studies of metal mobility in lake sediments strongly suggest that cadmium binds directly to hydroxyl surface groups of iron and manganese oxyhydroxides such as ferrihydrite and lepidocrocite under circumneutral conditions (Randall et al., 1999). 3.2. Growth of microorganisms Proliferation of bacteria inhabiting the SS soil and those introduced to the PIGS systems as soil constituents occurred during the 48-h incubation with RF. While no increase in the cfu number of soil fungi was observed, T. koningii introduced to the systems as a soil constituent was multiplied very intensively (from 7 · 102 ± 0.9 · 102 cfu ml 1 up to 9 · 104 ± 5.6 · 104 cfu ml 1). Enrichment of the soil extract with glucose had no effect on the cfu number of microorganisms introduced to the systems as soil constituents and soil fungi. Addition of glucose to RF resulted in an increase in the cfu number of soil bacteria from 1 · 107 ± 7.4 · 105 to 3 · 107 ± 2.1 · 106 cfu ml 1 in PIGS I; and, conversely, a decrease from 8 · 107 ± 3.1 · 106 to 1 · 107 ± 3.8 · 106 cfu ml 1 in PIGS II.
727
Chemical analyses of RFG at time 0 and after incubation indicated that the content of glucose (or reducing sugars) decreased by 40%. 3.3. Cd mobilization in PIGS Soil extracts circulating in the PIGS systems mobilized from 20% to 43% of total Cd from the SS soil (Fig. 1a). The amounts of Cd mobilized from the SS soil by RFG were significantly larger than those with RF. There were no significant differences in the amounts of Cd mobilized from the SS soil by RF when soil constituents were placed in the chambers individually (PIGS I) or all together as a mixture (PIGS II). However, the disposal of soil constituents in the multi-chamber system affected the amounts of Cd mobilized from the SS soil by RFG (Fig. 1a). More Cd was mobilized when soil constituents were introduced to the chambers one by one [PIGS I] (43% ± 3.7%) than when they were introduced as a mixture [PIGS II] (36% ± 2.0%). Chemical analysis of soil extracts circulating in PIGS after incubation indicated that enrichment of RF with glucose and the disposal of soil constituents had an effect on their pH values (Fig. 1c) and content of other tested compounds such as citric and acetic acids, and Fe(III)-chelators. The experimental conditions in PIGS I and PIGS II were different with respect to total weight of solid-to-circulating-extracts volume ratio. The value of this ratio was 20 g l 1 in PIGS I but 47 g l 1 in PIGS II. This fact was one of the reasons for higher concentrations of the tested metabolites in extracts circulating in PIGS II than in PIGS I (date not shown). An addition of glucose to RF resulted in an enhanced release of metabolites to the aqueous phase, e.g., in PIGS II concentrations of citric acid increased from 24 to 72 lg ml 1, acetic acid from 0 to 28 lg ml 1, total Fe(III)-chelators from 100 to 200 lg ml 1, and catechole siderophores from 5 to 20 lg ml 1. Glucose was added to RF in order to stimulate microbial growth and activity. Enrichment of soil solution with low molecular nutrient substrates originating from root exudates is natural to plant rhizosphere (Huang and Germina, 2002). The enhanced Cd-mobilizing efficiency of RFG could be related to its lower pH and higher contents of microbial products synthesized during incubation compared to RF. The more effective release of Cd in PIGS I in comparison with PIGS II was probably connected with a lower solid/solution ratio in PIGS I which guaranteed an easier contact between the sorbed metal and the metabolites present in the solution. Sequential extraction of Cd remaining in the SS soil after its mobilization by soil extracts indicated that the mobilizing efficiency of RFG was twice as high as that of RF; and RFG mobilized Cd mostly from the operational fraction bound to Fe and Mn oxides (Fig. 2). The experiments indicated that RF, and even more so, RFG circulating in the PIGS systems were rich in metal complexing compounds including siderophores and organic acids. Both LMWOAs identified in RF and RFG, besides lowering the
M. Majewska et al. / Chemosphere 67 (2007) 724–730
% of total Cd in SS soil
728
100 PIGS I
PIGS II
80 60 40 20 0 RF
RF
RFG remaining in soil mobilized
[
RFG in aqueous phase
+
]
resorbed
% of mobilized Cd
70 60
RF
50 40
RFG
30 20 10 0
Bacterial biomass
Fungal biomass
Humic acids
Montmorillonite
GFe
8 PIGS I
7
PIGS II
6
pH
5 4 3 RF
RFG
RF
RFG
Fig. 1. Cadmium mobilization from the SS soil and total resorption by soil constituents expressed as percent of Cd introduced with the SS soil (a); resorption of mobilized Cd by individual soil constituents (b); changes in pH (c) after 48-h incubation with RF or RFG.
pH of the soil solution, are also known to be able to form complexes with metals. Siderophores – highly specific chelators of Fe3+ – were produced by microorganisms and released into the soil extract during incubation. This was the probable cause of the dissolution of soil iron minerals mobilizing Cd bound to them. Dissolution of iron oxides in the presence of siderophores is well documented (Violante et al., 2002). 3.4. Resorption of mobilized Cd Cadmium released from the SS soil to the aqueous phase was in part resorbed by soil constituents introduced into side chambers (from 15% to 36%), and some of it (from 0% to 7%) remained soluble in soil extracts and can be considered bioavailable (Fig. 1a). The metal released from the SS soil by RF (pH 6.4) was resorbed in PIGS I mostly by montmorillonite (66%) and bacterial biomass (16%). Less Cd was resorbed by other abiotic components: GFe (9%) and humic acids (6%). Fungal biomass was a less efficient sorbent than bacterial biomass (Fig. 1b). Cadmium sorption efficiency of some of the tested soil constituents
changed dramatically when Cd was released from the SS soil by RFG (pH 5.0): the percentage of Cd resorbed by humic acids increased to 46%, while the percentage sorbed by montmorillonite decreased to 19%. The resorption efficiency of fungal biomass was also significantly altered. Biomass of T. koningii sorbed 19% of Cd immobilized by soil constituents compared to only 2% sorbed from RF (Fig. 1b). Participation of bacterial biomass and GFe in resorption of Cd released from the SS soil was independent of the soil extract used (RF or RFG). The simultaneous sorption of Cd by soil constituents from a solution containing Zn, Cd, and Hg as chlorides had been studied by Ledin et al. (1996, 1999). These authors used Pseudomonas putida or Bacillus subtilis and Trichoderma harzianum biomass as biotic soil constituents, kaolinite, Al2O3, quartz and goethite as inorganic soil constituents, and peat as an abiotic organic soil component. The results of both of those works, and the present study alike, indicate that the pH, the chemical composition of the medium circulating in PIGS, as well as the type and proportions of the soil constituents used in a study can significantly affect metal sorption efficiency by individual soil
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729
Fig. 2. Changes of Cd speciation in the SS soil incubated for 48-h in the PIGS systems with RF (a) or RFG (b). Inserts represent speciation of Cd mobilized from the SS soil by RF or RFG and resorbed by the mixture of soil constituents. Bars marked with the same letters are not significantly different (P > 0.05).
constituents. However, while Ledin et al. (1999) found a significant increase in sorption efficiency of Zn and Hg, but not Cd, by T. harzianum in response to the lowering of the pH of the medium circulating in PIGS, the present study indicates that also immobilization of Cd by proliferating fungal biomass increases when RF (pH 6.4) is replaced by RFG (pH 5.0). The differing effects of low pH on Cd sorption efficiency by fungal biomass found in the present study and that by Ledin et al. (1999) may be connected with differences in the composition of the solution circulating in PIGS, as well as the physiological state and species of fungus used in the studies. In spite of the experimental differences between the study by Ledin et al. (1999) and the study by the present authors, the importance of microorganisms for sorption of Cd at various pH values, despite their much lesser content in soil as compared to other solid components, is clearly evidenced. Microbial biomass resorbed 19% of Cd at pH 6.4 and 26% at pH 5.0 (Fig. 1b). The two microorganisms, T. harzianum and B. subtilis used by Ledin et al. (1999), which together constituted 1.7% of solid soil mass, accumulated under various conditions from 1% to 16% of added Cd. PIGS II, in which all the tested soil constituents were placed together as a mixture in one chamber, was also used in the experiments. It was found that the metal mobilized
from the SS soil was resorbed evenly into all operational fractions separated from the mixture of soil constituents; however, the mixture resorbed three times more Cd mobilized by RFG than that mobilized by RF (Fig. 2). In natural soil, all solid phase constituents are in contact. Interactions between soil constituents and microorganisms are bidirectional. Soil particles influence the survival and biological activity of microorganisms while microorganisms affect soil particles by modifying their arrangement and producing a new surface with specific properties (Chenu and Stotzky, 2002). The composition of the soil extract circulating in PIGS and the disposal of soil constituents in the system affected the size of the bioavailable Cd pool. The percentage of Cd remaining in the RFG circulating in PIGS I (bioavailable) was significantly higher than those with RF. Yet in PIGS II, the percentage of bioavailable Cd decreased significantly when RFG was used instead of RF (Fig. 1a). The results of the present studies indicate that low pH values and high contents of metal chelators in RFG circulating in PIGS II do not inhibit resorption of Cd mobilized from the SS soil by soil constituents. This finding contradicts the generally accepted opinion that both these factors enhance mobilization of heavy metals from soil and prevent their immobilization (Huang and Germina, 2002). We did not determine Cd forms present
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