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Published in J. Environ. Qual. 33:154-162 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Heavy Metals in the Environment

Sorption and Biodegradability of Sludge Bacterial Extracellular Polymers in Soil and Their Influence on Soil Copper Behavior

L. X. Zhou*, S. G. Zhou and X. H. Zhan

College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China

* Corresponding author (lxzhou{at}njau.edu.cn).

Received for publication August 7, 2002.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Bacterial extracellular polymers (BEP) affect the translocation and fate of organic and inorganic pollutants in terrestrial and aquatic ecosystems. In this study, BEP from activated sludge was compared with sludge dissolved organic matter (DOM) in terms of behavior and effects on the mobilization and bioavailability of Cu in a well-aged Cu-contaminated orchard sandy loam. Addition of sludge BEP (10–200 mg dissolved organic carbon [DOC] L–1) to the soil resulted in 1.6- to 12.8-fold-higher soil soluble Cu concentration over the control and 1.3- to 2.2-fold over sludge DOM of the same concentration. Consequently, the Cu uptake by the ryegrass (Lolium perenne L., cv. Target) grown in the soil was increased by 31% due to interval watering of 100 mg DOC L–1 of sludge BEP solution in a 35-d period. The influence of sludge BEP on mobilizing soil Cu could be maintained as long as 60 d or more, depending on BEP biodegradation status. The findings that sludge BEP promoted Cu mobilization and bioavailability could be attributed to less adsorption of BEP by soil, slow degradation, and higher affinity with Cu. For example, after 3 wk of aerobic incubation, the soluble Cu present in the sludge DOM-treated soil was reduced to about the level of the control, while the concentration of soluble Cu in BEP-treated soil was 6.2 times higher than that in the control. Therefore, sludge BEP could act as a facilitated-transport carrier of Cu. The environmental risk of Cu should receive much attention if BEP is incorporated into soils.

Abbreviations: BEP, bacterial extracellular polymer • DOC, dissolved organic carbon • DOM, dissolved organic matter • EDTA, ethylenediaminetetraacetic acid • MBC, maximum binding capacity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
COPPER CONTAMINATION in soil has received much attention due to the increased application of animal manure and urban wastes on farmland and repeated application of Cu-containing pesticides on orchards in China. Concentrations as high as 200 to 1000 mg Cu kg–1 in poultry litter and pig manure are often observed due to the historically common practice of adding Cu to animal feed (Van der Watt et al., 1994; Giusquiani et al., 1998). In many older orchard soils, the Cu level exceeds more than 300 mg kg–1 in soil due to application of Bordeaux mixture as a pesticide for decades (Aoyama, 1998). The mobility and translocation of Cu in contaminated soil is of environmental concern because of phytotoxicity and possible migration into ground water (Chen, 1996; Nowack et al., 1997).

Numerous investigations have revealed that dissolved macromolecules or colloidal particles in soils can act as a potential "carrier" to facilitate the transport of heavy metals through the formation of soluble metal–organic complexes (Lamy et al., 1993; McCarthy et al., 1993), especially for Cu (Temminghoff et al., 1997; Guibaud et al., 2003; Zhou and Wong, 2003). Dissolved macromolecules in soil systems include humic substances and bacterial polymers (Chen et al., 1995a).

Organic materials, such as green manure, crop residue, animal manure, and undigested activated sewage sludge, are often applied to the orchard soils to maintain soil fertility and to provide nutrients for plant growth in the northern part of Jiangsu Province, China. Different organic materials had markedly different effects on nutrient cycling and heavy metal mobility, which may be related to the DOM released from these organic materials (Zsolnay and Gorlitz, 1994), especially during the first few weeks following their application (Lamy et al., 1993; Baham and Sposito, 1983), and dissolved macromolecules such as BEP of sludge or soil microorganism origins (Allison, 1968; Martens and Frankenberger, 1992).

Bacterial extracellular polymers are composed of polysaccharides, protein, RNA, and DNA. They exhibit a high affinity toward certain metal ions (Geesey et al., 1989; Brown and Lester, 1982; Rudd et al., 1984; Mitteleman and Geesey, 1985), resulting in enhanced metal transport in the environment (Chen et al., 1995b). Activated sludge is typically composed of 85% bioflocculation (bacteria); therefore, BEP production is a common phenomenon by activated sludge microorganisms (MacNicol and Beckett, 1989; Hejzlar and Chudoba, 1986; Zhou et al., 2000). Application of the undigested activated sewage sludge on land results in release of sludge BEP into the soil environment.

Biodegradation of BEP in soil affects metal mobility. Low biodegradability can make BEP persist sufficiently long to permit transport and removal of BEP-bound metals. Martens and Frankenberger (1992) found that soil bacterial polymers (mainly monosaccharides) present in the added polymers were rapidly decomposed and the saccharide content of the polymer-treated soil returned to the level of the control after 2 wk of incubation. Similar results have also been reported by Martin and Richards (1963): extracellular polymers of the soil bacteria were degraded 50% after 1 wk and 70% after 4 wk. However, in the same experiment, it was found that the exopolymer of Chromobacterium violaceum was rather resistant to degradation in soil: after 1 wk, 10% of the polymer was mineralized to CO2 and after 8 wk, 53% of the polymer had been degraded. All factors affecting soil microbial activity will affect polymer degradation. For example, under aerobic upland conditions, sludge BEP or other dissolved organic matter applied to soil might exhibit drastically different biodegradation and metal dissolution from that under waterlogged conditions. However, there has been little research in this area. Knowledge of the behavior of sludge BEP incorporated into soil, especially its persistence in soil and the effect on metal mobility, can greatly improve risk assessment for agricultural application of sewage sludge. However, little is known about sludge BEP biodegradability in a contaminated soil and subsequent dynamic effect on metal dissolution. There is little evidence of an increase of metal uptake by plants in the presence of sludge BEP added into a contaminated soil. Hence, the aims of this study are to determine sludge BEP ability to dissolute soil Cu, especially the dynamic effect of sludge BEP degradability on Cu dissolution in contaminated soil under aerobic and anaerobic incubations, and bioavailability of Cu facilitated by BEP through a pot experiment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil
The soil used in this study was collected from the topsoil (0–15 cm) of the 40-yr-old Dashahe orchard farm in the northern part of Jingsu Province, China. The soil, originated from Yellow River alluvium, is sandy silty loam with a high pH and is contaminated with Cu from the application of Bordeaux pesticide used in the cultivation of apple trees. One subsample was air-dried and ground to pass through a 1-mm sieve for subsequent laboratory trials. The other was air-dried to pass through a 4-mm sieve for the experiment with ryegrass. The soil was classified as fine loam, Typic Hapludalfs. Details on soil properties are given in Table 1.


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  		Table 1. Preliminary soil properties.{dagger}

 
Preparation of Sludge Bacterial Extracellular Polymers and Dissolved Organic Matter
Bacterial extracellular polymers and DOM were extracted and prepared from activated sludge and anaerobically digested sludge of the same origin as described previously (Zhou and Wong, 2001; Zhou et al., 2001). Activated sludge was collected from Suojincun Sewage Treatment Plant in Nanjing, China and was rinsed twice with double-distilled water. Crude sludge BEP was harvested by centrifuging the rinsed activated sludge for 20 min at 32000 x g at 4°C to effect shearing of polymeric materials from the microbial surface. The supernatant was placed in 8000 molecular weight cut-off dialysis tubing (Spectra/Por 7; Spectrum Laboratories, Rancho Dominguez, CA) and dialyzed against double-distilled water with constant mixing for 3 d at 4°C. Eight water exchanges were made to remove low molecular weight impurities from the interior solution of dialysis tubing. After dialysis, the polymer solution was lyophilized and stored at 4°C in a sealed glass tube before use. The dissolved organic matter derived from anaerobically digested dewatered sludge of the same sources was also collected through extracting sludge with double-distilled water at 10:1 (v/w) for 16 h followed by centrifuging at 12500 x g for 20 min, filtered with 0.45-µm membrane, and then directly lyophilized. However, in the trial of the binding of DOM with Cu, the DOM was extracted, dialyzed, and lyophilized using the same procedure mentioned above. The DOC of BEP and DOM was determined with a TOC analyzer (TOC-5000A; Shimadzu, Kyoto, Japan).

Copper Dissolution and Bacterial Extracellular Polymer Sorption Experiment
The stock solution of BEP or DOM with about 350 mg DOC L–1 was prepared by dissolving lyophilized BEP and DOM in double-distilled water. The stock solutions of BEP and DOM were diluted with double-distilled water to six to seven different initial concentrations ranging from 10 to about 200 mg DOC L–1, representing the range of DOC commonly existing in soils amended with organic wastes (Gregorich et al., 1998). Triplicate 20-mL BEP or DOM solutions were added to 5.00 g of soil and shaken at 22°C in a reciprocal shaker at 200 rpm for 2 h. A shaking period of 2 h was adequate for achieving static adsorption equilibrium without any significant biodegradation of DOM (Zhou and Wong, 2000). After shaking, the suspensions were centrifuged at 12500 x g for 10 min, and then filtered through 0.45-µm membrane filters. The filtrates were analyzed for DOC by TOC analyzer and for soluble Cu by flame atomic absorption spectrophotometer (FAAS) (Z-8000; Hitachi, Tokyo, Japan). The soluble Cu concentrations of the initial 200 mg DOC L–1 of DOM and BEP solution analyzed with a graphite furnace atomic absorption spectrophotometer (GFAAS) (HGA-600; PerkinElmer, Wellesley, MA) were 48 and 10.6 µg L–1, respectively, which were much lower than the soluble Cu content of the contaminated soil (Table 1). Hence, the influence of soluble Cu from BEP or DOM itself on Cu solubility experiment should be negligible.

Batch Experiments for Bacterial Extracellular Polymer Biodegradability
The relative decomposition rate of sludge BEP and consequent effect on Cu dissolution in comparison with sludge DOM was determined by periodically measuring soluble Cu and residual DOC in the soil amended with BEP and DOM under aerobic and waterlogged conditions. In the aerobic incubation, about 2 mL of about 2100 mg DOC L–1 of BEP and DOM stock solutions (made by dissolving lyophilized BEP and DOM in double-distilled water) were mixed with 10 g of soil–sand mixture (soil to sand = 1:1 w/w) in a 50-mL plastic bottle to produce an initial concentration of 900 mg DOC kg–1 soil. This application rate was chosen because Martens and Frankenberger (1992) found in an incubation study that the application rate of 1 mg extracellular polymer (EP) C g–1 soil (equivalent to 1000 mg EP C kg–1 soil) was similar to the EP concentration existing in the soils receiving organic manures. Double-distilled water was then added to obtain soil moisture of 70% of field water holding capacity (about 25% of the soil–sand mixture weight), which was maintained throughout a 9-wk aerobic incubation by periodically weighing. In the aerobic incubation, the incorporation of fine acid-washed silica sand with 0.42- to 0.59-mm particle diameters into the soil was to maintain near-optimum aeration conditions and help keep soil moisture uniform in the case of the addition of double-distilled water in the mixture for supplementing water lost during incubation. The beaker was covered by parafilm with some small holes to allow air exchange. At the same time, a control was conducted using the same method except for no addition of BEP or DOM into soil–sand mixture. All samples were incubated in the dark at 22°C. Three replications of each treatment were extracted with double-distilled water (soil–sand mixture to water = 1:4 w/v) for 2 h at 200 rpm at 0, 0.5, 1, 2, 3, 5, 7, 10, 14, 21, 35, 49, and 63 d after incubation. After the soil suspension was centrifuged at 12500 x g for 10 min, the supernatant was filtrated using a 0.45-µm membrane. Dissolved organic C and Cu were determined in the filtrates. In the anaerobic procedure, the aerobic procedure was followed excepted that 5.00 g soil (no sand) was used and 10 mL double-distilled water was added in addition to the 2 mL BEP or DOM stock solution to keep a 1-cm water layer over the soil surface. The percentage of BEP or DOM decomposition was calculated by the percent proportion of the loss of DOC (subtracting DOC in the filtrates from total initial DOC) in relation to total initial DOC.

Binding of Sludge Bacterial Extracellular Polymers with Copper by Dialysis Method
The complexation of sludge BEP with Cu was determined as described by Truitt and Weber (1981). The BEP or DOM stock solutions with 350 mg DOC L–1, 0.1 M KCl solution as background electrolyte for maintaining stable ionic strength, and 2.5 x 10–3 M of CuCl2 were adjusted separately to pH 5 with 0.1 M HCl or 0.1 M NaOH before the complexing reaction. The above solutions and supplemented double-distilled water were then mixed in 50-mL plastic centrifuge tubes to produce a series of 10-mL mixing solutions consisting of 100 mg DOC L–1 of BEP or DOM, 0.01 M KCl, and 1.5 x 10–5 to 1.25 x 10–3 M of Cu2+. The complexing experiment was conducted by shaking the tubes at 200 rpm and 25°C for 2 h. The solution then was transferred to 8000 molecular weight cut-off dialysis tubing and dialyzed by suspending the dialysis tubing in double-distilled water at 4°C for 2 d with constant stirring and water change every 6 h to remove the free Cu. In our preliminary experiment, 48 h was found to be enough for this dialysis method to remove nonbound Cu. The solution in the dialysis tubing was drawn out and determined for bound Cu by FAAS. The free Cu was calculated from the difference between initial total Cu and bound Cu. The conditional stability constant of metal–BEP or metal–DOM complex and effective complexion capacity of BEP or DOM can be obtained from the graphical solution of the linear relationship described by Mitteleman and Geesey (1985). The similar trials at pH = 4 and 6 also were conducted with the same procedure described above except that initial Cu concentration range varied from 1.5 x 10–5 to 0.2 x 10–3 M in the trial at pH 6 only.

Ryegrass Bioassay
One kilogram of the soil sieved through a 3-mm screen was transferred to each plastic pot. The basal fertilizers at a rate of 0.15 g N kg–1, 0.10 g P2O5 kg–1, and 0.15 g K2O kg–1 were incorporated evenly into the soil in each pot, and then the soils were watered with double-distilled water to 80% of field water holding capacity. Thirty ryegrass seeds were sown in each pot. In early stage of seed germination and seedling growth, double-distilled water was added as needed to maintain soil moisture until the height of ryegrass seedling was about 5 cm (about 10 d). This was followed by interval-watering the same amount of water or a specific DOC solution for different treatments (double-distilled water, ethylenediaminetetraacetic acid [EDTA], stock sludge BEP, and stock sludge DOM solutions) with four pots for each treatment. The pH values of EDTA, stock DOM, and stock BEP solutions were adjusted to the same level as double-distilled water (pH = 6.0) with 1 M HCl or 1 M NaOH, and their concentrations were fixed to 100 mg DOC L–1, as determined with a TOC analyzer. The aboveground portion and roots of the ryegrass from each treatment were harvested and weighed separately 45 d after planting. About 480 mL of double-distilled water, EDTA, DOM, or BEP solutions were used to water each pot. The plant samples were oven-dried at 70°C, ground, wet-ashed using nitric and perchloric acids, and analyzed for Cu by FAAS. At the time of harvest, 30-g field-moist samples of the soil were taken from each pot and pH and soluble Cu concentration were determined.

Statistical Analysis
We used SAS for all statistical procedures (SAS Institute, 1996). General linear regression analysis was used to obtain the DOC sorption isotherm and the initial mass isotherm and to determine the relationship between water-soluble Cu and added DOC. The analyses of variance mean separation tests were also used to detect the significant treatment effect using least significance difference (LSD) at p = 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Sorption and Biodegradability of Bacterial Extracellular Polymers in Soil
The behavior of BEP in soil is an important factor affecting the mobility of metals. Bacterial extracellular polymers in the mobile form by soil are believed to enhance the transport of the associated contaminants through porous media (Newman et al., 1993). Figure 1 depicts the sorption isotherms for the BEP and DOM onto the sandy loam. The initial mass (IM) isotherm (Nodvin et al., 1986) can be used to describe a linear regression of sorption against DOC concentration with a higher coefficient of determination:

[1]
where RE is the adsorption of DOC onto soil (mg kg–1), which is obtained operationally through deducting DOC of equilibrium solution in the DOC-treated soil from the initial DOC solution; m is the regression coefficient; Xi is the initial concentration of DOC in soil suspension, expressed as mg kg–1 soil; and b is the intercept (mg kg–1).



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  		Fig. 1. The initial mass isotherm of sludge bacterial extracellular polymers (BEP) and dissolved organic matter (DOM) onto the Cu-contaminated sandy loam at 22°C with an equilibrium time of 2 h.

 
The distribution coefficient (Kd), an index of the affinity of BEP or DOM for soil, can be calculated according to Nodvin et al. (1986):

[2]

The IM approach has been shown to be a useful tool for describing the sorption of BEP or DOM in soils because it takes into consideration the release of indigenous DOC from soil (Nodvin et al., 1986; Kaiser and Zech, 1998). A significant net release of DOC was observed for the soil receiving DOC at a concentration of <160 mg DOC L–1 (Fig. 1). As the amount of added DOC increased, the release of DOC from the soil decreased. A net sorption was observed at a concentration of >160 mg DOC L–1 for DOM and >190 mg DOC L–1 for BEP. This demonstrated that DOC had higher mobility in the soil matrix and less adsorption by the soil. The alkaline pH (7.53) of the selected soil might be responsible for a decreased sorption of DOC by oxidic surfaces (Tipping, 1981; Jardine et al., 1989). Lion et al. (1988) reported that at low pH, surface sorption between BEP with mineral surfaces occurred more frequently by electrostatic attraction, ligand exchange with hydroxyl groups on the surface, or ternary surface co-adsorption of metal ions and BEP. However, BEP had a lower slope (m = 0.143) and distribution coefficient (Kd = 0.669) compared with DOM (m = 0.161, Kd = 0.771), indicating that the former exhibited a lower adsorption onto or higher mobility in the soil than the latter, although low net sorption for both was observed within the low DOC concentration ranges studied.

The differences in sorption behavior of BEP and DOM may be due to their differences in the relative proportion of hydrophobic and hydrophilic compounds. We found a higher proportion of hydrophilic fractions in BEP (65%) compared with DOM (53%) (Zhou et al., 2001). Numerous studies have shown that the mineral soil exhibits a strong retention of hydrophobic DOC and little retention of hydrophilic compounds (Jardine et al., 1989; Gu et al., 1995; Guggenberger and Zech, 1993; Kaiser and Zech, 1998).

The biodegradability of sludge BEP is another factor affecting the environmental behavior of metals. Biodegradation of BEP in the soil must be understood before the effect of polymers on metal mobility can be predicted. As shown in Fig. 2 , the degradation of BEP and DOM was characterized by a rapid decomposition, which plateaued after about 3 wk of incubation under aerobic (Fig. 2A) or anaerobic (Fig. 2B) conditions. Bacterial extracellular polymers were rather resistant to degradation: after 1, 3, and 9 wk, 45, 65, and 82% of DOC was mineralized, respectively, under aerobic conditions, values that were lower than DOM by about 20% on average. Under anaerobic incubation BEP also was relatively persistent in soil because of its low degradation rate (Fig. 2B). Only 28, 38, and 68% of BEP–DOC was mineralized at 1, 3, and 9 wk under anaerobic incubation, respectively. The degradability of the BEP from activated sludge in soil is lower than that from soil bacteria as reported by Martin and Richards (1963), who found that the extracellular polymers of the soil bacteria were degraded more readily: 50% after 1 wk and 70% after 4 wk. Martens and Frankenberger (1992) also noted that the exopolymers (from Arthrobacter viscocus, Azotobacter indicus, Bacillus subtilus, C. violaceum, and three Pseudomonas strains) were degraded relatively quickly in soil. Undoubtedly, the rate of polymer degradation varies with the type of microorganism that produced the polymer and the type of environment in which the polymer is degraded. Pavoni et al. (1972) indicated that the polymers produced by activated sludge organisms were found to have a biological oxygen demand (BOD5) (in wastewater) to chemical oxygen demand (COD) ratio of 0.10, implying a low level of biodegradability.



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  		Fig. 2. Degradation of sludge bacterial extracellular polymers (BEP) and dissolved organic matter (DOM) in the Cu-contaminated sandy loam under (A) aerobic and (B) anaerobic incubation at 22°C.

 
The relatively low biodegradability of BEP compared with DOM under two incubation conditions might be attributed to the specific chemical nature (such as large molecules and the presence of uronic acid) of BEP and the formation of additional bonds between BEP with Cu in the tested Cu-contaminated soil (Czajka et al., 1997). Indeed, Francis et al. (1992) found that the tridentate citrate complex with Cu was completely resistant to degradation, and that this resistance was caused by the chemical nature of the complex, not by the toxicity of the metals.

The consistently high DOC concentrations in BEP- or DOM-treated soils under waterlogged incubation were probably caused by a lack of strong sorption in the reduced mineral soil resulting from the reductive dissolution of Fe oxides (Jardine et al., 1989; Moore et al., 1992; Hagedorn et al., 2000). In addition, incomplete decomposition of organic substances under reducing conditions in contrast to the soil under aerobic incubation may be another reason for the high concentration of DOC under waterlogged incubation.

Lower biodegradability of BEP, especially under waterlogged conditions, could make it persist sufficiently in the soil environment to permit transport and removal of BEP-bound metals if this carrier was applied to the soil.

Soil Copper Dissolution by Bacterial Extracellular Polymers
The addition of BEP or DOM increased the water-soluble Cu level in Cu-contaminated calcareous soil significantly (p < 0.01) (Fig. 3) . The soil Cu dissolution was positively, linearly related to DOC concentration with higher correlation coefficients of determination (R2 = 0.992 for BEP and R2 = 0.990 for DOM). In the DOC range studied, water-soluble Cu in the soil receiving BEP was 2.3 times more than that receiving DOM as indicated by their slopes (0.066 L kg–1 for BEP and 0.029 L kg–1 for DOM) in Fig. 3. Soluble Cu contents increased 1.6- to 12.8-fold and 1.2- to 5.9-fold at DOC concentrations ranging from 10 to 200 mg L–1 for BEP and DOM, respectively. The significant difference (p < 0.01) between BEP and DOM in dissolving the soil Cu might be attributed to composition and chemical structure. In fact, as mentioned before, BEP exhibited less sorption by soil than DOM. Similar results had also been observed in a study of BEP mobility in aquifer sands by Chen et al. (1995b), who found that BEP was relatively mobile and, as a result, a greater than 90% reduction in metal adsorption was achieved at a BEP concentration of 10.6 mg L–1. However, soluble Cu level in the BEP- and DOM-amended soil decreased rapidly in the first 5 d after applying DOC solution followed by slow reduction, especially under aerobic incubation conditions (Fig. 4) . Soluble Cu was reduced by 77.5 and 46.3% for DOM- and BEP-amended soil, respectively, under aerobic incubation, and correspondingly 51.4 and 28.7% under waterlogged incubation conditions.



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  		Fig. 3. Water-soluble Cu in the Cu-contaminated soil as affected by different concentrations of sludge bacterial extracellular polymers (BEP) and dissolved organic matter (DOM).

 


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  		Fig. 4. The change of soluble Cu in Cu-contaminated sandy loam amended with sludge bacterial extracellular polymers (BEP) and dissolved organic matter (DOM) with the increase of (A) aerobic and (B) waterlogged incubation time.

 
In the period of incubation experiments (0–63 d), BEP-treated soil consistently produced more soluble Cu than DOM-treated soil (Fig. 4). After 3 wk of aerobic incubation, the soluble Cu present in the DOM-treated soil was reduced close to the level of the control, while the concentration of soluble Cu in the BEP-treated soil was on average 6.2 times higher than that of the control. Under anaerobic incubation the soils receiving two water-soluble organic materials had much higher soluble Cu levels than the corresponding treated soils under aerobic incubation. The higher mobility of Cu by BEP in the treated soil was maintained for more than 60 d, especially under anaerobic conditions, which should be sufficiently long to permit Cu uptake and translocation into plants. In contrast, a higher soluble Cu level above the control facilitated by DOM only persisted for about 21 d under aerobic conditions and 35 d under anaerobic conditions. It was noted that under aerobic incubation conditions soluble Cu reduction always lagged behind DOC biodegradation, especially after 3 d of incubation (Fig. 5) . This phenomenon was more apparent in the soil receiving BEP (Fig. 5B) than DOM (Fig. 5A), implying that the soluble Cu derived from the breakdown of mobile BEP–metal or DOM–metal complexes in the soil suspension due to BEP or DOM biodegradation was not adsorbed promptly by soil solid. Consequently, the released soluble Cu could persist longer in this environment. The findings suggested that BEP, to a certain extent, was capable of mobilizing soil Cu and that its effect on the environmental hazard of Cu should not be underestimated.



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  		Fig. 5. The relative comparison of soluble Cu and dissolved organic carbon (DOC) levels in the soil receiving sludge (A) dissolved organic matter (DOM) and (B) bacterial extracellular polymers (BEP) as sludge DOM and BEP biodegradation under aerobic incubation conditions at 22°C.

 
For DOC of the same origin, the concentration in soils played a predominant role in mobilizing Cu in spite of aerobic or waterlogged incubation conditions, as indicated by a similar slope derived from the relationship between DOC and soluble Cu level for different incubation conditions (Fig. 6) . Therefore, the reduction of soluble Cu in BEP- or DOM-treated soils with the increase of incubation time and/or the consistently higher soluble Cu levels in the BEP-treated soil compared with the DOC-treated soil regardless of aerobic or anaerobic incubation conditions could be mainly attributed to DOC concentration remaining in the soils.



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  		Fig. 6. The correlation between Cu dissolution from Cu-contaminated sandy loam and remaining dissolved organic carbon (DOC) in the soil under aerobic and waterlogged incubation conditions.

 
Binding of Bacterial Extracellular Polymers by Copper
Bacterial extracellular polymers exhibited higher affinity for Cu than DOM in terms of the sorption isotherm plot obtained at pH = 5 and 10°C (Fig. 7) . Binding constants could be experimentally derived from the Langmuir linear expression (Mitteleman and Geesey, 1985):

[3]
where [M] is the free Cu2+ concentration, [Mx] is moles of Cu bound per unit weight of metal binding component, MBC is the maximum binding capacity, and K is the conditional stability constant.



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  		Fig. 7. Adsorption isotherms of sludge bacterial extracellular polymers (BEP) and sludge dissolved organic matter (DOM) with Cu at pH = 5 and 25°C.

 
It was found that BEP had a higher MBC and stability constant than DOM at pH = 5 (p < 0.05) (Table 2). Maximum binding capacities were 324 µmol Cu g–1 of DOC for BEP and 210 µmol Cu g–1 for DOM. The MBC value of BEP was higher than that reported by Romkens and Dolfing (1998), who found that MBC was 250 µmol Cu g–1 of DOC for high molecular weight components extracted from arable soils. The stability constant (log K = approximately 4) of BEP–Cu complex was much lower than that of Cu-synthesized organic chelating agents (EDTA, DTPA, EDDHA, CDTA, etc.; log K > 18), but higher than that of some naturally occurring organic acids such as fulvic acid (log K = 3.5) (Yuan, 1990). Logan et al. (1997) also observed that the binding constants for Cu–HA complex were log K = 2.63 at pH = 5 and log K = 4.87 at pH = 6. Relatively higher affinity of BEP for Cu could be attributed to the strong binding of the functional groups of BEP such as hydroxyl groups of hexose or pentose in neutral polysaccharides and carboxyl groups in anionic polysaccharides (Brown and Lester, 1979).


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  		Table 2. Maximum binding capacities (MBC) and conditional stability constants (K) of sludge bacterial extracellular polymers (BEP) and dissolved organic matter (DOM) with Cu at pH = 5.

 
pH played an important role in Cu binding to BEP or DOM. Conditional stability constants increased with the decrease of hydrogen ion concentration over the pH range 4.0 to 6.0 (Table 3). This variation caused by pH might be due to a reduction in competition at Cu binding sites by H+ ions. Displacement of H+ ions by Cu was supported by potentiometric titration of the exopolysaccharide in the presence and absence of the metal. The concomitant decrease in concentration of H+ ions competing with Cu at the binding site resulted in a more stable interaction (higher affinity) between the reactive Cu species and the exopolymers (Mitteleman and Geesey, 1985). On the other hand, BEP exhibited the capacity to bind Cu ions with high affinity with the increase of pH, which could be contributed to ionized carboxyl groups on the BEP participating in cupric ion binding (Geesey et al., 1989).


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  		Table 3. Maximum binding capacities (MBC) and conditional stability constants (K) of sludge bacterial extracellular polymers (BEP) with Cu at pH = 4, 5, and 6.

 
In contrast to the conditional stability constant, MBC increased initially with increasing pH from 4 to 5 and then decreased when pH was 6. The pH-dependent MBC increase might be due to a greater negative charge of BEP caused by the increase of pH, which could consequently adsorb more Cu2+. Bacterial extracellular polymers, composed mainly of polysaccharides, usually were acidic, typically containing between 5 and 25% uronic acid residues (Czajka et al., 1997). Acidic polymers readily bind metals since negatively charged polymers are likely to exhibit anion-like adsorption behavior onto the surfaces of porous media. The presence of adsorbed polymers might enhance the adsorption of metals to surfaces at low pH. At higher pH values, bacterial polymers would be expected to weakly adsorb the porous media and might bind trace metals in the aqueous phase, thus reducing the adsorption of metals to the solid phase. But when pH was greater than 6, the decrease in MBC with increasing pH could result from a reduction in the concentration of free Cu since Cu hydroxide formation was favored under higher pH condition (pH > 5.8) (Zhou and Wong, 2001). The pH effects on MBC of BEP for Cu and conditional stability constants found in this study were similar to those reported for the interactions of Cu–humic acids and Cu–exopolymers from a freshwater–sediment bacterium (Mitteleman and Geesey, 1985).

Metal Uptake of Ryegrass in the Presence and Absence of Bacterial Extracellular Polymers
Ryegrass bioassay further showed a higher Cu concentration especially in plant roots in the presence of 100 mg L–1 of applied DOC for EDTA and BEP only (Table 4). Among soluble organic matters studied, EDTA exhibited the strongest ability to increase Cu uptake with a value 2.2 times greater than the control. Consequently, excess Cu uptake of ryegrass in the EDTA-treated soil produced Cu phytotoxicity with 17% reduction of ryegrass biomass and leaf chlorosis (yellowish leaf).


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  		Table 4. Copper concentrations in ryegrass grown in the contaminated soil receiving bacterial extracellular polymers (BEP), dissolved organic matter (DOM), and ethylenediaminetetraacetic acid (EDTA).

 
Copper concentration in roots and uptake by the ryegrass grown in the BEP-treated soil were increased 1.45- and 1.31-fold above the control in this study although the ability of BEP in enhancing the Cu bioavailability was much lower than EDTA. There was no statistical difference between sludge DOM and the control treatments in Cu concentration and Cu uptake of ryegrass. The biodegradability of various soluble organic materials and their affinity with Cu could be responsible for the difference of Cu uptake as affected by DOC. Therefore, as a naturally occurring soluble organic matter, bacterial extracellular polymers are also capable of enhancing Cu bioavailability in the Cu-contaminated soil through mobilizing Cu. Consequently, the risk of the increasing phytotoxicity of Cu in the Cu-contaminated soil in the presence of sludge BEP could not be excluded.

In the last decade, there has been a growing interest in adopting phytoremediation as a soil cleanup technology (Leeson and Alleman, 1999; Blaylock et al., 1997). Some researchers argued that BEP might act as a potential agent or surfactant for enhancing the remediation efficiency of the contaminated soils in engineered phytoremediation or bioremediation application because BEP exhibited an obvious affinity with heavy metal or hydrophobic organic pollutants. Furthermore, BEP occurs naturally, and application may be more acceptable to the public than the application of synthetic chelating agents or surfactants (Chen et al., 1995a, 1995b; Liu et al., 2001; Dohse and Lion, 1994; Czajka et al., 1997). However, in this study, BEP derived from activated sludge appeared to be of little value in remediating Cu-contaminated soils because its higher biodegradability and much lower ability to increase Cu uptake by plants than common synthetic chelating agents such as EDTA.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Compared with sludge DOM, sludge BEP had higher mobility in the selected soil and less adsorption by soil as shown by the BEP adsorption initial mass isotherm with a lower slope (m = 0.143) and distribution coefficient (Kd = 0.669). Bacterial extracellular polymers were found to be of a relative long persistence in soil because of the low degradation rate compared with DOM. Only 28, 38, and 68% of BEP–DOC was mineralized at 1, 3, and 9 wk after anaerobic incubation, respectively, values that were lower than those of DOM by about 20% under the same conditions, respectively. The degradation of BEP led to a significant decrease of soluble Cu in the BEP-treated soil. The affinity of BEP for Cu in terms of maximum binding capacity (324 µmol g–1 DOC) and conditional stability constant (1.02 x 104) at pH = 5 was higher than that of DOM and also naturally occurring organic matters such as fulvic acid. As a result, the dissolution of Cu in the contaminated soil could be greatly enhanced and higher water-soluble Cu could be maintained for a long period due to the addition of BEP compared with DOM. Furthermore, BEP significantly increased the Cu uptake by the ryegrass grown in the Cu-contaminated soil compared with DOM. Therefore, it was concluded that BEP could act as a facilitated-transport carrier of Cu due to its ability to mobilize Cu. Consequently, the environmental risk of Cu leaching to ground water in the Cu-contaminated soils might increase if BEP were incorporated into the soils.


   ACKNOWLEDGMENTS
 
This study was supported jointly by the National Natural Scientific Foundation of China (20007001, 29777022) and International Scientific Foundation (C/2669-2). The authors would like to thank Dr. William Berti and Dr. Jonathan W. Wong for their valuable comments on this manuscript.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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