Environ. Sci. Technol. 2009, 43, 5884–5889
In Vivo Studies to Elucidate the Role of Extracellular Polymeric Substances from Azotobacter in Immobilization of Heavy Metals PRACHI M. JOSHI AND ASHA A. JUWARKAR* Environmental Biotechnology Division, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur-440020, India
Received January 15, 2009. Revised manuscript received June 3, 2009. Accepted June 18, 2009.
The role of extracellular polymeric substances (EPS) produced by the heavy metal-resistant strain of Azotobacter spp. in restricting the uptake of cadmium (Cd) and chromium (Cr) by wheat plants cultivated in soils contaminated with the respective heavy metals has been demonstrated. A heavy metalresistant strain of Azotobacter spp. was isolated and identified. Minimum inhibitory concentrations (MIC) of Cd2+ and CrO42were determined to be 20 and 10 mg L-1, respectively. Under in vitro conditions, the EPS produced by the strain could bind 15.17 ( 0.58 mg g-1 of Cd2+ and 21.9 ( 0.08 mg g-1 of CrO42-. Fourier transform infrared spectra of the EPS revealed the presence of functional groups like carboxyl (-COOH) and hydroxyl (-OH), primarily involved in metal ion binding. Under pot culture experiments, the isolated strain of Azotobacter was added to the metal-contaminated soils in the form of free cells and immobilized cells. The total Azotobacter count and plant metal concentrations under different treatments showed a negative coefficient between the Azotobacter population and plant Cd (-0.496) and Cr (-0.455). Thus it could be inferred that Azotobacter spp. is involved in metal ion complexation either through EPS or through cell wall lipopolysaccharides (LPS).
Introduction Azotobacter are Gram negative, aerobic, cyst-forming, freeliving nitrogen-fixing bacteria with an ability to excrete ammonia and amino acids into the liquid media (1, 2). Inoculation of soil with Azotobacter has beneficial effects on crop yields due to the increase of fixed nitrogen content in soil (3-7) and also due to the microbial secretion of stimulating hormones like gibberellins, auxins, cytokinins (8-10), and siderophores (11). Hence, these bacteria are widely used as inoculants, especially for agricultural crops (12). At present, strains belonging to the species of A. vinelandii and A. chroococcum are proficiently employed as soil inoculants in rainy areas and in warm and alkaline soils (13). In agricultural soils contaminated with heavy metals, the Azotobacter population becomes affected, leading to the complete loss of nitrogen-fixing activity (14). This consequently affects the availability of nitrogen to the crop plants. One of the greatest concerns is thus establishment and activity * Corresponding auther phone: (O) (+91) 0712 - 2249764; fax: (+91) 0712 - 2249900; e-mail:
[email protected]. 5884
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 15, 2009
of these nitrogen-fixing bacteria under heavy metal stress. In order to cope with the situation, bacteria have evolved certain metal resistance strategies. Intracellular metal resistance mechanisms include efflux, complexation, or reduction of metal ions to a less toxic form (15); whereas chelation of metal ions by certain polymeric substances produced extracellularly by the bacterial cells is an extracellular mechanism for protection of the cells. Like other soil bacteria, Azotobacter species are also reported to be resistant to heavy metals (16, 17), and this property is known to be conferred by plasmids (18, 19). However, particularly in case of Azotobacter species before the metal ions enter the cell, they come in contact with extracellular polymeric substances (EPS), which are produced in copious amounts by this organism (20). EPS thus act as the first and foremost barriers that chelate the metal ions and prevent their entry into the bacterial cells. In general, EPS of bacteria are a complex mixture of high molecular weight polymers (Mw > 10000) produced from lysis and hydrolysis of adsorbed organic matters from the medium (21). The physiological functions of EPS produced by bacteria in nature have been widely discussed (22-27). In short, they can protect the cells from the harsh external environment, provide the bacterial cells with energy and a carbon source when substrate is in short supply, and influence the surface physicochemical properties, which are of considerable importance in governing bacterial flocculation and adhesion. In spite of many reports available on the various functions of EPS, including their role in binding heavy metal ions and protection of bacterial cells, they are limited to the laboratory scale (in vitro). However, to the best of our knowledge any in vivo study on bacterial EPS and their relation to the heavy metal ion concentration in the plants grown in metalcontaminated soils has not been reported so far. Hence, in the present study, an attempt has been made to elucidate the role of EPS produced by the heavy metal-resistant strain of Azotobacter spp. in uptake of Cd and Cr by wheat plants cultivated in the soils contaminated with the respective heavy metal ions.
Materials and Methods Isolation and Identification of Azotobacter Species. Soil samples from the vicinity of a manganese mine spoil dump near Gumgaon, India, were collected and screened for the presence of Azotobacter spp. using the soil paste method. Bacterial colonies tentatively identified to be of genus Azotobacter were purified, and further identification of the species level was done based on morphological, cultural, and biochemical characteristics (28). Determination of the Minimum Inhibitory Concentration (MIC) of Cd2+ and CrO42-. Minimum inhibitory concentration (MIC) of the metal ions was determined by the agar diffusion method (29). Stock solutions of Cd2+ and CrO42were prepared using CdCl2 and K2Cr2O7, respectively, in various concentrations ranging from 10-50 mg L-1. Two different media, namely, Jensen’s medium (30) and HEPESMES (HM) medium (31) were used. In this study, sucrose (a more suitable carbon source for Azotobacter) was used instead of arabinose used in the original HM medium. Culture Conditions for EPS Production. Jensen’s medium was used for EPS production (32). Bacterial cells were incubated at 30 °C at 200 rpm for 120 h. Recovery of Bacterial Cells and EPS. Methods given by Mullen et al. (33) and by Kim et al. (34) for recovery of bacterial cells and EPS, respectively, were employed with slight 10.1021/es900063b CCC: $40.75
2009 American Chemical Society
Published on Web 07/08/2009
modifications. Briefly, the broth culture was centrifuged at 10000 rpm. Supernatant was collected for extraction of EPS. The cell pellet was treated with 1 mM EDTA to extract cellbound (capsular) EPS and recentrifuged; the supernatant fraction was pooled with the fraction obtained in the previous step. The cell pellete was washed twice with cold 10 mM Ca(NO3)2 (pH 6.0), and the harvested biomass was dried at 60 °C for 24 h in an oven. For extraction of EPS, cold acetone (1:2 v/v) was added to the supernatant, and the mixture was shaken vigorously. The precipitated EPS were washed twice with cold acetone and then redissolved in distilled water. The EPS solution was dialyzed with distilled water for desalting. Purified EPS were obtained by freeze-drying of the dialyzed EPS solution. The dried bacterial cells and EPS were separately suspended in 10 mM Ca (NO3)2 (pH 6.0) at a final concentration of 12 mg (dry weight) mL-1 and 17 mg (dry weight) mL-1, respectively. Characterization of EPS. The protein content of EPS was determined by the Lowry protein assay (35) with bovine serum albumin as the standard. The carbohydrate content was determined by the anthrone reagent, using pure glucose as standard (36). Further analysis such as uronic acid, amino sugars, sulfate content, etc. was not carried out. EPS were also characterized by Fourier transform infrared (FT-IR) spectroscopy. The infrared spectrum of the dry EPS sample was recorded in a Bruker-Vertex 70-22 FT-IR spectrometer (Brukerophics, Germany). The samples were prepared as KBr discs. Biosorption of Cd2+ and CrO42-. Biosorption studies of Cd2+ and CrO42- were conducted separately using whole cells as well as EPS. The metal sorption (q) was determined using the following equation (37) q ) V(C i - C f)/1000W where V is the volume of solution in tube, W is the mass weight of adsorbent (whole cells or EPS) (g), and Ci and Cf are the initial and final concentration of metal in solution (mg L-1), respectively. Pot Culture Studies. Pot culture studies were carried out at three different concentrations of each metal (at MIC, half of the MIC, and above the MIC) in order to see the effect of increasing metal concentration on the total Azotobacter population. The following concentrations of the metals were selected: Cd, 10 mg kg-1 (half of the MIC); 20 mg kg-1 (at MIC); and 30 mg kg-1 (above the MIC); Cr, 5 mg kg-1 (half of the MIC); 10 mg kg-1 (at MIC); and 15 mg kg-1 (above the MIC). The soil used for the pot culture experiments was a sandy loam with pH 7.8 and organic carbon content (0.28%). Wheat, Triticum aestivum var. AKW1071, was used as a test crop. The soil was artificially contaminated with the solutions of CdCl2 (Cd2+) and K2Cr2O7 (CrO42-). Sufficient water was added to bring the soil to 50% of its water-holding capacity. The soil was preincubated with the respective metals for 2 weeks before sowing. An extra set of pots in which heavy metal was not added served as the uncontaminated control. Cultures of Azotobacter spp. were added as free cells (FC) and as immobilized cells (IC) at a concentration of ∼150 × 106 cfu g-1 each (38) in the respective treatments at the time of sowing. Azotobacter Count under Different Treatments. The total Azotobacter count from different treatments was estimated using the standard plate count method (39) at an interval of 30 days throughout the life cycle of wheat. Concentration of Cd and Cr in Wheat Plants. After harvest, the plant material was dried at 70 °C, and the metal concentration was determined following Misra and Chaturvedi (40), using an atomic absorption spectrophotometer (Varian AA-10). Statistical Analysis. All of the experiments were performed in triplicate, and the data represent the mean of three values
TABLE 1. Determination of Minimum Inhibitory Concentration (MIC) of Selected Heavy Metals (Cadmium and Chromium) against Azotobacter on Different Growth Supporting Media (N = 6) heavy metal cadmium
chromium
metal concentration (mg L-1) 10 20 30 50 10 20 30 50
zone of Inhibition (mm) Jensen’s medium
HM medium
MIC (ppm)
P
resistant resistant 20 ( 3 25 ( 1 resistant 17 ( 1 20 ( 2 25 ( 1
resistant resistant 25 ( 1 30 ( 2 resistant 17 ( 3 25 ( 1 23 ( 2
20
>0.05
10
>0.05
( standard deviation. Results of the MIC and metal ion biosorption studies were analyzed using students t- test. Results of the plant metal concentration and Azotobacter population under various treatments were analyzed by ANOVA. The correlation coefficient between the Azotobacter count and metal concentration in wheat plants was also calculated. All of the analyses were carried out using SPSS (11.0) software.
Results and Discussion Identification of Azotobacter Species. Slimy, glistening colonies, which appeared after 3-7 days of incubation at 27-30 °C on soil paste plates and turned brown with aging, were tentatively identified to be of Azotobacter spp. Morphologically, the cells were Gram negative, blunt, rod-shaped, and motile. Results of morphological, cultural, and biochemical analysis clearly indicated that the isolated strain was of Azotobacter spp. (28). The soil paste method employed in this study for preliminary isolation of Azotobacter from the mixed population of microorganisms in soil samples was found to be selective for the isolation of Azotobacter spp. as reported by Aquilanti et al. (41). The method is not only rapid and feasible but is also reliable and selective as compared to the other methods for the isolation of Azotobacter from soil samples such as (1) streaking of serial soil dilutions on plates containing Brown N free medium (42, 43) and (2) enrichment in Winogradsky solution for 7-14 days (44, 45) followed by streaking onto Brown agar. Determination of the Minimum Inhibitory Concentration (MIC) of Cd2+ and CrO42-. The concentration of the metal that permitted the bacterial growth and beyond which there was no growth (30) after 24-48 h of incubation was considered as the MIC of the metal for the given strain. The purpose of determining the MIC was to identify the actual concentration of Cd2+ and CrO42- that is tolerable by the strain so as to decide the concentrations of the respective metal ions for pot culture studies. In order to finalize the growth medium for further studies, a comparative assessment between the Jensen’s medium and HM medium was made. As presented in Table 1, the zone of inhibition recorded from the two media did not reveal any statistically significant difference (P > 0.05) in the case of the metal ions, and the MIC of Cd2+ and CrO42- was found to be 20 and 10 mg L-1, respectively, for the Azotobacter spp. It was considered that the MICs of selected heavy metals were the same irrespective of the medium used. Hence, Jensen’s medium can be used for further metal related studies. From the data of MIC studies, it is clear that CrO42- is more toxic to the strain than Cd2+. The results are in contrast with those reported by Ather and Ahmed (14) where the Azotobacter population in the heavy metal-contaminated soil VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
5885
was found to be more sensitive to Cd2+ than CrO42-, among a wide range of metals tested. Aleem et al. (30) carried out metal resistance studies on Azotobacter chroococcum; the MICs reported for Cd2+ as well as CrO42- were higher than those obtained in the present study. From the above observations, it is clear that heavy metal resistance in Azotobacter spp. is variable, which may depend on the species and growth conditions as well as the chemical composition of the soil from where the strain has been isolated, and no generalizations in this context can be made. Many laboratory media contain metal-binding (e.g., yeast extract) and metal-precipitating (e.g., phosphate or sulfate salts) components that can reduce solution-phase metal concentrations (46-49). Jensen’s agar medium, which contains dipotassium phosphate and ferrous sulfate, may precipitate the metal ions thus rendering them unavailable for the organism and yielding higher MIC. However, the toxicity test in such solid media could be useful in the evaluation of metal toxicity in contaminated soil, where conditions of diffusion, complexation, and availability of metal are different from those observed for liquid media (50). Another advantage to using nitrogen-free Jensen’s agar medium for the metal toxicity test for Azotobacter lies in the fact that the growth of the organism in this medium ensures its retention of nitrogen fixation potential at that particular metal concentration. HM medium used by Cole and Elkan (31) to study transmissible resistance to penicillin G., neomycin, and chloramphenicol in Rhzobium japonicum was also employed in this study to determine the MIC of Cd2+ and CrO42-. Because this medium contains HEPES and MES buffers, the pH of the medium is maintained around 6.6, at which the metal ions added in the medium are bioavailable. Other mineral salts used in the medium being in micro quantities were sufficient enough for the proper growth of the microorganisms but are not expected to bind the metal ions significantly. Characterization of EPS. Biochemical studies revealed that the EPS contained 0.0525 mg mg -1 of protein and 0.045 mg mg -1 of carbohydrates. Proteins and carbohydrates contribute the carboxyl (-COOH) and hydroxyl (-OH) moieties, which impart the negative charge to the EPS molecules. The negative charge gives the molecules a “sticky” quality. The “stickiness” is important in terms of the affinity of these EPS for binding to cations such as dissolved metals (18). The EPS from Azotobacter spp. were characterized by FTIR spectroscopy as shown in Figure S1 of the Supporting Information. The band at 3548.79 cm-1 could be assignable to the OH stretching frequency. A sharp peak at 1646.16 cm-1 assignable to the CdO vibration of carboxylic acid particularly shows that internal hydrogen bonding was observed. Peaks observed in the region between 1000-1200 cm-1 correspond to the presence of carbohydrates (51). The IR spectra of the EPS thus evidenced the presence of carboxyl and hydroxyl functional groups, which are known to be primarily involved in metal ion binding by forming coordination bonds (52-55). It is however difficult to confirm the presence of phosphate and sulfate groups of the EPS (which may also be present and involved in metal ion binding) in the IR spectra because the corresponding vibrations appear in a region where there is interference and overlapping of other functional groups (56). Biosorption Studies. Results of the biosorption studies of Cd2+ and CrO42- by whole cells and by EPS are shown in Figure 1. The data clearly indicated that the whole cells could not bind any of the metals significantly, whereas the EPS sorbed 15.17 ( 0.58 mg g-1 Cd2+ and 21.9 ( 0.08 mg g-1 CrO42-. The biosorption values obtained for Cd2+ in this study corroborated with that reported for other cations like Cu2+, 5886
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 15, 2009
FIGURE 1. Biosorption of cadmium and chromium by cells and EPS. Error bars represent ( standard deviation. Zn2+, and Fe2+ and are 15.5, 20, and 25 mg g-1 EPS, respectively (32). For copper, the maximum biosorption values of 1602 mg g-1 EPS by Paenibacillus polymyxa (57) and 320 mg g-1 EPS by a copper-resistant strain of Pseudomonas aeruginosa and 270 mg g-1 EPS by a copper-sensitive strain of P. aeruginosa (58) have been reported. Maximum uptake capacity of 154 mg Cd g-1 EPS was observed in the case of Alteromonas species (59). In contrast to the results obtained by Emtiazi et al. (32), Acosta et al. (57), and Kazy et al. (58), whole cells used in this study were not able to bind any of the metal ions significantly. The reason for such an unusual observation could be that in the case of the Azotobacter cells, which have been freed from capsular EPS by EDTA treatment, the cell wall LPS is the first target for cation binding (60). The cells were suspended in a Ca(NO3)2 solution, and there is a possibility that the Ca2+ ions had more affinity and hence preoccupied the binding sites on the LPS, thus resulting in insignificant metal ion (Cd2+ and CrO42-) binding. Whereas in the case of EPS material [which was also suspended in a Ca(NO3)2 solution], there is a possibility that the polysaccharides were different in the chemical composition (from cell wall LPS) and hence had more affinity for Cd2+ ions as compared to Ca2+ ions. Thus, in this case, the Cd2+ ions might be outcompeting Ca2+ ions for binding sites on EPS. Alternatively, protein moieties of the EPS could be the main macromolecules involved in metal ion binding, and lack of binding of metal ions by whole Azotobacter cells could be due to the fact that the cell wall LPS is mainly composed of polysaccharides. Similar findings have been reported by Sheng et al. (61), wherein under toxic conditions the protein/ carbohydrate ratios of EPS was greatly influenced. The ratios in the presence of toxic substances were always higher than that of the control, suggesting that extracellular proteins could protect cells against toxic substances. The above discussion on metal ion chelation by the negatively charged EPS stands true for Cd2+ ions. Binding of CrO42- ions to the EPS was supposed to have been accomplished through a well-known “calcium bridging mechanism” (62), wherein Ca2+ ions act as a bridge for the adsorption of two negatively charged moieties (in this case CrO42- ions and EPS). The findings, however, require further investigations to discover the exact mechanism of metal ion binding. Biosorption studies for the whole cells as well as for EPS were carried out in a Ca(NO3)2 solution, which was maintained at pH 6. In a Ca(NO3)2 matrix at pH 4.0, metals are predicted to be in the free ionic form (>97% in all cases) (33). However, in this study, pH of the Ca(NO3)2 solution was adjusted to pH 6 taking into account the activity and stability of functional groups of EPS with respect to metal ion binding (63). Biosorption investigations in the acidic pH range have demonstrated a reduction in the available metal-binding sites
because of protonation (64) or interaction between cations (H+) and negative charges of acidic functional groups of polysaccharide (65). However, as the pH increases the H+ ion concentration falls, and the heavy metal ions can compete to a greater extent at the binding sites on the EPS. The sorption reaction was given an equilibration time of 2 h. The time course of the metal adsorption has been reported to be complete after 2 h incubation at 25 °C for both cells (34) as well as EPS (57), and hence, the reaction was carried out for 2 h. Pot Culture Studies. Among heavy metals, Cd2+ and CrO42- have received considerable attention over the past few years because of their deleterious effects on the overall biota. These heavy metals have been of special interest as far as agricultural soil is concerned as the two are most frequently added to the soil through fertilizers, pesticides, and wastewater from tanneries, etc. Once in the soil, the heavy metals accumulate preferentially in the parts of the soil where the plant roots are concentrated and in forms easily accessible for plants (66). These heavy metals are subsequently taken up by the plants and thus enter the food chain. Estimation of Azotobacter Population under Different Treatments. The data presented in Tables S1 and S2 of the Supporting Information show the range of the Azotobacter count (min-max) in different treatments of Cd- and Crcontaminated soils, respectively. Upon first exposure (in the initial samples) to the heavy metals, the overall Azotobacter population declined gradually and significantly (P < 0.01) with increasing metal concentrations, irrespective of the treatment. In Cd-contaminated soil, the total Azotobacter count varied in the range of 2.4-2.5 × 1004, 1.3 × 1004, and 1.0-1.1 × 1004 cfu g-1 at 10, 20, and 30 mg kg-1 (dry weight), respectively. At 5, 10, and 15 mg kg-1 Cr, the total Azotobacter count varied in the range of 3.3-3.5 × 1004, 2.8-3.0 × 1004, and 2.5-2.9 × 1004 cfu g-1, respectively. The control sample (uncontaminated) showed the Azotobacter count in the range of 3.7-3.8 × 1004 cfu g-1. With time, the total Azotobacter count improved significantly (P < 0.01) in the treatments with the free (FC) and immobilized Azotobacter cells (IC) as compared to that of their respective metal-contaminated controls. This marked increase in the total number of Azotobacter in the treatments with the added heavy metalresistant strain of Azotobacter (in the form of FC and IC) indicated the survival and proliferation of this population under metal stress. Where the Azotobacter population in Cd-contaminated soil decreased in the post harvest samples (as compared to that of 40 and 80 DAS), the same in Cr-contaminated soil showed a gradual decline from 80 DAS to post harvest in all treatments. This observation suggested that the toxicity of Cr to the Azotobacter population was time dependent. Results of the pot culture study obtained in the present investigation are in agreement with those reported by the other authors who found that the number of indigenous asymbiotic nitrogen-fixing bacteria was adversely affected due to the heavy metal contamination of the soil (67) (14). Concentration of Cadmium and Chromium in Wheat Plants. Results of heavy metal concentration in wheat plants in Cd- and Cr-contaminated soils are depicted in Figures 2 and 3, respectively. It was observed that the metal content of the plants increased with increase in the total metal content of the soil. Plants grown in the Cd-contaminated soil amended with the heavy metal-resistant strain of Azotobacter showed reduction in Cd concentration by 35% (FC), 39.63% (IC) at 10 mg kg-1; 47.84% (FC), 49.28% (IC) at 20 mg kg-1; and 27.9% (FC), 16.91% (IC) at 30 mg kg-1 Cd. The percent reduction in plant metal ion concentration in the treatments with added Azotobacter cells was significant (P < 0.01) as compared to that of their respective (metal contaminated) controls. Surprisingly, in plants grown in Cr-contaminated
FIGURE 2. Cadmium concentration in wheat plants under different treatments. Error bars represent ( standard deviation. GS, garden soil; Cd 1, Cd (10 ppm); Cd 2, Cd (10 ppm) + free cells; Cd 3, Cd (10 ppm) + immobilized cells; Cd 4, Cd (20 ppm); Cd 5, Cd (20 ppm) + free cells; Cd 6, Cd (20 ppm) + immobilized cells; Cd 7, Cd (30 ppm); Cd 8, Cd (30 ppm) + free cells; and Cd 9, Cd (30 ppm) + immobilized cells.
FIGURE 3. Chromium concentration in wheat plants under different treatments, Error bars represent ( standard deviation, GS, garden soil; Cr 1, Cr(5 ppm); Cr 2, Cr (5 ppm) + free cells; Cr 3, Cr (5 ppm) + immobilized cells; Cr 4, Cr(10 ppm); Cr 5, Cr (10 ppm) + free cells; Cr 6, Cr (10 ppm) + immobilized cells; Cr 7, Cr (15 ppm); Cr 8, Cr(15 ppm) + free cells; and Cr 9: Cr (15 ppm) + immobilized cells. soils amended with free (FC) as well as immobilized Azotobacter cells (IC), the metal was not detected up to 10 mg kg -1. Any significant difference between the metal concentrations in plants grown with free (FC) and immobilized cells (IC) was not seen. The negative correlation coefficient between Azotobacter count and the plant metal (Cd, -0.496; Cr, -0.455; the correlation is significant at P < 0.01) suggested that as the Azotobacter population increased the metal concentration in the wheat plants decreased. The data presented for the Azotobacter population and plant metal concentration reveals the role of added Azotobacter spp. in restricting the uptake of metal ions by wheat plants; the underlying mechanism for the observed phenomenon is not certain. We believe that the EPS (the production of which has been reported to be stimulated in the presence of toxic substances, including heavy metals (61)) produced by the Azotobacter cells were involved in metal ion chelation (based on the findings of in vitro studies), thus rendering them unavailable to plants. However there is no direct evidence to claim the hypothesis, and a possibility that the bacterial cell surface (LPS), which otherwise could not sorb the metal ions (in vitro), could have been modified in the soil environment (contaminated with heavy metals) thus promoting the metal ion complexation (66) cannot be VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
5887
overlooked. Further research in this area could explore a new facet of EPS in bioremediation of metal contaminated soils. The overall findings confirm that a heavy metal-resistant strain of Azotobacter spp. has a high capacity to bind Cd and Cr not only in vitro but also in vivo and thus holds considerable promise for restricting their uptake by wheat plants grown in heavy metal-contaminated soils.
Acknowledgments The authors are thankful to Dr. Suresh Dhopte and Mrs. Sera Das (Instrumentation Division, NEERI, Nagpur) for their help in the heavy metal analysis and EPS characterization studies. The authors are also indebted to Ms. Radha Rani Mewaram (SRF, NEERI) for her help in revision of the manuscript.
Supporting Information Available FT-IR spectra of the EPS from Azotobacter spp. (Figure S1), tables representing the effect of different treatments on the Azotobacter population under cadmium and chromium contaminated soils (Table S1 and S2, respectively). This material is available free of charge via the Internet at http:// pubs.acs.org.
Literature Cited (1) Narula, N.; Lakshminarayana, K.; Tauro, P. Ammonia excretation by Azotobacter chroococcum. Biotechnol. Bioeng. 1981, 23, 467– 470. (2) Gordon, J. K.; Jacobson, M. R. Isolation and characterization of Azotobacter vinelandii mutant strains with potential as bacterial fertilizer. Can. J. Microbiol. 1983, 29, 973–978. (3) Gouri, P. S. V. M.; Jagasnnatathan, R. Biotechnology in organic farming. Biotechnol. Dev. Rev. 1995, 5, 34–47. (4) Maltseva, N. N.; Nadkernichnaya, E. V.; Kanivets, N. A. Associations of Nitrogen-Fixing Bacteria with Winter Rye; Proceedings of the 10th International Congress on Nitrogen Fixation; St. Petersburg, Russia, 1995, Vol. 614. (5) Mrkovackˇi, N.; Mezei, S.; Kovaceˇv, L. Effect of Azotobacter inoculation on dry matter mass and nitrogen content in the hybrid varieties of sugar beet. Period. Sci. Res. Field Veg. Crops. 1996, 25, 107–113. (6) Zahir, Z. A.; Arshad, M. Effectiveness of Azotobacter inoculation for improving potato yield under fertilized conditions. Pak. J. Agric. Sci. 1996, 33, 1–5. (7) Zahir, Z. A.; Arshad, M.; Hussain, A.; Sarfraz, M. Improving wheat yield by inoculation with Azotobacter under optimum fertiliser application. Pak. J. Agric. Sci. 1996, 11, 129–131. (8) Azcon, R.; Barea, J. M. Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandii and Azotobacter beijerinckii related to effects produced on tomato plants. Plant Soil 1975, 43, 609–619. (9) Salmeron, V.; Martinez-Toledo, M. V.; Gonzalez-Lopez, J. Nitrogen fixation and production of auxins, gibberellins and cytokinin by Azotobacter chroococcum strain isolated from root of Zea mays in presence of insoluble phosphate. Chemosphere. 1990, 20, 417–422. (10) Gonzales-Lopez, J.; Martinez-Toledo, M. V.; Reina, S.; Salmeron, V. Root exudates of maize on production of auxins, gibberellins, cytokinins, amino acids and vitamins by Azotobacter chroococcum chemically defined media and dialysed soil media. Toxicol. Environ. Chem. 1991, 33, 69–78. (11) Suneja, S.; Narula, N.; Anand, R. C.; Lakshminarayana, K. Relationship of Azotobacter chrooccum siderophores with nitrogen fixation. Folia Microbiol. 1996, 41 (2):), 154–158. (12) Mrkovacki, N.; Milic, Y. Use of Azotobacter chroococcum as potentially useful in agricultural application. Ann. Microbiol. 2001, 51, 145–158. (13) Pandey, A.; Sharma, E. S.; Palni, L. M. Influence of bacterial inoculation on maize in upland farming systems of the Sikkim Himalaya. Soil Biol. Biochem. 1998, 30, 379–384. (14) Ather, R.; Ahmed, M. Heavy metal toxicity: Effect on plant growth and metal uptake by wheat and on free living Azotobacter. Water, Air, Soil Pollut. 2004, 138, 165–180. (15) Nies, D. H. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol. 1999, 51 (6), 730–750. 5888
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 15, 2009
(16) Foster, J. Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev. 1983, 47, 361– 409. (17) Rouch, D.; Camakaris, J.; Lee, B. T.; Luke, R. K. Inducible plasmidmediated copper resistance in Escherichia coli. J. Gen. Microbiol. 1985, 131 (4), 939–943. (18) Sadoff, H. L.; Shimel, B.; Ellis, S. Characterization of Azotobacter vinetandii deoxyribonucleic acid and folded chromosomes. J. Bacteriol. 1979, 138, 871–877. (19) Robson, R.; Chesshire, J. A.; Wheeler, C.; Jones, R.; Woodley, P. R.; Postgate, J. R. Genome size and complexity in Azotobacter chroococcum. J. Gen. Microbiol. 1984, 130, 1603–1612. (20) Gorin, P. A. J.; Spencer, J. F. T. Exocellular alginic acid from Azotobacter vinelandii. Can. J. Chem. 1966, 44, 993–998. (21) Wingender J.; Neu, T. R.; Flemming, H. C. Microbial Extracellular Polymeric Substances: Characterization, Structures and Function. Springer: New York, 1999; Vol. 130. (22) Whitfield, C. Bacterial extracellular polysaccharides. Can. J. Microbiol. 1988, 34, 415–420. (23) Sutherland, I. W. Bacterial exopolysaccharides. Adv. Microbial Physiol. 1972, 8, 143–213. (24) Costerton, J. W.; Cheng, K. J.; Geesey, G. G.; Ladd, T. I.; Nickel, J. C.; Dasgupta, M. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 1987, 41, 435–464. (25) Dudman, W. F. The Role of Surface Polysaccharides in Natural Environments. In Surface Carbohydrates of the Prokaryotic Cell; Sutherland, I. W., Ed.; Academic Press: New York, 1977; Vol. 35, pp 7-414. (26) Postgate, J. R. Evolution within nitrogen fixing systems. Symp. Soc. Gen. Microbiol. 1974a, 24, 263–292. (27) Leigh, J. A.; Coplin, D. L. Exopolysaccharides in plant bacterial interactions. Ann. Rev. Microbiol. 1992, 46, 307–346. (28) Garrity, G.; Brenner, D. J.; Krieg, N. R.; Staley, J. T., Eds. Part C: The Proteobacteria; Part B: Gammaproteabacteria. In Bergey’s Manual of Systematic Bacteriology; Springer-Verlag: Berlin, 2005; Vol. 2. (29) Thompson, G. A.; Watling, R. J. A simple method for determination of bacterial resistance to metals. Bull. Environ. Contam. Toxicol. 1983, 31, 705–711. (30) Aleem, A.; Isar, J.; Malik, A. Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chroococcum isolated from rhizospheric soil. Bioresour. Technol. 2003, 86 (1), 7–13. (31) Cole, M. A.; Elkan, G. H. Transmissible resistance to penicillin G, neomycin. and chloramphenicol in Rhlizobiin japonicumn. Antimicrob. Agents Chemother. 1973, 4, 248–253. (32) Emtiazia, G.; Ethemadifara, Z.; Habibi, M. H. Production of extracellular polymer in Azotobacter and biosorption of metal by exopolymer. Afr. J. Biotechnol. 2004, 3 (6), 330–333. (33) Mullen, M. D.; Wolf, D. C.; Ferris, F G.; Beveridge, T. J.; Fleming, C. A.; Bailey, G. W. Bacterial sorption of heavy metal. Appl. Environ. Microbiol. 1989, 55 (12), 3143–3149. (34) Kim, S. Y.; Kim, J. H.; Kim, C. J.; Oh, D. K. Metal adsorption of the polysaccharide produced from Methylobacterium organophilum. Biotechnol. Lett. 1996, 18, 1161–1164. (35) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 1951, 193, 265–274. (36) Herbert, D.; Phipp, P. J.; Strange, R. E. Methods in Microbiology; Norris, J. R., Ribbons, D. W., Eds.; Academic Press: London, 1971; Vol. 5b, pp 266-269. (37) Volesky, B.; May-Phillips, H. A. Biosorption of heavy metals by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 1995, 42, 797–806. (38) Joshi, P.; Rayalu, S.; Bansiwal, A.; Juwarkar, A. A. Surface modified zeolite: A novel carrier material for Azotobacter chroococcum. Plant Soil 2007, 296, 151–158. (39) Peech, M. Hydrogen Ion Activity. In Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. Black, C. A., Ed., Agronomy Series No. 9; American Society of Agronomy: Madison, WI, 1965, pp 914-925. (40) Misra, V.; Chaturvedi, P. K. Plant uptake/bioavailability of heavy metals from the contaminated soil after treatment with humus soil and hydroxyapatite. Environ. Monit. Assess. 2007, 133, 169– 176. (41) Aquilanti, l.; Mannazzua, I.; Papaa, R.; Cavalcab, L.; Clementia, F. Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: A contribution to the study of these free-living nitrogen-fixing bacteria. J. Microbiol. Methods 2004, 57, 197–206. (42) Brown, M. E.; Burlingham, S. K.; Jackson, R. M. Studies on Azotobacter species in soil. I. Comparison of media and
(43) (44) (45) (46) (47) (48)
(49)
(50) (51) (52) (53) (54) (55)
(56)
techniques for counting Azotobacter in soil. Plant Soil 1962, 17, 309–319. Knowles, R. Free-Living Dinitrogen-Fixing Bacteria. In Methods of Soil Analysis; Black, C. A. , Ed.; Soil Science Society of America: Madison, WI, 1982; pp 10071-11077. Augier, J. A “propos de la nume” ration des Azotobacter en milieu liquide. In Masson et CIE; Annales de l’Institut Pasteur: Paris, 1956; Vol. 759, p 765. Pochon, J.; Tardieux, P. Techniques d’analyse en microbiologie du sol. De la Tourelle: St. Mande’, France, 1962. Hughes, M. N.; Poole, R. K. Metal Toxicity. In Metals and Microorganisms; Hughes, M. N., Poole, R. K., Eds.; Chapman and Hall: New York, 1989; Vol. 25, pp 2-302. Poulson, S. R.; Colberg, P. J. S.; Drever, J. I. Toxicity of heavy metals (Ni, Zn) to Desulfovibrio desulfuricans. Geomicrobiol. J. 1997, 14, 41–49. Bryan, G. W.; Langston, W. J. Bioavailability, accumulation, and effects of heavy metals in sediments with special reference to United Kingdom estuaries: A review. Environ. Pollut. 1992, 76, 89–131. Llanos, J.; Capasso, C.; Parisi, E.; Prieur, D.; Jeanthon, C. Susceptibility to heavy metals and cadmium accumulation in aerobic and anaerobic thermophilic microorganisms isolated from deep-sea hydrothermal vents. Curr. Microbiol. 2000, 41, 201–205. Hassen, A.; Saidi, N.; Cherif, M.; Boudabous, A. Resistance of environmental bacteria to heavy metals. Bioresour. Technol. 1998, 64, 7–15. Geesey, G. G.; Bremer, P. J. Laboratory-based model of microbiologically induced corrosion of copper. Appl. Environ. Microbiol. 1991, 57, 1956–1962. Cozzi, D.; Desideri, P. G.; Lepri, I. The mechanism of ion exchange with alginic acid. J. Chromatogr. 1969, 40, 130–137. Geddie, J. L.; Sutherland, I. W. Uptake of metals by bacterial polysaccharides. J. Appl. Bacteriol. 1993, 74, 467–472. Norberg, A. B.; Persson, H. Accumulation of heavy metal ions by Zoogloea ramigera. Biotechnol. Bioeng. 1984, 26, 239–246. Brown, M. J.; Lester, J. N. Role of bacterial extracellular polymers in metal uptake in pure bacterial culture and activated sludge. I. Effects of metal concentration. Water Res 1982, 16, 1539– 1558. Bellamy, L. J., Ed. The Infrared Spectra of Complex Molecules; John Wiley and Sons: New York, 1975; pp 311-365.
(57) Acosta, M. P.; Valdman, E.; Leite, S. G. F.; Battaglini, F.; Ruzal, S. M. Biosorption of copper by Paenibacillus polymyxa cells and their exopolysaccharide. World J. Microbiol. Biotechnol. 2005, 21, 1157–1163. (58) Kazy, K.; Sar, P.; Singh, S. P.; Sen, A. K.; D’Souza, S. F. Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: Synthesis, chemical nature. and copper binding. World J. Microbiol. Biotechnol. 2002, 18, 583– 588. (59) Loaec, M.; Olier., R.; Guezennec, J. Uptake of Pb, Cd, and Zn by a novel bacterial exopolysaccharides. Water Res. 1997, 31, 1171–1179. (60) Hagge, S. V.; Hammer, M. U.; Wiese, A.; Seydel, U.; Gutsmann, T. Calcium adsorption and displacement: characterization of lipid monolayers and their interaction with membrane-active peptides/proteins. BMC Biochem. 2006, 7 (15). (61) Sheng, G. P.; Yu, H. Q.; Yue, Z. B. Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances. Appl. Microbiol. Biotechnol. 2005, 69 (2), 216–222. (62) Ashwood, P.; Thompson, R. P. H.; Powel, J. J. Fine particles that adsorb lipopolysaccharide via bridging calcium cations may mimic bacterial pathogenicity towards cells. Exp. Biol. Med. 2007, 232, 107–117. (63) Sag, Y.; Kutsal, T. The use of Zoogloea ramigera in waste water treatment containing Cr(V1) and Cd(I1) ions. Biotechnol. Lett. 1989, 11, 145–148. (64) Savvaidis, I.; Hughes, M. N.; Poole, R. K. Copper biosorption by Pseudomonas cepacia and others strains. World J. Microbiol. Biotechnol. 2003, 19, 117–121. (65) Salehizadeh, H.; Shojaosadati, S. A. Removal of metal ions from aqueous solution by polysaccharide produced from Bacillus Firmus. Water Res. 2003, 37, 4231–4235. (66) Wilcke, W.; Mosbach, J.; Kobza, J.; Zech, W. Distribution of Al and heavy metals in bulk soil and aggregates at three sites contaminated by the emissions of a central Slovak Al smelter. Water, Air, Soil Pollut. 1998, 106, 389–402. (67) Ahmad, I.; Hayat, S.; Ahmad., A.; Samiullah., I. Effect of heavy metal on survival of certain groups of indigenous soil microbial population. J. Appl. Sci. Environ. Manage. 2005, 9 (1), 115–121.
ES900063B
VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
5889
