Grantee Research Project Results
Final Report: Development of Novel Bioadsorbents for Heavy Metal Removal
EPA Grant Number: R827227Title: Development of Novel Bioadsorbents for Heavy Metal Removal
Investigators: Chen, Wilfred , Mulchandani, Ashok , Mehra, Rajesh
Institution: University of California - Riverside
EPA Project Officer: Aja, Hayley
Project Period: December 1, 1998 through November 30, 2001
Project Amount: $360,818
RFA: Exploratory Research - Environmental Engineering (1998) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Sustainable and Healthy Communities , Land and Waste Management
Objective:
The overall objective of this research project was to develop high-affinity microbial bioadsorbents for heavy metal removal. Genetically engineered Escherichia coli with surface-expressed peptide analogues (ECs) of phytochelatin were utilized as microbial bioadsorbents for the removal of heavy metals such as Cd, Hg, and Pb. A series of synthetic genes encoding ECs ranging from 2 to 20 cysteine was expressed on the cell surface, enabling metal sequestration in the absence of uptake. Recombinant bacteria that exhibit the best selectivity and affinity for heavy-metal accumulation were co-expressed with the cellulose-binding domain (CBD) protein on the cell surface and used for developing bioadsorbent columns with cellulose-based support. The overall objective was realized by research directed towards the following specific objectives: (1) expression of various ECs on the cell surface and selection of the best EC peptide(s) for heavy metals, Cd, Hg, and Pb, removal; (2) co-expression of the optimal EC(s) and CBD on the cell surface; (3) selection of cellulose material for cell immobilization; (4) development of cellulose-based bioadsorbent column for heavy-metal removal; and (5) characterization and optimization of bioadsorbent column for continuous operation and test with real samples.
Summary/Accomplishments (Outputs/Outcomes):
Enhanced Cadmium Removal by Cell-Surface-Expressed Synthetic Phytochelatins
Four different synthetic phytochelatins ranging from 7 to 20 cysteines (EC7, EC8, EC11, and EC20) were fused with Lpp-OmpA and expressed in JM105. The synthetic genes for ECs were introduced into plasmid pOP131 under control of the lac promoter to form plasmids pEC7, pEC8, pEC11, and pEC20, respectively. The high cysteine content of the fusion proteins, when labeled with 35S cysteine, enables their ready detection by autoradiography. In the presence of 1 mM IPTG, protein bands of 19-21 kDa were detected, indicating the synthesis of full-size fusions.
To test the metal-binding capability of synthetic phytochelatins, maltose-binding protein (MBP)-EC20 fusion proteins were purified from cultures of JM105(pM20) grown in the presence of Cd2+ using an amylose resin affinity column. The purity of the fusion protein was confirmed through SDS-PAGE and less than one equivalent of Cd2+ was found to associate with the purified MBP-EC20 fusions. A significantly higher stoichiometric ratio was obtained for the MBP-EC20 fusions when the proteins were reconstituted with Cd2+ after treatment with dithiothreitol (DTT). The ratios of Cd2+ to MBP-EC20 were determined to be 9.9, 10.1, and 9.8 in fractions 6, 7, and 8, respectively. The concentration of MBP-EC20 was determined by both the thiol assay and the Bradford method to ensure that the thiols in fractions 6-8 are not from the added DTT. Free DTT and DTT-Cd2+ complex were eluted in fractions 12-15. Because there is no cysteine residue in MBP, this result reflected the Cd2+ binding stoichiometry to EC20. It is generally accepted that mammalian metallothioneins (MTs) have a stoichiometric ratio of 7 for Cd2+ and Zn2+ and 12 for copper; our results demonstrated that EC20 has 40 percent higher Cd2+ binding capacity than that of MTs.
To test the ability of E. coli expressing ECs on surface in enhancing heavy metal adsorption, we monitored the binding of cadmium to E. coli transformed with pEC7, pEC8, pEC11, and pEC20 through atomic absorption spectrometry. E. coli carrying pUC18 was used as a control. Cells were grown in MJS medium supplemented with 1 mM cadmium, and metal binding was monitored 5 hours and 16 hours after induction. Strains displaying the ECs on the surface accumulated a substantially higher amount of Cd (up to 50 nmol/mg of dry weight of cell) than cells carrying pUC18. From this result, it is clear that synthetic phytochelatins with up to 20 cysteines can bind metal with very high affinity. In fact, the amount of Cd2+ accumulated increases with increasing cysteine residues on the ECs. Cells with EC20 expressed on the surface accumulated almost twice the amount of Cd as compared to cells expressing EC7 or EC8. This result is consistent with the increasing number of metal-binding centers present.
Enhanced Hg2+ Accumulation by Genetically Engineered E. coli
Overexpression of metal-binding proteins such as MTs in bacterial cells resulted in enhanced Hg2+ accumulation and thus offers a promising strategy for the development of microbial-based biosorbents for the removal and recovery of Hg2+ from contaminated water or soil. However, Hg2+ removal by intracellular accumulation has been problematic because of the limited metal uptake. This uptake limitation could be potentially overcome either by co-expressing an Hg2+ transport system or by anchoring the metal-binding proteins directly on the cell surface. To facilitate the transport of Hg2+ across the cell membrane, the Hg2+ transport proteins, MerP and MerT, were co-expressed with MBP-EC20. Plasmid pCLTP, containing the merT and merP genes, were co-transformed with pMC20. Transformed cells were selected on (LB) plates containing ampicillin and spectinomycin. For comparison, E. coli strain JM109 carrying only pMC20 also was used. An alternate strategy to bypass Hg2+ uptake is to directly anchor EC20 on the cell surface. Plasmid pLO20, expressing the Lpp-OmpA-EC20 fusion, was used in this study.
To investigate the Hg2+ binding stoichiometry of EC20, MBP-EC20 fusion proteins were purified from cultures of JM109(pM20) using an amylose resin affinity column (New England BioLabs). Five nmol of the purified fusion protein was resuspended in 50 mM Tris-Cl buffer (pH 7.4) supplemented with 5 mM DTT and incubated with 1 to 1,200 nmol Hg2+ for 2 hours. Hg(II)-glutathione complexes were used instead of HgCl2 to prevent precipitation as reported previously. The protein-Hg2+ complex was recovered using a Microcon centrifugal filter membrane (Millipore) and the amount of bound Hg2+ was measured by cold-vapor atomic absorption spectroscopy (Coleman Model 5B Mercury Analyzer System). A saturating ratio of 20 Hg2+ per MBP-EC20 was obtained, a value much higher than the typical ratio of 7 reported for MTs. A similar binding experiment was conducted with purified MBP with no significant binding of Hg2+ observed.
To investigate the effect of uptake on bioaccumulation of Hg2+, the binding capability of various E. coli strains was compared. Overnight cultures grown in LB medium at 37°C were harvested, washed with distilled water twice, and resuspended to a final OD600 of 1.0 in LB medium containing 5 µM Hg2+. The Hg2+ content was determined after 1 hour. E. coli strain JM109/pUC18 accumulated a very low level of Hg2+. The intracellular accumulation of Hg2+ increased by sixfold for cells over-expressing MBP-EC20 (JM109/pMC20). By eliminating Hg2+ uptake, cells with EC20 anchored on the cell surface (JM109/pLO20) accumulated about threefold more Hg2+ than cells with EC20 expressed in the cytoplasm. This threefold improvement is in good agreement with our earlier observation with Cd2+ accumulation using cells with surface-expressed EC20. In the presence of the Hg2+ transporters (JM109/pCLTP/pMC20), intracellular accumulation of Hg2+ also increased significantly. The level of Hg2+ accumulation was similar to cells expressing EC20 on the surface. In both cases, 100 percent of the added Hg2+ was removed after 1 hour. These results indicate that uptake is indeed the rate-limiting step for the intracellular accumulation of Hg2+.
To determine the benefits on the rate of Hg2+ bioaccumulation, a time-course assay was conducted. Overnight cultures were harvested, washed with distilled water twice, and resuspended to a final OD600 of 1.0 in LB medium containing 5 µM Hg2+. Cells expressing only MBP-EC20 (JM105/pMC20) accumulated Hg2+ at a very slow rate with less than 20 percent removed after 20 minutes. Co-expression of the Hg2+ transporters and MBP-EC20 (JM105/pCLTP/pMC20) improved the bioaccumulation rate significantly with 95 percent of the added Hg2+ removed within 20 minutes. These results again confirmed that Hg2+ uptake is the rate-limiting step in the bioaccumulation of Hg2+. However, the rate of bioaccumulation was further improved for JM105/pLO20 cells with EC20 expressed on the surface; more than 95 percent of the added Hg2+ was removed within 1 minute. It appears that the introduction of EC20 on the cell surface is even more effective in eliminating the uptake limitation, resulting in virtually instantaneous removal of Hg2+.
Enhanced Heavy Metal Removal by Genetically Engineered Moraxella sp
Because lab-born E. coli strains are not suitable for in situ soil remediation, a more realistic approach is to engineer soil bacteria that are known to survive in contaminated environments for an extended period. Particularly interesting are Pseudomonas and related species such as Moraxella sp. The initial challenge is to develop an efficient system to target EC20 on the surface of these gram-negative bacteria. Ice-nucleation protein (INP) is an outer membrane protein from Pseudomonas syringae that acts as a template for ice nucleation. Both INP and the truncated version of INP containing only the N- and C- terminal portion (INPNC) can be used to target proteins to the cell surface of E. coli, Salmonella, and Moraxella sp. We recently demonstrated that expression of organophosphorus hydrolase on the cell surface was more efficient in Moraxella sp. than in E. coli using the INP anchor. This strategy could be used to further improve the whole cell accumulation of heavy metal with surface-expressed EC20.
For expression of INPNC-EC20 in Moraxella sp., plasmid pINP20, carrying the INPC-EC20 fusion was constructed by inserting the INPC-EC20 fragment into a shuttle vector pVLT33. Expression of the INPNC-EC20 fusion was under control of a tac promoter. Induced cultures of Moraxella sp. were viable during prolonged incubation for 24 hours. Although EC20 was expressed in both E. coli and Moraxella sp. as demonstrated by Western blotting with the INPNC antiserum, the level of expression was about threefold higher in Moraxella sp., judging from the intensity of the band corresponding to INPC-EC20. To investigate whether the INPC-EC20 fusion proteins were displayed on the bacterial surface in a stable conformation, immunofluorescence microscopy was used. Cells were probed with rabbit anti-INPNC serum as a primary antibody and then fluorescently stained with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG antibody. Cells harboring pINP20 were brightly fluorescent, indicating that the INPNC fusions was successfully displayed on the surface. Cells carrying only pVLT33 were not stained at all with the FITC-labeled secondary antibody.
The Hg2+ bioaccumulation capability of whole cells expressing EC20 was tested by monitoring the binding of Hg2+ by cold-vapor atomic absorption spectroscopy (Coleman Model 5B Mercury Analyzer System). Strains expressing EC20 on the cell surface accumulated a significantly higher amount of Hg2+ than cells carrying pVLT33. The amount of Hg2+ accumulated was almost tenfold and threefold higher than that of E. coli expressing INPC-EC20 or Lpp-OmpA-EC20, respectively. This result is consistent with the higher level of surface-expressed EC20 in Moraxella sp.
Heavy-Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose
Great success in heavy-metal removal by microbial-based systems has been achieved by using organisms overexpressing either MTs or ECs. However, peptide-based systems have so far failed to attract much attention due to the tedious protocol and the high cost associated with purification. In recent years, several affinity-tag systems such as hexahistidines, glutathione S-transferase, or MBP have been used for one-step purification and immobilization of enzymes or proteins. These systems, unfortunately, require costly affinity matrices and are too expensive for large-scale applications.
CBDs, which bind specifically to cellulose, have been isolated from a variety of cellulolytic bacteria. Because of their high affinity towards cellulose, CBD has been exploited as an affinity tag for the purification and immobilization of heterologous fusion proteins onto cellulose supports. Fusion proteins containing a CBD moiety could be constructed so that little or no alternation in the properties of the fusion partner is observed. The CBD cellulose-affinity system is attractive because it does not require a derivatized matrix, and cellulose is available in a variety of inexpensive forms, such as preformed microporous beads, highly adsorbent sponge, cloth, or microcrystalline powders.
The potential of using immobilized CBD-EC20 fusion proteins for heavy-metal removal was examined. Purification and immobilization of the CBD fusions were achieved by incubating cell extracts with different amounts of Avicel A1 (Sigma) in the presence of 0.1 percent Triton-X100. After extensive washing, the bound and unbound fractions were subjected to SDS-PAGE analysis. The percentage of CBDclos-EC20 bound increased with increasing amount of Avicel present. With 240 mg Avicel added, more than 95 percent of the soluble CBDclos-EC20 fusions was removed from the supernatant and was found to bind to Avivel. Remarkably, nearly all CBD-EC20 fusions produced were both soluble and functional. Approximately 22 mg/L of functional CBD-EC20 fusion was produced under these conditions.
To illustrate the metal-binding functionality of the immobilized CBDclos-EC20, Avicel-bound CBDclos-EC20 was prepared as described above. Consistent with our reported binding stoichiometry for EC205, a ratio of approximately 10 Cd2+ per immobilized CBDclos-EC20 was observed. In contrast, essentially no Cd2+ removal was observed with the Avicel-bound CBDclos, indicating that the functionality of the EC20 alone was responsible for Cd2+ removal. The bound Cd2+ could be removed by the addition of ethylenediaminetetraacetic acid (EDTA); more than 99 percent removal was achieved by incubating with 25 mM EDTA within 30 minutes. Regenerated sorbents again retained the same metal-binding capability with a similar metal-binding ratio of approximately 10 Cd2+ per immobilized CBDclos-EC20 for two additional cycles.
The immobilization of CBDclos-EC20 onto CF11 cellulose was used to investigate the possibility of removing very low levels of Cd2+ in repeated operations. A 0.8-cm diameter column was prepared by loading 560 µg of CBDclos-EC20 onto 0.32 mg of CF11 cellulose. One-mL fractions of 0.9 ppm solution of CdCl2 then were added to the column, and the solution eluted out of the column was collected and analyzed for Cd2+. One hundred percent removal of Cd2+ was retained for the first 21 fractions, followed by a gradual increase in the effluent Cd2+ concentration. Complete breakthrough was not detected until the 43rd fraction. The total amount of Cd2+ removed was determined to be 25.6 µg by integrating the breakthrough curve. This corresponds to a ratio of approximately 11 Cd2+ per EC20, a value consistent with the expected stoichiometry. In contrast, breakthrough occurred after only one fraction for a similar column with only CBDclos immobilized. These results clearly indicated the high affinity of EC20 for Cd2+ even at the ppm level. This is precisely what is needed for this strategy to be useful as an efficient polishing process for heavy-metal removal. Again, regeneration of the immobilized sorbents was accomplished by adding 25 mM EDTA. All bound Cd2+ was removed after the third washing.
Factors Influencing Whole-Cell Binding to Cellulose Materials
The binding of CBD to cellulose materials has been reported to be strongly affected by temperature, pH, and ionic strength. Previous studies employing CBD as an affinity tag for enzyme immobilization also have shown the same influences. Although whole-cell immobilization via the action of surface-expressed CBD has been demonstrated, the precise conditions for optimal binding have not been elucidated. Our focus in this part of the project was to identify the optimal whole-cell immobilization conditions for future bioreactor applications.
The effect of pH on whole-cell immobilization was first investigated. Previous reports have shown that the binding of CBDcex is strongly affected by pH with optimal binding at pH 7. Binding affinity decreases dramatically at lower pH. To investigate the effect of pH on cell binding, 150 mg of wet cells with or without CBD on the cell surface were incubated with a 4 x 4 cm cellulose fiber at room temperature for 24 hours at a pH ranging from 4-9.5. After the fiber was carefully removed from the cell suspension, the amount of cells (based on optical density, protein, and wet-weight measurements) remaining in the suspension was measured. Cells with CBD on the surface bound much stronger to the cellulose fiber than the control cells at a pH higher than 6.0. Although cell binding also was significant at a pH of 4.0, this mostly wasdue to nonspecific interaction between the cells and the fiber as even the control cultures showed very significant binding. These results agreed very nicely with the pH binding profile of CBDcex.
The effect of temperature on cell binding was investigated. Cell binding was determined as before. For CBDcex or CBD fusion enzymes, binding affinity has always been shown to be the highest at 4°C. However, our results indicated that binding at 37°C actually provided the highest whole-cell immobilization. This observation was unexpected; even others have reported their initial whole-cell binding experiments at 4°C. Our aim is to eventually operate the immobilization cell bioreactor for the degradation of organophosphates. Therefore, these results suggest that 37°C may be an ideal temperature not only for the metal removal, but also for cell immobilization.
For CBD fusion enzymes, immobilized enzymes were found to leak from the column at a slow rate. This leakage could be overcome by using two CBDs in the fusion enzymes instead of one. Similarly, we expect the number of CBD molecules anchored on the cell surface also could influence that whole-cell binding to cellulose. To investigate this effect, a regulated expression system was employed to fine-tune the expression of CBD to the cell surface. Clearly, the amount of CBD anchored on the surface has a direct correlation with cell immobilization. Whole-cell binding increased with higher CBD expression until the binding was saturated. This is very important, because one of the major objectives is the coexpression of CBD and ECs on the cell surface at the same time. Our result suggests that even a low level of CBD may be sufficient to provide maximum cell-immobilization efficiency.
Journal Articles on this Report : 10 Displayed | Download in RIS Format
Other project views: | All 18 publications | 10 publications in selected types | All 10 journal articles |
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Type | Citation | ||
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Bae W, Chen W, Mulchandani A, Mehra RK. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and Bioengineering 2000;70(5):518-524. |
R827227 (2000) R827227 (Final) |
not available |
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Bae W, Mehra RK, Mulchandani A, Chen W. Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury. Applied and Environmental Microbiology 2001;67(11):5335-5338. |
R827227 (Final) |
not available |
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Bae W, Mulchandani A, Chen W. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. Journal of Inorganic Biochemistry 2002;88(2):223-227. |
R827227 (Final) |
not available |
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Bontidean I, Ahlqvist J, Mulchandani A, Chen W, Bae W, Mehar RK, Mortari A, Csoregi E. Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics 2003;18(5-6):547-553. |
R827227 (Final) |
not available |
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Chen W, Georgiou G. Cell surface display of heterologous proteins: From high-throughput screening to environmental applications. Biotechnology and Bioengineering 2002;79(5):496-503. |
R827227 (Final) |
not available |
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Shimazu M, Mulchandani A, Chen W. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology Progress 2001;17(1):76-80. |
R827227 (Final) |
not available |
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Shimazu M, Mulchandani A, Chen W. Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp with surface-expressed organophosphorus hydrolase. Biotechnology and Bioengineering 2001;76(4):318-324. |
R827227 (Final) |
not available |
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Wang AA, Mulchandani A, Chen W. Whole-cell immobilization using cell surface-exposed cellulose-binding domain. Biotechnology Progress 2001;17(3):407-411 |
R827227 (2000) R827227 (Final) |
Exit Exit |
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Wang AJA, Mulchandani A, Chen W. Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase. Applied and Environmental Microbiology 2002;68(4):1684-1689. |
R827227 (Final) |
not available |
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Xu ZH, Bae W, Mulchandani A, Mehra RK, Chen W. Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose. Biomacromolecules 2002;3(3):462-465. |
R827227 (Final) |
not available |
Supplemental Keywords:
environmental biotechnology, bioremediation, pollution prevention, waste reduction., Scientific Discipline, Air, Toxics, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, National Recommended Water Quality, Environmental Chemistry, Bioremediation, Engineering, Chemistry, & Physics, Mercury, fate and transport, bioadsorption, aquatic, wastewater treatment, bioremediation model, waste reduction, chemical speciation, phytochelation, cellulose binding, lead, chemical transport, metal binding, biological attenuation, bioadsorbent, organophospherous hydrolase, synthetic genes, water quality, wetland, cadmium, heavy metals, mercury concentrationsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.