Grantee Research Project Results
1999 Progress 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 Period Covered by this Report: December 1, 1998 through November 30, 1999
Project Amount: $360,818
RFA: Exploratory Research - Environmental Engineering (1998) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Land and Waste Management , Sustainable and Healthy Communities
Objective:
The overall objective of this research is to develop high-affinity microbial bioadsorbents for heavy metal removal. Genetically engineered E. coli with surface-expressed peptide analogues (ECs) of phytochelatin will be utilized as microbial bioadsorbents for the removal of heavy metals such as cadmium (Cd), mercury (Hg), and lead (Pb). A series of synthetic genes encoding ECs ranging from 2 to 20 cysteine will be 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 will be co-expressed with the cellulose binding domain protein on the cell surface and used for developing bioadsorbent columns with cellulose-based support. This objective will be realized through research directed towards the following goals: (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 cellulose-binding domain (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 columns for continuous operation and testing with real samples.
Progress Summary/Accomplishments: 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 labelled with 35S cysteine, enables their ready detection by autoradiography. In the presence of 1 mM isopropyl-?-d-1-thiogalactopyranoside (IPTG), protein bands of 19?21 kDa were detected, indicating the synthesis of full-size fusions.
To test the ability of E. coli expressing ECs on surface in enhancing heavy metal adsorption, the binding of cadmium to E. coli transformed with pEC7, pEC8, pEC11, and pEC20 was monitored 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 did cells expressing EC7 or EC8. This result is consistent with the increasing number of metal-binding centers present.
To co-express CBD simultaneously, a compatible low copy number vector was used to target CBD to the surface. The lpp-ompA-cbd fragment was PCR-amplified from pTX210, and subcloned into pK184 to give pKCBD. Expression of Lpp-OmpA-CBD can be induced easily by IPTG. Production of Lpp-OmpA-CBD fusion protein was verified by immunoblotting with CBD antiserum. The localization of Lpp-OmpA-CBD in the membrane fraction from the fractionation experiment was verified by immunoblotting. After incubation with proteinase K for 1 hour at 30 C, no Lpp-OmpA-CBD fusions were detected by immunoblotting
To demonstrate that the whole cell expressing CBD on the surface can bind tightly on cellulose material, the cell suspensions were incubated with whatman filter paper and a cellulose matrix at room temperature for 1 hour. After the matrix was carefully removed from the cell suspension, cell density remaining in the suspension was measured. More than 40 percent of the cells were removed from the cell resuspensions of XL1-blue (pKCBD), while only 4 percent were removed from XL1-blue after incubation, suggesting the tight attachment of cells onto cellulose materials through the specific binding of CBD.
In summary, novel bioadsorbents based on recombinant E. coli with surface-expressed ECs (synthetic phytochelatins) have been developed with high specificity and affinity for cadmium. Co-expression of the cellulose binding domain on the cell surface enables the bioadsorbents to immobilize tightly to cellulose materials. In fact, the co-expression of two proteins on the cell surface with distinct functionalities has been demonstrated for the first time. The resulting bioadsorbent column can be applied continuously with other existing technologies for bulk heavy metal removal to comply with regulatory standards.
Future Activities:
In the second year, the focus will be on the selection of cellulose materials for cell immobilization. In addition, co-expression of CBD and EC20 on the cell surface will be investigated. The goal is to fine tune the ratio of CBD and EC20 to achieve the optimal cell binding and metal binding capability.Journal Articles:
No journal articles submitted with this report: View all 18 publications for this projectSupplemental Keywords:
treatment, bioadsorbant, heavy metal removal , genetic engineering., Air, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Water, Waste, Toxics, National Recommended Water Quality, Engineering, Mercury, Environmental Chemistry, Engineering, Chemistry, & Physics, Bioavailability, Bioremediation, heavy metals, wetland, bioadsorbent, phytochelation, regulatory standards, water quality, organophospherous hydrolase, lead, bioremediation model, cadmium, biological attenuation, aquatic, bioadsorption, fate and transport, synthetic genes, wastewater treatment, waste reduction, chemical transport, chemical speciation, cellulose binding, 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.