Grantee Research Project Results
2003 Progress Report: Nanoscale Biopolymers with Tunable Properties for Improved Decontamination and Recycling of Heavy Metals
EPA Grant Number: R829606Title: Nanoscale Biopolymers with Tunable Properties for Improved Decontamination and Recycling of Heavy Metals
Investigators: Chen, Wilfred , Mulchandani, Ashok , Matsumoto, Mark
Institution: University of California - Riverside
EPA Project Officer: Aja, Hayley
Project Period: February 1, 2002 through January 31, 2005
Project Period Covered by this Report: February 1, 2002 through January 31, 2003
Project Amount: $390,000
RFA: Exploratory Research: Nanotechnology (2001) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The objectives of this research project are to: (1) express various synthetic genes coding for tunable, metal-binding biopolymers with a wide range of transition temperatures; (2) investigate the ability of these biopolymers to self-assemble as aggregates under different pH and temperature; (3) elucidate the metal binding capability and selectivity of the various biopolymers toward mercury and arsenic; and (4) demonstrate the performance and durability of these tunable biopolymers in repeated batch operations for heavy metal removal. Conventional technologies such as precipitation-filtration, ion exchange, reverse osmosis, oxidation-reduction, and membrane separation often are inadequate to reduce heavy metal concentrations in wastewater to acceptable regulatory standards, and recent research has been focusing on the development of novel materials with increased affinity, capacity, and selectivity for target metals. Tunable biopolymers based on repeating elastin units offer an easy and flexible way to generate metal-binding materials for this application. The effectiveness of these tunable biopolymers for large-scale application will be evaluated.
Progress Summary:
Research Accomplishment in Year 2 of the Project
Construction of Elastin Biopolymers for Mercury Removal. Elastin-like polypeptide (ELP)-based biopolymers have been generated previously using a polyhistidine tag as the metal chelating domain, demonstrating the possibility of easy purification and regeneration for many repeating cycles. The use of histidine clusters, however, offers no selectivity, low affinity, and a narrow working pH range. To address these problems, we exploited the high specificity and affinity of metalloregulatory proteins toward their cognate heavy metal species as a novel chelating domain for an improved biopolymer design. Because the transition properties of ELP are a strong function of the chain length, initial designs were focused on obtaining biopolymers that would remain soluble under the desired temperature and pH conditions for mercury removal, but aggregate in response to a small environmental stimulus (either salt addition or an elevated temperature). To satisfy these requirements, ELP consisting of 153 VPGVG repeats was selected based on their estimated Tt of 33oC. A mercury-responsive metalloregulatory protein, MerR, was fused to the C-terminal to generate the biopolymer ELP153MR.
Biopolymers were produced using a modified induction procedure as described previously. Up to 800 mg/L purified biopolymers were obtained by cultivation in a rich nutrient broth medium for 48 hours without the addition of isopropyl-beta-D-thiogalactopyranoside. Because of the temperature responsive properties of the ELP domain, the biopolymers were easily purified by inverse temperature cycling to homogeneity, as judged by the presence of a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The correct molecular weights of the purified biopolymers were independently verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (data not shown).
Because the ELP concentration is known to affect the inverse phase transition behaviors (Prabhukumar and Matsumoto, 2003), the transition temperatures of the biopolymers were characterized as a function of concentration to determine the conditions that will provide a desired transition around 30oC for easy aggregation and recovery of biopolymer-mercury complexes. Turbidity measurements were used to determine the onset of folding and aggregation, and the value of Tt was defined as the temperature at which 50 percent turbidity occurred (see Figure 1). The Tt for ELP153MR was in line with the working temperature range of interest.
Figure 1. Transition Temperature as a Function of the ELP153MR Biopolymer Concentration
In addition to temperature, the pH behaviors of the biopolymers were investigated. Most industrial wastewaters generated from plating operations are acidic in nature, with pH in the range of 3 to 5. The ELP153MR biopolymer remained soluble from pH 4 to 7 (data not shown), and aggregation could be induced by increasing the temperature, demonstrating the adaptability of the biopolymers even to acidic environments. A similar binding stoichiometry of 0.5 mercury per biopolymer was observed at all tested pH values (from 4 to 8), a result consistent with the binding capacity of the native MerR protein, which is one mercury per MerR dimer. This result demonstrates that the mercury-binding function and the dimer formation of MerR are not affected by fusion to the ELP domain. Because similar binding behaviors were observed at all pH values, further experiments were focused on acidic contaminated water at pH 4.0.
To test the kinetics of mercury binding, stoichiometric amounts of ELP153MR and mercury were mixed and incubated from 0 to 60 minutes. After recovery by precipitation, the amount of Hg2+ bound to the aggregates was measured. No difference in the amount of mercury accumulated by ELP153MR was detected between the 0-minute and 60-minute samples, showing that mercury binding to MerR occurred in less than the few seconds required for sample mixing.
To demonstrate the in situ binding selectivity of the biopolymers, we investigated whether ELP153MR could bind mercury with the same stoichiometry in the presence of other heavy metals. ELP153MR was mixed with stoichiometric amounts of mercury and up to 100-fold molar excess of cadmium and zinc. In both cases, mercury binding is very specific; only mercury was bound in notable amounts and the extent of mercury binding was unaffected by the competing heavy metals (see Figure 2). The low levels of binding for cadmium at 100-fold excess likely is because of binding to other cysteine residues that are not involved in mercury binding.
Figure 2. Selectivity of the ELP153MR Biopolymer. Binding of mercury by ELP153MR in the presence of competing heavy metals. The amount of (O) mercury and (?) competing metals bound to the biopolymer is reported.
The ultimate use of the tunable biopolymers is in a recyclable system of continual mercury binding and extraction. Rapid regeneration of the mercury binding sites is essential in this case. Previously, we have demonstrated that sequestered cadmium can be removed from polyhistidines either by lowering the pH or by treating it with ethylenediaminetetracetic acid (EDTA). Because of the high affinity of MerR towards mercury, even 100 mM EDTA was unable to extract mercury from the Hg2+-biopolymer complexes at all pH values tested (4 to 8.8). Only the use of a strong complexing agent, 2-mercaptoethanol, at 50 mM concentration and pH 4 could effectively remove the bound mercury after two rounds of extraction. The regenerated biopolymer aggregates were resolubilized below 25oC, and they remained fully functional even after four repeating cycles. In each cycle, the original 44 ppb mercury added was reduced to less than or equal to 1.6 ppb (see Figure 3), a concentration below the required drinking water limit of 2 ppb.
Figure 3. Recycling of ELP153MR. In each cycle, 50 nmol of the protein were mixed with 4.36 nmol mercury. After the first heat precipitation, the amount of residual mercury (a) in the supernatant, the amount of mercury subsequently extracted from the biopolymers by the first extraction (b) and second (c) extraction step, and the amount of mercury remaining on the biopolymers after two extractions (d) are shown for four repeating cycles. The original amount of mercury added in each cycle (e) is shown on the right.
Future Activities:
During Year 3 of the project, we will focus on investigating the possibility of differentially precipitating different metal-binding biopolymers. Our objective is to demonstrate the powerful utility of these biopolymers for the differential sequestration and recovery of mixed metal wastes. We also will conduct additional experiments for soil flushing and batch reactor studies.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 15 publications | 8 publications in selected types | All 8 journal articles |
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Type | Citation | ||
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Kostal J, Mulchandani A, Gropp KE, Chen W. A temperature responsive biopolymer for mercury remediation. Environmental Science & Technology 2003;37(19):4457-4462. |
R829606 (2003) R829606 (Final) |
not available |
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Kostal J, Mulchandani A, Chen W. Affinity purification of plasmid DNA by temperature-triggered precipitation. Biotechnology and Bioengineering 2004;85(3):293-297. |
R829606 (2003) R829606 (Final) |
not available |
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Prabhukumar G, Matsumoto M, Mulchandani A, Chen W. Cadmium removal from contaminated soil by tunable biopolymers. Environmental Science & Technology 2004;38(11):3148-3152. |
R829606 (2003) R829606 (Final) |
not available |
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Shimazu M, Mulchandani A, Chen W. Thermally triggered purification and immobilization of elastin-OPH fusions. Biotechnology and Bioengineering 2003;81(1):74-79. |
R829606 (2003) R829606 (Final) |
not available |
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Stiborova H, Kostal J, Mulchandani A, Chen W. One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation. Biotechnology and Bioengineering 2003;82(5):605-611. |
R829606 (2003) R829606 (Final) |
not available |
Supplemental Keywords:
elastin-like polypeptide, EIP, environmental biotechnology, bioremediation, pollution prevention, waste reduction, nanotechnology, pollutants, toxics, sustainable industry, business, waste, water, analytical chemistry, arsenic, biochemistry, chemicals, chemistry, chemistry and materials science, engineering, environmental chemistry, environmental engineering, mercury, innovative technologies, physics, remediation, sustainable environment, technology for a sustainable environment, wastewater, water pollutants, arsenic removal, bioengineering, biodegradation, biopolymers, biotechnology, cadmium, decontamination, decontamination of heavy metals, environmental sustainability, environmentally applicable nanoparticles, heavy metal recycling, heavy metals, industrial wastewater, nanocatalysts, nanoparticle-based remediation, nanoparticle remediation, nanoscale biopolymers, remediation technologies., RFA, Scientific Discipline, Waste, Water, POLLUTANTS/TOXICS, Sustainable Industry/Business, Remediation, Wastewater, Environmental Chemistry, Sustainable Environment, Arsenic, Chemicals, Technology for Sustainable Environment, Analytical Chemistry, Environmental Monitoring, Bioremediation, New/Innovative technologies, Water Pollutants, Engineering, Environmental Engineering, Mercury, heavy metal recycling, decontamination, nanoparticle remediation, biopolymers, industrial wastewater, nanoscale biopolymers, bioengineering, nanotechnology, biodegradation, remediation technologies, environmental sustainability, arsenic removal, bio-engineering, nanocatalysts, biotechnology, environmentally applicable nanoparticles, decontamination of heavy metals, biochemistry, sustainability, cadmium, innovative technologies, heavy metals, bioploymersProgress 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.