Grantee Research Project Results
Final 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 Amount: $390,000
RFA: Exploratory Research: Nanotechnology (2001) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
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 focused on the development of novel materials with increased affinity, capacity, and selectivity for target metals. Tunable biopolymers based on repeating elastin (ELP) 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 were evaluated. The objectives of this research project were 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.
Summary/Accomplishments (Outputs/Outcomes):
Construction of Elastin Biopolymers for Mercury Removal
ELP-based biopolymers using a polyhistidine tag as the metal chelating domain have been generated previously, 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 (IPTG). Thanks to 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 sulfate-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, et al., 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 30°C 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 (Figure 1). The Tt for ELP153MR was inline with the working temperature range of our 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 min. 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 a 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 (Figure 2). The low level of binding for cadmium at 100-fold excess is likely because of binding to other cysteine residues 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 with 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 25°C and remained fully functional even after four repeating cycles. In each cycle, the original 44 ppb mercury added was reduced to ≤ 1.6 ppb (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.
Cadmium Removal from Contaminated Soil by Tunable Biopolymers
The feasibility of using the ELP biopolymers as an effective extractant for heavy metal
removal from contaminated soil. The soil used in the study was coarse, slightly acidic, subsurface soil obtained from the City of San Bernardino Rapid Infiltration and Extraction wastewater treatment facility. The soil was found to be mostly sand, 91 percent, with low amounts of silts and clay. Other soil characteristics were as follows: pH 6.5, 0.27 percent total organic carbon, and a cation exchange capacity of 10.1 meq 100 g-1.
A biopolymer (ELPH12) composed of 78 ELP repeats and a tandem hexahistidine cluster (His-tag) was used in batch soil washing experiments to evaluate the efficiency of removing cadmium from contaminated soil. The affinity of ELPH12 toward cadmium was investigated by determining the stability constant (KL) of the biopolymer-metal complex in aqueous solution. After incubating different amounts of ELPH12 with 50 nmol of cadmium, the cadmium-biopolymer complex was recovered by precipitation and the amount of bound cadmium was measured. The stability constant (KL) was determined from the slope of a straight line relationship obtained by plotting the moles of cadmium bound per mole of ELPH12 versus the free metal concentration in solution. A log KL value of 6.8 was obtained, which is comparable to that obtained with a rhamnolipid biosurfactant previously used successfully for cadmium removal from soil (Prabhukumar, et al., 2003; Kostal, et al., 2005). When comparing to the values for cyanide (5.3), citrate (5.0), and EDTA (18.2), all of which are considered to be strong chelators for Cd(II), our result indicates very strong complexation between ELPH12 and cadmium.
Artificially contaminated soil was prepared by soaking a sandy soil in 1 mM cadmium nitrate for 3 months. The final cadmium content was determined to be 118 mg/kg soil. To evaluate the feasibility of extracting soil-bound cadmium with the ELPH12 biopolymers, contaminated soil was incubated overnight with various concentrations of ELPH12 dissolved in water. The pH of the wash solution remained near neutral even without the addition of buffer. As shown in Figure 4, cadmium removal increased with increasing ELPH12 concentrations; 38, 44, and 55 percent of the soil-bound cadmium was removed by 1.25, 2.5, and 5 mg/mL ELPH12 solutions, respectively. For comparison, soil washing with distilled water or with 1.25 mg/mL (0.036 mM) of ELP biopolymers containing no His-tag removed only 8 percent of the bound cadmium, indicating that the histidine cluster on ELPH12 is mainly responsible for the improved metal chelation. More importantly, the fraction of ELPH12 adsorbed onto soil was less than 10 percent in all soil washing experiments, a value slightly lower than soil not contaminated with cadmium. This reduction in sorption is important as a significantly lower concentration of ELPH12 (0.036 mM as compared to 5-10 mM of biosurfactants) was required to achieve similar extraction efficiencies when comparing to biosurfactants.
To compare the efficiency of the ELPH12 biopolymers with a commonly used chelating agent, ethylenediaminetetraacetic acid (EDTA), batch washing experiments also were performed using 0.036 mM EDTA. The amount of cadmium removed was only 50 percent of the value obtained using a similar concentration of ELPH12, suggesting that the ELPH12 biopolymer may be more efficient as a chelator for soil washing than EDTA on a molar basis.
To explore the possibility of sequential extraction using the minimum amount of biopolymers, three batches of soil washing with 1.25 mg/mL of ELPH12 were performed. The extent of cadmium removed increased to 55 percent in the second wash to a level comparable with the removal by 5 mg/mL ELPH12 in a single batch washing (Figure 4). Sequential washes with water, in contrast, did not improve the removal efficiency. Any additional wash, however, did not yield any higher level of cadmium removal. This may result from the presence of cadmium inside the pores of soil particles minimizing complexation with the biopolymers.
Figure 4. Comparison of Batch Soil Washings by ELPH12 and Various Extractants. (1) A single Batch washing with distilled water; (2) two sequential batch washings with distilled water; (3) a single batch washing with 1.25 mg/mL ELP without the histidine tag; (4) a single batch washing with 0.036 mM EDTA; (5) a single batch washing with 1.25 mg/mL ELPH12; (6) a single batch washing with 2.5 mg/mL ELPH12; (7) a single batch washing with 5 mg/mL ELPH12; and (8) two sequential batch washings with 1.25 mg/mL ELPH12.
Improvement Cadmium Soil Remediation by ELPEC20
We have successfully generated ELP biopolymers containing a polyhistidine tail (ELPH12) as the metal chelating domain, and demonstrated the feasibility of easy extraction of cadmium from contaminated soil in ex situ soil washing. Although the result was quite encouraging, the overall level of extraction was still lower than the potentially extractable fraction. Moreover, the nonselective nature of the polyhistidine domain resulted in significant coremoval of zinc, rendering it difficult to recover the extracted cadmium by simple precipitation. It should be noted that the flexibility of tailoring the desired metal-binding domain in the ELP biopolymer is a unique property that could be exploited easily for improved affinity and specificity for the target metals. Naturally occurring metal-binding peptides, such as metallothioneins are the main metal sequestering molecules used by plants or animals to immobilize metal ions, offering selective, high-affinity binding sites for cadmium as a result of the repetitive cysteine residues. Recently, a new class of metal-binding peptides known as synthetic phytochelatins (ECs) with the repetitive metal-binding motif (Glu-Cys)nGly were shown to have improved Cd2+ binding capability over that of metallothioneins. Incorporation of ECs into the ELP biopolymer is expected to significantly enhance its ability to extract cadmium from contaminated soil. The aim in the second year of the project was to present the use of a new generation of ELP biopolymer by taking advantage of the improved affinity and selectivity of synthetic phytochelatin (EC20), and to demonstrate the utility of ELPEC20 for enhanced extraction of cadmium in soil washing experiments.
The binding property of ELPEC20 was evaluated by incubating the biopolymers with a range of cadmium concentrations. After 1 hour incubation, the biopolymer-cadmium complex was recovered by precipitation and subjected to cadmium analysis. Cadmium binding increased linearly at lower concentrations with essentially all added cadmium removed under these conditions. A maximum binding ratio of 8.2 Cd2+ per ELPEC20 was observed, which is substantially higher than the ratio of 2.1 observed for ELPH12. This value also is consistent with the previously reported binding stoichiometry for EC20. More importantly, a similar maximum binding ratio also was obtained using size-exclusion chromatography, indicating that precipitation of the biopolymer-metal ion complex has no effect on cadmium binding. The stability constant (KL) of the biopolymer-cadmium complex was determined using a Ruzic plot. The log KL value of 5.2 for ELPEC20 is one order of magnitude higher than that obtained with ELPH12 (4.0) under the same binding condition. As a result, the use of ELPEC20 offers not only improved binding stoichiometry but also improved affinity over that of ELPH12.
One of the major limitations of the ELPH12 biopolymers is the lack of specificity. Even the presence of a low concentration of zinc was shown to inhibit and displace cadmium from the polyhistidine domain. To investigate whether use of the EC20 moiety affords improved selectivity, a competitive cadmium binding experiment was conducted in the presence of equal concentrations (molar) of either Ca2+, Mg2+, Fe2+, Zn2+, or Al3+. In all cases, no decrease in cadmium binding was detected. Even in the presence of 10-fold excess of these same metals, only Zn2+ reduced the Cd2+ binding ratio by 20 percent, indicating the selective nature of ELPEC20 towards cadmium (data not shown). This in combination with the improved affinity and binding ratio makes this biopolymer an attractive candidate for soil washing applications.
To test the efficiency of the ELPEC20 biopolymer in soil washing experiments, artificially contaminated sandy soil was used. The final cadmium content was determined to be 297 (± 18) mg/kg with 48.6 (± 7.6%), 29.1 (± 4.5%), and 11.2 (± 0.80%) of the bound cadmium in the exchangeable, oxidizable (bound to organic matter), and residual fraction, respectively. The cadmium content for the contaminated soil used in this study is three times higher than the soil used in our previously study using ELPH12. In addition, it has a higher total organic matter content, which leads to the lower level of easily exchangeable fraction.
Soil washing using either distilled water or buffer as controls removed only 2 percent of bound cadmium (Figure 5). The use of 50 μM ELPEC20 increased the extraction efficiency substantially with 25 percent of the bound cadmium removed (Figure 4). This doubled the amount extracted using the same concentration of ELPH12. Increasing the ELPEC20 concentration to 75 μM further improved the extraction efficiency to 40 percent, whereas virtually no increase in efficiency was observed with ELPH12. When the actual cadmium extracted that was associated with the ELP biopolymers was analyzed, 20 percent of extracted cadmium was associated with the ELPH12 biopolymers and the remainder was found in solution. Comparatively, 82 percent of extracted cadmium was complexed with the ELPEC20 biopolymer, a result consistent with the selective nature of the EC20 domain towards cadmium.
To explore whether the cadmium removal efficiency could be further improved at higher ELPEC20 concentrations, batch soil washing experiments were repeated using 150, 200, and 250 µM biopolymer. As shown in Figure 4, cadmium removal increased with increasing ELPEC20 concentration and up to 62 percent removal was achieved at a biopolymer concentration of 250 µM. This corresponds to 100 percent removal of the ion-exchangeable fraction and 45 percent of the tightly bound, less mobile/labile fraction bound to organic matter. These results are comparable to those reported with the use of either EDTA or ethylenediamine disuccinic acid (EDDS) for the extraction of soil-bound Cu2+ or Zn2+, which were shown to remove mostly exchangeable fraction and some of the organic-bound fraction. The major advantage of the ELPEC20 biopolymers, however, is the use of a significantly lower concentration (250 µM) of biopolymer as compared to 4 mM of EDTA or EDDS used in the other study, enabling the treatment of more contaminated soil with the same amount of ELPEC20 biopolymer.
The kinetics of the soil washing was investigated to determine whether 24 hours incubation is necessary for maximum cadmium extraction. Cadmium extraction by ELPEC20 showed a very rapid initial increase with more than 90 percent of the maximum extraction occurring within 1 hour. In comparison, less than 70 percent extraction was achieved with ELPH12 during the same duration. Complete extraction was observed within 4 hours of contact, demonstrating that this could be a very rapid technology with very minimum possibility for the degradation of biopolymers. The improved affinity and selectivity of ELPEC20 resulted in cadmium removal not only from the exchangeable fraction but also the organic-bound fraction within 1 hour, suggesting that this could be a rapid technology with minimum possibility for the biodegradation of biopolymers.
Figure 5. The Efficiency of Batch Soil Washing by ELPEC20 and ELPH12. Experiments were carried out with 1 g of soil at a constant 1:10 (wt/vol) soil to solution ratio. The percentage of cadmium removed was calculated based on an initial cadmium content of 296.9 mg/kg.
Journal Articles on this Report : 8 Displayed | Download in RIS Format
Other project views: | All 15 publications | 8 publications in selected types | All 8 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Chen W, Mulchandani A, Deshusses MA. Environmental biotechnology: challenges and opportunities for chemical engineers. AIChE Journal 2005;51(3):690-695. |
R829606 (Final) |
not available |
|
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 |
|
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 |
|
Kostal J, Prabhukumar G, Lao UL, Chen A, Matsumoto M, Mulchandani A, Chen W. Customizable biopolymers for heavy metal remediation. Journal of Nanoparticle Research 2005;7(4-5):517-523. |
R829606 (Final) |
not available |
|
Lao UL, Chen A, Matsumoto MR, Mulchandani A, Chen W. Cadmium removal from contaminated soil by thermally responsive elastin (ELPEC20) biopolymers. Biotechnology and Bioengineering 2007;98(2):349-355. |
R829606 (Final) |
|
|
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 |
|
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 |
|
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:
environmental biotechnology, bioremediation, pollution prevention, waste reduction, nanotechnology, arsenic removal, biodegradation, bioengineering, bioploymers, biopolymers, biotechnology, decontamination, decontamination of heavy metals, environmental sustainability, environmentally applicable nanoparticles, heavy metal recycling, heavy metals, industrial wastewater, innovative technologies, nanocatalysts, nanoparticle based remediation, nanoparticle remediation, nanoscale biopolymers, nanotechnology, remediation technologies,, RFA, Scientific Discipline, Water, Waste, POLLUTANTS/TOXICS, Sustainable Industry/Business, Wastewater, Sustainable Environment, Environmental Chemistry, Remediation, Chemicals, Arsenic, Technology for Sustainable Environment, Analytical Chemistry, Environmental Monitoring, Water Pollutants, New/Innovative technologies, Bioremediation, 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.