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Ohio Sea Grant College Program
and Stone Laboratory

Ohio Sea Grant and Stone Laboratory

Biocomplexation of Heavy Metals by Engineered Algae

Project Number: R/BT-004, Progress Report

Start Date: 3/1/1998

Completion Date: 2/28/2000

Revision Date: 3/12/2012

Principal Investigator(s)1.Richard T. Sayre, Biochemistry and Plant Biology The Ohio State University*
Co-Principal Investigator(s)2.Terry Gustafson, Chemistry The Ohio State University*
3.Lada Malek, Biology Lakehead University*
4.Samuel J. Traina, The Ohio State University
This shows the current affiliation and may not match affiliation at time of participation. *

Funding Record

Source: Ohio Sea Grant College Program
Source FundState MatchPass Through
First Year$ 97,139.00$ 62,648.00$ 0.00
Second Year$ 99,604.00$ 64,328.00$ 0.00
Total$ 196,743.00$ 126,976.00$ 0.00

Objectives

The objective of our proposal is to develop a renewable biological system to selectively recover toxic heavy metals from contaminated sites and waste streams.

Abstract

On a global basis, trace metal pollution is among the most pervasive and serious environmental problem facing the biosphere. Trace metals pose particular problems for environmental control because of their chemical stability; they must be sequestered from the media they have contaminated. Traditional methods of sequestering trace elements have involved chemical engineering approaches in which the elements are precipitated or sorbed from the medium. These methods are rarely selective and result in large volumes of waste. In contrast, living organisms have been shown to selectively sequester heavy metals by a variety of methods including: charged groups (carboxylic acid, amino and sulfate groups) and heavy metal binding proteins (e.g., metallothioneins) The algae have many features which make them ideal candidates for the selective removal and concentration of heavy metals. Some of these features include:
  1. tolerance to heavy metals [100 µM]
  2. many can grow autotrophically as well as heterotrophically
  3. large surface area/volume ratios
  4. phototaxy (a useful feature for harvesting)
  5. expression of phytochelatins (heavy metal binding peptides)
  6. high non-specific metal binding capacities and
  7. they have the potential for genetic manipulation.
The objective of our proposal is to develop a renewable biological system to selectively recover toxic heavy metals from contaminated sites and waste streams. Our experimental strategy will be:
  1. to further characterize the chemistry of the heavy metal binding sites of the algae
  2. introduce genes and gene fusions encoding heavy metal binding domains into the model transformable alga, Chlamydomonas reinhardtii, and
  3. chemically cross-link and stabilize dried algal powders with enhanced heavy metal binding properties.
Ultimately, these "heavy metal sponges" will be used to bioremediate heavy metal contaminated sub-surface, surface, and waste stream sites, as well as recover heavy metals from aqueous waste effluents. We propose that a biologically renewable heavy metal recovery system will provide a less expensive and more selective alternative to the chemical based precipitation or ion exchange systems which are currently in use.

Rationale

On a global basis, trace metal pollution is among the most pervasive and serious environmental problem facing the biosphere. Trace metals pose particular problems for environmental control because of their chemical stability; they must be sequestered from the media they have contaminated. Traditional methods of sequestering trace elements have involved chemical engineering approaches in which the elements are precipitated or sorbed from the medium. These methods are rarely selective and result in large volumes of waste. In contrast, living organisms have been shown to selectively sequester heavy metals by a variety of methods including: Charged groups (carboxylic acid, amino and sulfate groups) and heavy metal binding proteins (e.g., metallothioneis). The algae have many features which make them ideal candidates for the selective removal and concentration of heavy metals. Some of these features include:
  1. tolerance to heavy metals [100 µM]
  2. many can grow autotrophically as well as heterotrophically
  3. large surface area/volume ratios
  4. phototaxy (a useful feature for harvesting)
  5. expression of phytochelatins (heavy metal binding peptides)
  6. high non-specific metal binding capacities
  7. they have the potential for genetic manipulation

Methodology

Our experimental strategy will be: 1) to further characterize the heavy metal binding sites chemistry of the algae, 2) introduce genes and gene fusions encoding heavy metal binding domains into the model transformable alga, Chlamydomonas reinhardtii, and 3)chemically cross-link and stabilize dried algal powders with enhanced heavy metal binding properties. Ultimately, these "heavy metal sponges" will be used to bioremediate heavy metal contaminated sub-surface, surface, and waste stream sites, as well as recover heavy metals from aqueous waste effluents. We propose that a biologically renewable heavy metal recovery system will provide a less expensive and more selective alternative to the chemical based precipitation or iron exchange systems which are currently in use.

Benefits & Accomplishments

  1. We have characterized the number and chemistry of the functional metal binding sites on freeze-dried Chlamydomonas cells for the following metals, cadmium, selenium, uranium, copper, chromate.The pkas of the fiunctional groups were determined as well as the binding capacity for the various metals. It also was demonstrated that cadmium binding was reversible as a function of the pH and that up to 16 cycles of metal binding and release had little effect on the total metal binding capacity. In addition, it was demonstrated that other monon- and divalent cations had little effect on cadmium binding capacity in mixed metal solutions.
  2. We have developed a new shuttle vector for nuclear transformation of Chlamydomonas. The vector has 16 multi-cloning sites, a strong B-tubulin promoter an E.coli origin of replication and ampicillin and bleomycin resistance selectable markers.
  3. Genes controlling cysteine and proline synthesis were introduced in to Chlamydomonas using the above vector. The transformed algae are resistant to 5-times the lethal cadmium concentration for the non-transformants.The cells expressing higher levels of cysteine bind 50% more metal at cadmium concentration that induce phytochelatin synthesis. This is the first gene that increase the binding capacity of the cells at cadmium concentrations that induce phytochelatin synthesis.Currently, we are measuring intra-cellualr proline and cysteine concentration in these transformants to determine if the levels of these amino acids is altered in the transformants and is correlated with the heavy metal content. This phase of the project should be completed by the end of June, 2000.
  4. Insertional mutants have been generated that have apparent interruptions in the gene(s) encuding the glutathione conjugate export pump. This pump has been shown to regulate steady-state levels of metal and organic pollutants in the cells. These mutants accumulate cadmium at cadmium levels in the growth medium that typically do not induce phytochelatin expression. In addition the mutants only have a verapamil insensitive xenobiotic export pathway.These results suggest that the expoort of xenobiotic- or Cd-phytochelatin conjugates by a vereapmil sensitive ABC transporter has been blocked. The interrupted gene is currently being identified.
  5. We have generated transgenic Chlamydomonas expressing synthetic genes encoding a plasmamembrne anchoring protein fused to monomers or polymers (1-5) of the alpha- or beta-domain of mettalothionein-II (MT-II), or the complete and intact MT-II. These cells are tolerant to very toxic concentrations of cadmium. Currently, we are determining the cadmium binding capacity and site of cadmium sequestration in these transformants. This phase of the project will be completed in three months.
  6. We have characterized the chemistry of cadmium binding sites including bond distances and ligand identity by EXAFS spectroscopy at the Stanford Synchrotron.

Publications & Media

Peer-reviewed reprints
Peer-reviewed reprintsCai, X-H., Bown, C., Adihya, C., Traina, S., and R.T. Sayre. 1999, Growth and Heavy Metal Binding Properties of Transgenic Algae (Chlamydomonas reinhardtii) Expressing a Foreign Metallothionein Gene
Int. J. Phytoremediation 1: 53-65.
Peer-reviewed reprintsAdhiya, J., Cai, X-H., Traina, S. and R. T. Sayre. 1999, Binding of Aqueous Cadmium by the Lyophilized Biomass of Chlamydomonas reinhardtii
Journal of Colloid and Interface Science. RS-357
Peer-reviewed reprintsCai, X-H., Brown, C. Adihuya J. Traina, S. and R.T. Sayre. 1998, Heavy Metal Binding Properties of Wild Type and Transgenic Algae (Chlamydomonas sp.)
Pgs. 199-202. In New Developments in Marine Biotechnology. Le Gal, Y. and Halvorson, H. eds.; Plenum Press.
Conference, symposia, or workshop proceedings, and summaries
Conference, symposia, or workshop proceedings, and summariesSiripornadulsil, S. and R.T. Sayre. 1999, pSSCR7 a Transformation Vector for Expression of Foreigh Genes in the Nuclear Genome of Chlaydomonas reinhardtii
Northeast Algal Society Meeting, Plymouth, MA.
Conference, symposia, or workshop proceedings, and summariesSayre, RT 2000, Applications of genetically altered algae for the recovery of toxic trace metals from the environment
US-China Chemical engineering: Environmental Symposium, Beijing, Sept 25-28, 2000.
Conference, symposia, or workshop proceedings, and summariesCai, X-H, Brown, C., Malek, L. and R.T. Sayre. 1998, Charaterization of Heavy Metal Detoxification Systems in Wild Type and Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene
8th International Conference on the Cell and Molecular Biology of Chlamydomonas; Lake Tahoe, CA.
Conference, symposia, or workshop proceedings, and summariesSayre RT, Siripornadulsil S and Pal Verma, DP. 2000, Enhancement of the heavy metal binding capacity of Chlamydomonas reinhardtii cells by expression of foreign genes regulating cysteine and proline synthesis
9th International Conf. On the Cell and Molec. Biol. Chlaymdomonas. Amsterdam, Netherlands.
Conference, symposia, or workshop proceedings, and summariesOriecuia, V., Malek, L., Adhiya, J., Traina, S. and R. Sayre. 1999, Heavy Metal Binding by Chlamydomonas reinhardtii
Northeast Algal Society Meeting, Plymouth, MA.
Conference, symposia, or workshop proceedings, and summariesSayre RT. 2000, Heavy metal binding capacity of Chlamydomonas cells expressing genes encoding synthetic heavy metal binding proteins
Biotechnology of Microalgae Conference Potsdam, Germany.
Conference, symposia, or workshop proceedings, and summariesSayre RT. 2000, Metal grabbing algae: bioengineering algae to cleanup heavy metal pollution
Department of Biology, Lakehead University, Canada. Also presented at Environmental Education Council of Ohio, Newark, OH.
Conference, symposia, or workshop proceedings, and summariesRubinelli, P., Chulikorn, E., Malek, L. and R.T. Sayre. 1999, Modulation of ABC-transporter Activity in Chlamydomonas reinhardtii for Improved Bioremediation: Pilot Study of Organic Absorptioon and Controlled Efflux.
Northeast Algal Society Meeting, Plymouth, MA.

Supported Students

StudentSurasak Siripornadusil (Graduate)
The Ohio State University