Bioremediation of Radioactive Waste by Engineered D. radiodurans

Background. Radioactive waste has been produced in Canada since the early 1930s when the first uranium mine began operating at Port Radium in the Northwest Territories.

Since then, the uses of nuclear energy have become more diverse and widespread. Operation of the CANDU (Canada Deuterium Uranium) power reactors generates nuclear fuel waste or high-level waste which remains radioactive for thousands of years (1). At present, there is no permanent disposal facility for nuclear fuel waste. Nuclear fuel waste is kept in wet or dry storage at the reactor sites and at the Atomic Energy of Canada Limited (AECL) waste management facilities at Chalk River, Ontario. Ontario Hydro, operating 12 reactors produced the majority of waste (1,928 m3 per year) which accounted for around 86% of nuclear fuel waste of the country.

There are over two million fuel bundles now in storage, enough to fill five hockey rinks to the top of the boards (1). Because of the high costs and environmental disturbances associated with current cleanup technologies, bioremediation technology has the potential to be implemented in radioactive waste material management.

Bioremediation is a process that includes the use of organisms such as bacteria, fungi, and plants to remove, degrade, or detoxify pollutants from contaminated environmental sites. Previously, microorganisms such as Deinococcus radiodurans, Methanococcus jannaschii, and Geobacter sulfurreducens have been investigated for bioremediation of radioactive materials (2-4). Different bacteria use different mechanisms including biotransformation, bioaccumulation, and biosorption to remediate the radioactive waste (5, 6).

Extracellular enzymatic detoxification by microbes such as Desulfovibrio desulfuricans, Geothrix fermentans, Deltaproteobacteria, and Clostridium was found to be the mechanism of radionuclides biotransformation, including uranium (U), technetium (Tc), and chromium (Cr) (7,8). Oxidized forms of these radionuclides are highly soluble in aqueous media and mobile in ground water, whereas reduced forms are insoluble and often precipitable into the solution (9, 10). In bioaccumulation and biosorption however, radionuclides are transported into the cytoplasm through the cellular membrane of microbial cell such as Micrococcus luteus, Arthrobactor nicotianae, Bacillus megaterium, and Citrobacter sp. N14 (11, 12).

Bacillus sphaericus for example uses ionisable groups in the cell surface to accumulate uranium (13). The tiny bacterium Deinococcus radiodurans-first isolated from a meat tin during radiation sterilization experiments in the 1950s-has been engineered to express the genes for toluene degradation from common soil bacteria. Numerous existent microorganisms have the ability to bioremediate wastes; however, D. radiodurans can withstand the extreme environments that are found at most radioactive sites (14).

The recombinant organism is capable of surviving exposures to continuous high-level radiation and toxic solvents while degrading target compounds. Its radiation resistance however is not achieved by blocking penetration; rather, it rapidly repairs damage to its DNA (14). Moreover, D. radiodurans was successfully engineered to sequester or detoxify radioactive compounds such as Cr (VI) and Tc (VII), along with other toxic metals or organic chemicals (15, 16). In addition, D. radiodurans transformed with Escherichia coli gene (merA) was able to assimilate carbon and utilize the energy, driven from mercury and toluene (radioactive contaminants) catabolism (17).

Thus, engineered D. radiodurans can be a promising candidate for bioremediation of high-level radioactive wastes as well as mixed radioactive wastes containing both organic and metallic components. While there has been significant progress in engineering D. radiodurans for remediation of radioactive waste environments (14, 17, and 18), prospective treatment of contaminated sites with engineered D. radiodurans will be limited to temperatures below 39°C; its maximum growth temperature.

However, there is a need to develop bioremediating bacteria that are resistant to both radiation and high temperatures because of the existence of thermally insulated contaminated environments, where temperatures are elevated by the decay of long-lived radionuclides (e.g., 137Cs and 90Sr) (14).

Therefore, urgency arose to develop, a bacterium that can resist radiation and perform their metabolic activities at higher temperature. Moreover, wild-type D. radiodurans has also been shown to reduce limited amount of uranium only in the presence of humic acids under strict anaerobic conditions (16). Such restrictions effectively reduce the efficiency of microbes to detoxify the radionuclides, and have to be overcome by engineering new pathways in bacteria such as D. radiodurans.

In this proposal, I address the following question: how to overcome limitations such as high temperature and substrate-specific requirements in D. radiodurans? This study can develop into a larger program by transferring other bioremediation genes to sequester or detoxify radioactive compounds along with other toxic metals or organic chemicals in a complex radioactive waste site. Short-term objective: 1. Engineering of D. radiodurans for bioremediation of high-temperature radioactive waste environments.

2. Engineering of D. radiodurans for bioprecipitation of uranium from nuclear waste. Long-term objective: The study will develop into a long-term program to investigate genes involved in detoxifying radioactive compounds, toxic metals and organic chemicals in a complex radioactive waste site.

Following exploration of those genes, I plan to develop genetic engineered D. radiodurans which will be able to tolerate extreme environmental conditions and efficiently be able to bioremediate the complex radioactive waste. This will cut the cost currently applied in different remediation methods to treat mixed waste pollution. The goal is to use what we know from remediation of other types of wastes and extend them to the treatment of mixed wastes.

Individual research components and methodological approach 1. Engineering of D. radiodurans for bioremediation of high-temperature radioactive waste environments. i) Cloning of high-temperature resistance gene from tobacco (Nicotiana tabacum) into D. radiodurans. Under heat-shock conditions, all organisms synthesize several families of evolutionary conserved heat-shock proteins (HSPs).

Previously, TLHS1 which is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum), was successfully expressed in Escherichia coli (19). E. coli cells expressing TLHS1 were able to survive at elevated temperature (>50°C) and showed that plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature (19). E. coli strain DH5? containing pBADNH expression vector carrying TLHS1 shall be obtained from Dr. Choo’s lab.

Using specific primers, the gene will be amplified and sub-cloned into Escherichia coli-D. Radiodurans shuttle plasmid pMD66 will be obtained from Dr. Michael lab. pMD66 and its numerous derivatives have been used successfully to functionally express cloned genes in D. radiodurans growing under chronic irradiation (18, 20, and 21).

Different antibiotic resistance genes are provided in the plasmid for the bacteria selection. In addition, wild type D. radiodurans will be purchased from ATCC (ATCC® Number: 19172™). Wild type D. radiodurans will be transformed with pMD66 plasmid carrying TLHS1 based on common bacterial transformation techniques.

ii) Selection of the transformed D. radiodurans based on their ability to tolerate elevated temperature in the presence of radiation. Growth of the transformed cells in the presence of chronic ?-irradiation, 50 Gy/h (137Cs Gammacell 40 irradiation unit [Atomic Energy of Canada Limited]), will be carried out at different temperatures ranging from 30 to 55 °C.

For the radiation experiments, I will use the facilities at Northwestern Ontario Regional Cancer Center in Lakehead University. Wild-type E. coli and wild-type D. radiodurans will be used as negative and positive controls respectively. Survival rates will be determined by plating appropriate dilutions of irradiated cells and counting the colony forming units per plate.

In addition, the transformed cells will be selected based on their ability to survive at the presence of irradiation along with their resistance to the elevated temperature. These new engineered D. radiodurans will be used for further genetic engineering experiments, including transformation of other bioremediation genes such as bioprecipitation of uranium in a complex radioactive waste site.

2. Engineering of D. radiodurans for bioprecipitation of uranium from radioactive waste sites. i) Cloning of high efficient uranium precipitation gene into the recombinant D. radiodurans. Uranium U (VI) is the most common radionuclide contaminant at the sites of nuclear complexes. U (VI) can be immobilized microbiologically from groundwater by its reduction from UO22- to insoluble U (IV) oxide (22). It has been shown that nonspecific periplasmic acid phosphatase (PhoN) hydrolyzes organic phosphates, and the inorganic phosphate thus released, interacts with the metal and precipitates it on the cell surface as insoluble metal phosphate (23, 24).

One of the promising bacteria which have the gene and ability to precipitate uranium is Salmonella enterica serovar Typhi. However, the sensitivity of such bacteria to the adverse effects of radiation makes them unsuitable candidates for remediating radioactive waste. In this study, we will clone and express the phoN gene of Salmonella enterica serovar Typhi (to be purchased from ATCC [ATCC® Number: 6994™]) in D. radiodurans using common molecular biology techniques such as RT-PCR, PCR, and etc.

The primers for PCR amplification will be designed on the basis of the published sequence of the phoN gene of Salmonella enterica serovar Typhimurium (GenBank accession no. X59036).

ii) Selection of the transformed D. radiodurans based on acid phosphatase expression and their ability to precipitate uranium in the presence of radiation.

The recombinant D. radiodurans clones will be grown in the presence of chronic ?-irradiation as described before and the expression of the enzyme will be measured using the cell extracts. Uranium precipitation ability of D. radiodurans clones harbouring phoN will be tested by growing the cells in presence of uranyl nitrate. Aliquots will be taken at different time intervals and subjected to centrifugation. Residual uranium in the supernatant and uranium associated with the cell pellet will be estimated using the method of Fritz and Bradford (25). Lakehead University is getting a certificate for the radioactive experiments and we will set it up in our lab.

Significance of the proposal. Recombinant D. radiodurans will be capable to grow significantly at a higher temperature (55°C) and also have remarkable resistance against chronic irradiation (50 Gy/h). Moreover its ability to precipitate uranium, the most common radionuclide contaminant at the sites of nuclear complexes, may be a robust candidate for treatment of radioactive waste sites. The study may also have broader applications, such as transferring other genes to develop a bacteria strain which will be able to work in a complex of radioactive wastes/heavy metal environment.

Training throughout proposal. Training HQP will be the cornerstone of the research program. Graduate and undergraduate students working on this project will be exposed to a multidisciplinary research environment combining various approaches of biotechnology, molecular biology, microbiology and biochemistry.

The trainees will have excellent opportunities to learn and apply advanced techniques of biotechnology and molecular studies. Three MSc students will be working on the research program (one student during years 1-2, second during 3-4 and third student will be involved in studies during year 5 of the program). Training of undergraduate students will also be a priority during the research program (4th year research thesis projects and summer research students).

Their practical experience during the training will help to define their future scientific goals. The theoretical knowledge and practical skills gained by students may encourage them to pursue careers in biotechnological research and industry. HQP trained with a multidisciplinary approach will surely contribute exceptional and lasting benefits to Canada.