Team:Lyon-INSA-ENS/Project/Context

Cobalt Buster Project

• Overproduction of curli via a synthetic operon controled by a cobalt-inducible promoter Prcn-csgBAEFG


• Overexpression of curli genes via the superactivator OmpR234


• Engineering E. coli adhesion for improved bioremediation

Overproduction of curli via a synthetic operon controled by a cobalt-
inducible promoter Prcn-csgBAEFG

Overexpression of curli genes via the superactivator OmpR234

Engineering E. coli adhesion for improved bioremediation

Biofilms and depollution. Often associated to disease and unwanted surface fouling, biofilms are helpful in bioremediation, biocatalysis or as microbial fuel cells. Bioremediation processes use natural microbial ability to degrade organic substances or to modify metal speciation by immobilization or volatilization. Such properties are observed in natural ecosytems as in artificial systems used to clean solid or liquid waste. Intensity and quality of the microbial activities depend on local physical and chemical factors, and also on the way of life of microbes (biofilm or plankton). Biofilm formation is associated to resistance to most of biocides by diverse mechanisms. Adhesion is therefore a choice property in most remediation processes.

Strategy: boost natural abilities! Binding to extracellular matrix, efflux pumps and activation of transporters allow concentration and sequestration of biocides such as metals. Genetic engineering allows to boost these activities and to improve the treatment of metallic pollution, especially for toxic metals in low concentration. Classic chemical processes using ion-exchange resins are then economically inappropriate, and thanks to their high selectivity, micro-organisms appear very efficient.

OGM biofilters for nuclear liquid waste treatment. Treatment of nuclear waste is a promising application for biological treatment of metals contaminations. Confinement is indeed a major hindrance to the use of Genetically Modified Organisms for waste treatment. Since radioactive waste are submitted to a strict and regulated handling, use of GMO in this context should be well-accepted by the society. The activity of modern nuclear power plants with pressurized water reactors generates radioactive effluents that contain among others radioactive cobalt. The tubing of the cooling circuit is made of a steel alloy rich in cobalt and nickel. Under neutrons bombardment coming from the reactor, these stable metals change into radioactive isotopes.

Undergoing neutron bombardment coming from the reactor , stable metals change into 60Co (half-life = 5.3 years) and 58Co (half-life = 71 days). The capture of cobalt is interesting on a sanitary point of view, because it represents a danger under both its radioactive and stable forms (carcinogenic). It also represents an advantage on an environmental point of view, in order to avoid contamination of waters, soil and groundwater. Even with a short half life, cobalt 60 emits high intensity gamma rays, and decays to nickel, which is stable but polluting.

Corrosion results in solubilization of these activation products, and water contamination.

Selective cobalt capture. Controlled immobilization of radioactive cobalt is both an important sanitary and environmental issue. Activation products are routinely captured by using synthetic ion exchangers. This generates large volume of solid waste due to the nonspecific nature of ion sorption. In this context, a researcher from the Lyon INSA-ENS team has recently constructed an E.coli strain able to eliminate 85% of radioactive cobalt initially present as traces in a simulated nuclear effluent.

An efflux gene rcnA* knockout mutant of the E. coli was engineered to produce a transporter with preferential uptake for cobalt (NiCoT). The process that was developed by Agnès Rodrigue and her Indian colleagues ensures the decontamination of cobalt up to 0,5 ppm (8 nM in 100 000L) with only 4kg of bacteria as against 50kg with an unmodified bacterium or 8,000kg of an ion-exchange polymer in only twice one-hour incubations. This kind of process with modified bacteria will be a good value because the production of bacteria in a bioreactor is economical. (Appl Microbio Biotechnol 2009 81:571- 578).
* rcnA = resistance to cobalt and nickel

However, the recovery of cobalt-fixing bacteria has to be facilitated before to consider industrial application.

“Cobalt Buster” biofilter. Our objective is to facilitate the recovery of the metal-stuffed bacteria by inducing their fixation to a solid support. (France 3 movie?). We choose to engineer this sought-after adherence property by using the exceptional properties of the curli amyloid fibers. In a first approach, a synthetic operon comprising the absolutely required genes for curli production under control of a strong and cobalt-inducible promoter was designed and synthesized. This construct allows K12 E. coli (MC4100, MG1655, NM522…) to stick to polystyrene and glass. Adherence is reinforced by the presence of cobalt and should avoid free floating growth. In a second approach, a part allowing the constitutive overproduction of the curli superactivator OmpR234 was constructed. By activating the cryptic curli genes located in the core genome of K12 E. coli, this part allows to increase bacterial adherence to polystyrene and glass. Such results lead us to discuss of a possible industrialization with the ASSYSTEM company and of research and development perspectives with the EDF company.