Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme

Abstract

A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-def icient mutant accumulated little UOf+, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface-and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO,PO,AH,O. Nucleation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of NMR-silent ' 'l2Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.

Engineering ofDeinococcus radioduransR1 for Bioprecipitation of Uranium from Dilute Nuclear Waste

“…4B). Both clones removed more than 90% of the uranium from solutions containing 0.8 mM uranyl nitrate in about 6 h. This is much superior to the uranium precipitation ability reported for native Citrobacter strains (strain N14, PhoN activity of 400 to 500 U and 37 to 61% uranium precipitation in 6 h; strain dc5c, PhoN activity of 850 U and 58% uranium precipitation in 6 h) under similar experimental conditions (10,20) for an engineered and immobilized E. coli strain expressing PhoN (PhoN activity of 100 to 150 U and 98% uranium precipitation in 400 h) (1). While E. coli cells (both wild type and transformants) showed considerable radiosensitivity, the transfer of phoN to Deinococcus radiodurans did not compromise its radioresistance (Fig.…”

“…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 (12,19). Other organisms capable of precipitating metals up to nine times their biomass have been known (10). However, the sensitivity of such bacteria to the adverse effects of radiation makes them unsuitable candidates for remediating radioactive waste.…”

“…and recombinant Escherichia coli expressing multiple copies of a nonspecific periplasmic acid phosphatase (PhoN) (10) have been reported to precipitate heavy metals efficiently (1,19). 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 (12,19).…”

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

“…The periplasmic nonspecific acid phosphatases are known to concentrate at cell poles in Gram-negative bacteria (41)(42)(43). In accordance with this, the E. coli(pPN1) cells under GC1 displayed a periplasmic location of the precipitate at the poles (within the cells).…”

Genome Sequence of Citrobacter sp. Strain A1, a Dye-Degrading Bacterium

“…is a ubiquitous Gram-negative enteric coccobacillus from the family of Enterobacteriaceae. Despite the pathogenicity of Citrobacter sp., biotechnological significance of the bacterium was reported mainly in dye decolorization, heavy metal precipitation, and biohydrogen production (1,6,7,10). A local strain of Citrobacter sp., designated strain A1, was isolated from a sewage oxidation pond in the vicinity of Universiti Teknologi Malaysia in 1997 (N. A.…”