Mei-Fang Chien

Mercury removal and recovery by immobilized Bacillus megaterium MB1

From several mercury removing microorganisms, we selected Bacillus megaterium MB1, which is nonpathogenic, broad-spectrum mercury resistant, mercuric ion reducing, heat tolerant, and spore-forming, as a useful bacterium for bioremediation of mercury pollution. In this study, mercury removal performance of the immobilized B. megaterium MB1 was investigated to develop safe, efficient and stable catalytic bio-agent for mercury bioremediation. The results showed that the alginate gel immobilized B. megaterium MB1 cells efficiently removed 80% of mercury from the solution containing 10 mg/L mercuric chloride within 24 h. These cells still had high activity of mercury removal even after mercuric ion loading was repeated for nine times. The analysis of mercury contents of the alginate beads with and without immobilized B. megaterium MB1 suggested that a large portion of reduced metallic mercury was trapped in the gel beads. It was concluded that the alginate gel immobilized B. megaterium MB1 cells have potential to remove and recover mercury from mercury-containing water.

Mercury resistance transposons in Bacilli strains from different geographical regions

A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, Tn(d)MER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). Tn(d)MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from Tn(d)MER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn(d)MER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers.

Construction of a Cell Surface Engineered Yeast Aims to Selectively Recover Molybdenum, a Rare Metal

The depletion of rare metals is an issue of major concern since rare metals are limited in the abundance but essential for high technology industry. However, the present rare metal recovery technical by chemical methods has high environmental impact, poor selectivity, and is too expensive to be practical. To resolve these problems, this study aimed to create a rare metal recover system using yeast, and molybdenum was selected as the first target. A molybdenum binding protein, ModE, which was derived from Escherichia coli was selected. A fusion gene was generated by linking partial modE with a secretion signal and a domain of α-agglutinin to display the ModE on the surface of yeast cells. The expression of fusion protein on the cell surface was detected by immunofluorescence labeling. As for the recovery experiment, the engineered yeast cells were incubated in 10 mM of sodium molybdate solution for 2 h, and the recovery of molybdenum ion was measured by ICP-AES. The results of fluorescence micrographs showed that the designed fusion protein was successfully expressed on yeast cell surface. According to the results of ICP-AES, the cell surface engineered yeast adsorbed molybdenum and the cells after 72~84 h incubation gave the best adsorption. Besides, the results suggested that the optimization of each functional domain in the fusion protein was important. The selectivity and the lower limit of recoverable concentration are under investigation, while this study provides a preliminary result of bio-extraction technology using cell surface engineered yeast.