Separation and chemical analysis was investigated using bitumen samples from Athabasca oil sand in Alberta. Fractionation according to solubility and polarity has been used to separate bitumen into its fractions. The solvent de-asphaltening was performed by n-pentane solvent (solubility fractionation), and the polarity fractionation using Fuller's earth allows maltene to separate into SARA components (saturates, aromatics, resins and asphaltenes). The SARA components are analyzed comprehensively using elemental analysis (EA), Fourier-transformed infrared (FT-IR), ultraviolet-visible spectroscopy (UV-vis), high performance chromatography (HPLC) and thermogravimetric analysis (TGA). EA (C, H, N, S), heavy metals (Ni, V) concentrations, FT-IR and UV-vis tests provided the explanation of chemical composition. From IR spectra, maltene and saturates/aromatics (sat/aro) contained more aliphatic compounds than resin or asphaltene. Also, IR spectrum of sat/aro was similar to crude oil and VGO (vacuum gas oil). Different UV signal data clearly indicates the contribution of aromatic constituents in the fractions. Using optimized analysis conditions of HPLC, we successfully separated the peaks for bitumen and its fractions. The characteristic peak pattern of SARA (saturates, aromatics, resins, asphaltenes) fractions was observed, and also the peak pattern of sat/ aro was similar to that of crude oil and VGO. However, TGA results revealed that thermal behavior for sat/aro was similar to that of crude oil but different from that of VGO. Also, from the comparison between decomposition temperature of TGA and boiling point, their correspondence was found.

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Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (g-Glu-Cys) n moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and g-glutamyl cysteine (g-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3D strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n ¼ 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn 2þ enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

Phytochelatins (PCs) with good binding affinities for a wide range of heavy metals were exploited to develop microbial sorbents for cadmium removal. PC synthase from Schizosaccharomyces pombe (SpPCS) was overexpressed in Escherichia coli, resulting in PC synthesis and 7.5-times-higher Cd accumulation. The coexpression of a variant ␥-glutamylcysteine synthetase desensitized to feedback inhibition (GshI*) increased the supply of the PC precursor glutathione, resulting in further increases of 10-and 2-fold in PC production and Cd accumulation, respectively. A Cd transporter, MntA, was expressed with SpPCS and GshI* to improve Cd uptake, resulting in a further 1.5-fold increase in Cd accumulation. The level of Cd accumulation in this recombinant E. coli strain (31.6 mol/g [dry weight] of cells) was more than 25-fold higher than that in the control strain.Widespread pollution by heavy metals generated from various industrial and agricultural activities has serious adverse effects on human health and ecosystems (12). The increased public concern over these heavy metals has caused more-stringent control of the allowable limits in drinking water and soil. Although conventional technologies are adequate to remove the bulk of heavy-metal contamination, they are often inadequate to reduce heavy-metal concentrations to acceptable regulatory standards (8). Bioremediation based on genetically engineered bacteria is an emerging technology that is receiving more attention as an inexpensive and efficient way of cleaning up toxic-metal contamination (10). In particular, the production of metal-binding peptides such as metallothioneins and phytochelatins (PCs) has been shown to confer enhanced heavy-metal-binding capabilities (1,2,18).PCs are naturally occurring peptides consisting of the repeating ␥-Glu-Cys dipeptide unit terminated by a Gly residue (6,21). The presence of a ␥ bond between glutamic acid and cysteine indicates that the synthesis of PCs cannot occur via the ribosomes. PC biosynthesis, indeed, proceeds through the transfer of ␥-Glu-Cys from glutathione (GSH) to another GSH or other PCs (21) by the enzyme PC synthase (PCS) when this enzyme is activated by heavy metals such as Cd, Cu, Hg, and Pb. PCs are known to bind heavy metals such as Cd, Hg, As, and Pb, especially cadmium, with high affinity through thiolate complexes (7, 16). Recently, the genes coding for PCS have been cloned from plants and fungi and functionally expressed in Escherichia coli (15). The intracellular cadmium content of the E. coli strain expressing PCS increased more substantially than that of the control strain. A direct correlation between PC content and metal accumulation was observed, indicating that PC is primarily responsible for the metal sequestration.Although the initial results of the previous study (15) demonstrated the possibility of heavy-metal remediation using PCproducing cells, a severe drop in the GSH content suggests that the GSH supply may be the limiting step in PC synthesis. In E. coli, the biosynthesis of GSH proceeds via ...