Chemical/Genetical Modification Of Microbial Cell For Heavy Metal Separation And Analysis/Speciation

Bioaccumulation has a wide prospect in the field of heavy metal removal and analysis (speciation). As a novel material for heavy metal adsorption/extraction, biosorbent has a lot of advantages such as wide source, low cost, sufficient binding site, high adsorption capacity et al. However, the diversity of various binding sites leads to the low selectivity towards a certain metal ion, which become the bottle neck of bioaccumulation. Therefore, it is crucial to improve the selectivity of cells and develop new recognition methods based on the advantage of cell itself. Chemical modification and genetic engineering can artificially manipulate the component of the surface of cells, thus can effectively enhance the capacity and selectivity towards target metal. So far, however, the application of chemically/genetically modified cells in the field of heavy metal removal is just at the beginning, and has not been employed for the purpose of heavy metal analysis, thus has a prospective potential to be developed.The present work aims at improving the selectivity of microbial cells towards a certain metal via chemical modification method and genetic engineering technique. Meanwhile, various important issues during the process of cell accumulation of metal species and the accumulation mechanisms of these modified cells are also part of our concern. The final goal of the studies is to explore the potential of modified cells for the selective removal of heavy metal, as well as to develop selective preconcentration and speciation procedures for heavy metal species.Chapter 1 is a brief introduction of the feature, classification and mechanism of bioaccumulation, as well as its application in heavy metal remediation and trace metal preconcentration/speciation.In Chapter 2, Bacillus subtilis was loaded with Fe(III) by a simple method, and was then applied for the adsorption of As(III) and As(V). Incubating the bacteria with Fe(III) causes iron uptake (up to-0.5% w/w), and some of the iron attaches to the cell membrane as hydrous ferric oxide (HFO) with additional HFO as a separate phase. Remarkably,30% of the Bacillus subtilis cells remain viable after treatment by 8 mM Fe(III). At pH 10 both arsenic forms are sorbed, the As(V) sorption capacity of the ferrated Bacillus subtilis is at most 11 times higher than that of the native bacteria. At pH 8 (close to pH of most natural water), the arsenic binding capacity per mole iron for the iron loaded bacteria is greater than those reported for any iron containing sorbent. At pH 3, upon metallation, As(III) binding capacity becomes-0, while that for As(V) increases more than three times, offering an unusual high selectivity for As(V) against As(III).Based on the observation in Chapter 2, a sensitive inorganic arsenic speciation method was thus developed in Chapter 3. When pH is 3, the adsorption efficiency of As(V) onto iron loaded bacteria reached 96%, while that of As(III) is nearly zero. The retained As(V) can be quantitatively eluted by HNO3, and its concentration can be determined by GFAAS afterwards; the concentration of As(III) can be calculated by subtraction. By using 1 mL sample solution,100 μL eluent (0.8 mol L-1 HNO3), a detection limit of 0.08 μg L-1 (3a, n= 7) is achieved along with a precision of 4.0% RSD (1.0 μg L-1, n=9) within a linear range of 0.30-2.00 μg L-1. Meanwhile, multi spectra also revealed that replacement of Fe-bound-OH groups with-OAsO(OH)2 moieties and the formation of As-O-Fe linkage is the possible mechanism of interaction between As(V) and iron loaded cell.In Chapter 4, the arsenic regulatory protein ArsR was expressed in Escherichia coli by cell engineering, which significantly enhances the adsorption/accumulation capacity of methylated arsenic species. The ArsR expressed E. coli cells (denoted as E. coli-ArsR) give rise to 5.6-fold and 3.4-fold improvements on the adsorption/ accumulation capacity for monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) with respect to native E. coli cells. The uptake of MMA and DMA by the E. coli (ArsR) is a fast process which fitting Langmuir adsorption model. The competitively uptake of foreign metal ions suggests an improved selectivity towards MMA and DMA after ArsR expression. Furthermore, the accumulation of MMA and DMA is less sensitive to the variation of pH value and ionic strength changes with respect to the blank control cells. In addition, the effective removal of MMA and DMA at lower concentration illustrates a great potential for the E. coli-ArsR for selective remediation of methylated arsenic species in waters, even in the presence of high concentration of salts.In Chapter 5, the potential of selective cell-sorption for separation/ preconcentration of ultra-trace heavy metals was exploited by surface engineering of Saccharomyces cerevisiae cells. The general idea is to display the cadmium binding peptide on cell surface in order to enhance the covalent interaction between cadmium and the yeast cells. By immobilizing the surface engineered yeast cells onto micro-carrier beads Cytopore for cadmium adsorption, we demonstrated that with respect to the native yeast 600-fold and 25-1000 fold improvements were observed respectively for the tolerance of ionic strength and the tolerant capability toward various metal cations after surface engineering. Based on these observations, a novel procedure for selective cadmium preconcentration was developed with detection by graphite furnace atomic absorption spectrometry (GFAAS), employing the engineered S. cerevisiae cell loaded Cytopore beads as renewable sorption medium incorporated into a sequential injection lab-on-valve system. The cadmium retained on yeast cell surface was eluted with a small amount of nitric acid and quantified with GFAAS. Within a range of 5-100 ng L"1 and a sample volume of 1 mL, an enrichment factor of 30 was achieved along with a detection limit of 1.1 ng L-1, a sampling frequency of 20 h-1 and a precision of 3.3% RSD at 50 ng L-1. The procedure was validated by analyzing cadmium in certified reference materials and a series of environmental water samples.